Exploration of Microbially Inoculated Biochar for...

Exploration of Microbially Inoculated Biochar for

Plant Growth Promotion

By

Mazhar Rafique

Department of Plant Sciences

Faculty of Biological Sciences

Quaid-i-Azam University

Islamabad

2018

Exploration of Microbially Inoculated Biochar for


A thesis submitted in partial fulfillment of the requirements for the

degree of Doctor of Philosophy

In

Plant-Microbe Interactions

By

Mazhar Rafique

Department of Plant Sciences

Faculty of Biological Sciences

Quaid-i-Azam University

Islamabad

2018

CERTIFICATE

This is to certify that this thesis entitled as “Exploration of Microbially Inoculated

Biochar for Plant Growth Promotion” submitted by Mr. Mazhar Rafique is accepted

in its present form by the Department of Plant Sciences, Faculty of Biological Sciences,

Quaid-i-Azam University, Islamabad as satisfying the thesis requirement for the degree

of Doctor of Philosophy (PhD) in Plant Sciences.

SUPERVISOR _____________________________

Dr. Hassaan Javed Chaudhary

Dated:

Author’s Declaration I Mr. Mazhar Rafique hereby state that my PhD thesis titled “Exploration of

Microbially Inoculated Biochar for Plant Growth Promotion” is my own work and

has not been submitted previously by me for taking any degree from Quaid-i-Azam

University, Islamabad or anywhere else in the country/world.

At any time if my statement is found to be incorrect even after my graduate the university

has the right to withdraw my PhD degree.

Mazhar Rafique

Date: June 25, 2018

Plagiarism Undertaking

I solemnly declare that research work presented in the thesis titled “Exploration

of Microbially Inoculated Biochar for Plant Growth Promotion” is solely my

research work with no significant contribution from any other person. Small

contribution/helo wherever taken has been duly acknowledged and that complete thesis

has been written by me.

I understand the zero tolerance policy of the HEC and Quaid-i-Azam University

towards plagiarism. Therefore, I as an Author of the above-titled thesis declare that no

portion of my thesis has been plagiarized and any material used as a reference is properly

referred/cited.

I understand that if I am found guilty of any formal plagiarism in the above-titled

thesis even after award of Ph.D. degree, the University reserves the rights to

withdraw/revoke my Ph.D. degree and that HEC and the University has the right to

publish my name on HEC/University website on which names of students are placed who

submitted plagiarized thesis.

Mazhar Rafique

DEDICATED

TO

My Parents and

everyone who assisted me in

learning/progress

Table of Contents

Sr. # Title Page No.

Abbreviations i

List f Tables iv

List of Figures vii

List of appendices x

Abstract xi

Chapter 1

1 General Introduction and Review of Literature 1

1.1. Microbes 1

1.1.1. Plnt Growth Promoting Rhizobacteria (PGPR) 2

1.1.2. Phosphorus Solubilizing Bacteria (PSB) 2

1.1.3. Mycorrhizal fungi 4

1.1.4. Co-inoculation of PSB and AM fungi 5

1.2. Biochar 8

1.3. Biochar and microbial interaction for plant growth in

limited nutrients condition

8

1.4. Biochar and microbial interaction for plant growth

under heavy metal stress

12

1.5. Hypothesis 13

1.6. Overall objectives 15

Chapter 2

2.1. Introduction 16

2.2. Objective 18

2.3. Materials and methods 19

2.3.1. Biochemical characterization of bacteria 19

2.3.1.1. Catalase activity 19

2.3.1.2. Oxidase activity 19

2.3.1.3. Phosphate solubilization 19

2.3.1.4. N-fixation quality 19

2.3.1.5. Gelatinase activity 20

2.3.1.6. Citrate utilization test 20

2.3.1.7. Indole acetic acid 20

2.3.1.8. Hydrogen sulfide production 20

2.3.1.9. Urease activity 20

2.3.1.10 Antibiotic resistance test 20

2.3.2. Bacterial strains genetic identification 20

2.3.3. Phylogenetic analysis 22

2.3.4. Biochar preparation and analysis 22

2.3.5. Setting pot experiment and plant-soil analyses 23

2.3.6. Recovery of inoculated bacteria 25

2.3.7. Statistical analysis 25

2.4. Results 26

2.4.1. PSB strains characterization 26

2.4.2. Molecular characterization of PSB 27

2.4.3. Plant height 28

2.4.4. Plant nutrient concentration 30

2.4.5. Soil nutrient concentration 33

2.5. Discussion 36

2.6. Conclusion 39

Chapter 3


3.2. Objective 41


3.3.1. Biochar preparation 42

3.3.2. Electrical conductivity and pH 42

3.3.3. Moisture, volatile matrix and ash contents 42

3.3.4. Total nutrient analysis 43

3.3.5. Carbon analyses 43

3.3.6. Scanning electron microscopy 44

3.3.7. Fourier transform infrared spectroscopy (FTIR) 44

3.3.8. Thermal gravimetric analysis (TGA) 44

3.3.9. Statistical analysis 44

3.4. Results 46

3.4.1. pH and electrical conductivity 46

3.4.2. Moisture, volatile matrix and ash contents 46

3.4.3. Mean nutrient analysis 46

3.4.4. Carbon anlayses 48

3.4.5. Scanning electron microscopy 49

3.4.6. Fourier transform infrared spectroscopy 53

3.4.7. Thermal gravimetric analysis 57

3.4.8. Correlation 65

3.5. Discussion 67

3.6. Conclusion 71

Chapter 4


4.2. Objective 75


4.3.1. Nursery development and plant growth in greenhouse 76

4.3.2. Biochar preparation 77

4.3.3. Pot study setup 78

4.3.4. Plant harvesting and sample preparation 79

4.3.5. Chlorophyll fluorescence measuremet 79

4.3.6. Tissue nutrient analyses and AMF root colonization 80

4.3.7. Recovery of inoculated bacteria 80

4.3.8. Calculations and statistical analyses 81

4.4. Results 82

4.4.1. Chlorophyll fluorescence 82

4.4.2. Tissue macronutrient analyses 82

4.4.3. N- and P- uptake 86

4.4.4. Tissue micronutrient analysis 89

4.4.5. Root colonization and root traits 93

4.5. Dicussion 100

4.6. Conclusion 102

Chapter 5


5.2. Objectives 105


5.3.1. Experimental design and pot study setup 106

5.3.2. Harvest and sample preparation 107

5.3.3. Root characterizatoin 107

5.3.4. Tissue nutrient analyses and microbial root

colonization

107

5.3.5. Calculations and statistical analyses 108

5.4. Results 109

5.4.1. Root characteristics 109

5.4.2. Shoot and root dry weight 113

5.4.3. Root and shoot tissue nutrients analysis 114

5.4.4. Root colonization 116

5.5. Discussion 126

5.6. Conclusion 128

Chapter 6


6.2. Objective 132


6.3.1. Biochar preparation and soil collection 133

6.3.2. Experimental design 133

6.3.3. Gas exchange measurement 134

6.3.4. Tissue nutrient analyses and AMF root colonization 135

6.3.5. Cd extraction and determination in plant 136

6.3.6. Soil P analysis 136

6.3.7. Calculations and statistical anlayses 137

6.4. Results 138

6.4.1. Gaseous exchange 138

6.4.2. Shoot and root dry weight 140

6.4.3. Root colonization and characterization 141

6.4.4. Nutrients concentration in maize shoot and root 144

6.4.5. Cd concentration in plant 147

6.4.6. Soil P concentration 147

6.5. Discussion 149

6.6. Conclusion 152

Summary and Conclusion 153

Literature cited 155

LIST OF ABBREVIATIONS

Abbreviations Full Name

% Percent

A Net assimilation rate of CO2

AB-DTPA Ammonium bicarbonate-diethylenetriaminepentaacetic acid

AMF Arbuscular Mycorrhizal Fungi

ANOVA Analysis of variance

AW Animal waste

B1 Bacillus subtilis

B2 Lisinibacillus fusiformis

BC-1 Bagasse biochar

BC-2 Sawdust biochar

BLAST Basic Local Alignment Search Tool

BNF Biological nitrogen fixation

C Carbon

Ca3(PO4)2 Tri-calcium phosphate

CaCO3 Calcium carbonate

CEC Cation exchange capacity

CFU Colony forming unit

ChM Chicken manure

ChMB Chicken manure biochar

Ci Intercellular CO2

cm Centimeter

CM Cow manure

CMB Cow manure biochar

cmolc kg-1

Centi mole charge per kilogram

CO2 Carbon dioxide

D45

45 days

D65 65 days

DMRT Duncan Multiple Range Test

DNA Deoxyribonucleic acid

dS m-1

DeciSiemens per meter

DTA Differential thermal analysis

DT Differential thermal

E Transpiration rate

EC Electrical conductivity

EM Ectomycorrhizal

ERM Ericoid mycorrhizal

ESEM Environmental Scanning Electron Microscope

Eu Eucalyptus

EuB Eucalyptus biochar

F0 Minimal fluorescence in the dark-adapted state

FC Fixed carbon

FeCl3–HClO4 Ferric chloride–perchloric acid

Fm Maximal fluorescence in the dark-adapted state

Fv Difference between maximum fluorescence and minimum

fluorescence

FS Feedstock

FTIR Fourier transform infrared spectroscopy

g kg-1

Gram per kilogram

gsw Stomatal conductance to water vapor

H2O2 Hydrogen peroxide

ha-1 Per Hectare

hr Hour

IAA Indole acetic acid

ICP-OES Inductively coupled plasma optical emission spectrometry

kg Kilogram

LB Lauria-Bertani

M1 B. subtilis strain 18MZR + bagasse biochar

M2 L. fusiformis strain 31MZR + bagasse biochar

M3 B. subtilis strain 18MZR + sawdust biochar

M4 L. fusiformis strain 31MZR + sawdust biochar

MEGA Molecular Evolutionary Genetic Analysis

mg kg-1

Miligram per kilogram

MOP Muriate of Potash

mS Mili simens

MUSCLE Multiple Sequence Comparison by Log-Expectation

NaHCO3 Sodium bicarbonate

NCBI National Center for Biotechnology Information

NH4OAC Ammonium acetate

NIST National Institute of Standards and Technology

NO3-N Nitrate nitrogen oC Degree Celcius

OC Organic carbon

OD600 Optical density at 600-nanometer wavelength

PCR Polymerase chain reaction

PGP Plant Growth Promotion

PGPR Plant Growth Promoting Rhizobacteria

Ph Phragmites

PhB Phragmites biochar

Pi Orthophosphate

PSB Phosphorus Solubilizing Bacteria

PSF Phosphorus solubilizing fungi

PTE Potentially toxic element

rRNA Ribosomal ribonucleic acid

S Sludge

SAS Statistical Analysis System

SB Sludge biochar

SD Sawdust

SDB Sawdust biochar

SEM Scanning electron microscopy

SM Sheep manure

SMB Sheep manure biochar

SO2 Sulfur dioxide

SOM Soil organic matter

TC Total carbon

TGA Thermal gravimetric analysis

TG Thermal gravimetric

VM Volatile matrix

TABLES


2.1 Properties of Biochar used in study 23

2.2 Biochemical characteristics of bacteria used in study 26

2.3 Plant nutrients concentration at D45 and D65 harvesting 32

2.4 Soil nutrients concentration at D45 and D65 harvesting 34

2.5 Pearson‘s correlation coefficients among plant and soil

parameters at D45

35

2.6 Pearson‘s correlation coefficients among plant and soil

parameters at D65

35

3.1 pH, EC, proximate, ultimate analyses and nutrient

elements of biochar

47

3.2 Stable, unstable and calcium carbonate content in biochar 48

3.3 FTIR spectroscopy wave number (cm-1

) for feed stocks

and there respective biochar

55

3.4 Pearson‘s correlation values among biochar properties for

quality assessment

66

4.1 Soil properties before addition of chemical fertilizer and

biochar amendment

77

4.2 Properties of biochar used as soil amendment 78

4.3 Chlorophyll fluorescence ( Fv/Fm) of the onion plant

under different soil conditions

82

4.4 Concentration of N (%) in plant shoot under different soil

conditions and P application

84

4.5 Concentration of P (%) in plant shoot under different soil


85

4.6 Concentration of K (%) in plant shoot under different soil


87

4.7 Nitrogen and Phosphate uptake (%) under different soil


88

4.8 Concentration of Cu (ppm) in plant shoot under different

soil conditions and P application

90

4.9 Concentration of Mn (ppm) in plant shoot under different


91

4.10 Concentration of Zn (ppm) in plant shoot under different


92

4.11 p-values from analysis of variance for macronutrients of

shoot and root

98

4.12 p-values from analysis of variance for micronutrients of

shoot and root

99

5.1 Root length of maize plant influenced by biochar-

microbial interaction

110

5.2 Root surface area of maize plant influenced by biochar-


111

5.3 Root volume of maize plant influenced by biochar-


112

5.4 Concentration of P (%) in plant shoot under different soil


117

5.5 Concentration of K (%) in plant shoot under different soil


118

5.6 Concentration of Ca (%) in plant shoot under different


119

5.7 Concentration of Mg (%) in plant shoot under different


120

5.8 Concentration of Cu (ppm) in plant shoot under different


121

5.9 Concentration of Mn (ppm) in plant shoot under different


122

5.10 Concentration of Zn (ppm) in plant shoot under different


123

6.1 Soil properties before fertilization and biochar amendment 136

6.2 Root length, Root surface area and root volume of maize

plant in Cd-spiked soil treated with biochar and AMF.

Values are mean of three replicates

143

6.3 Nutrients concentration in maize shoot in Cd-spiked soil.

Values are means of three replicates

145

6.4 Nutrients concentration in maize root in Cd-spiked soil.

Values are means of three replicates

146

FIGURES


1.1 Schematic representation showing the impact of soil

microbes on the nutrient acquisition and plant productivity

in natural ecosystems

7

1.2 The Hypothetical model is showing mycorrhizal fungi

colonization of plants grown in nutrient-poor and nutrient-

rich soils

11

1.3 The hypothetical relationship between nutrient availability

and the microbial contribution to plant productivity.

12

2.1 Agarose gel electrophoresis of PCR product 16S rRNA for

strain 18 and 31

22

2.2 Phylogenetic tree showing inter-relationship of Strain

18MZR (KX710213) and 31MZR (KX710214).

28

2.3a Root and shoot length of maize plant after 45 days

harvesting for all treatments

29

2.3b Root and shoot length of maize plant after 65 days

harvesting for all treatments

30

3.1 SEM images of biochar obtained at different pyrolysis

temperatures and feedstock sources

52

3.2 Fourier transform infrared (FTIR) spectra of feedstock 54

3.3a TGA-DTA curves of various animal feedstock and their

biochar at 550oC

61

3.3b TGA-DTA curves of plant derived biochar at 550oC 62

3.3c TGA-DTA curves of plant derived biochar at 350oC 64

4.1 Root colonization in onion plants in soils under different

types of biochar Phragmites biochar ( PhB), Sawdust

biochar (SDB) and treatment as control (C), bacteria (B),

mycorrhuiza (M) and bacteria + mycorrhuiza (B+M)

93

4.2 Microscopic picture of root colonization in only mycoohizal 94

(M) inoculated plant and bacteria + mycorrhuiza (B+M)

inoculate the plant

4.3 Root length of onion plant in soils under different types of

biochar Phragmites biochar (PhB), Sawdust biochar (SDB)

and treatment as control (C), bacteria (B), mycorrhuiza (M)

and bacteria + mycorrhuiza (B+M)

95

4.4 Root surface of onion plant in soils under different types of

biochar Phragmites biochar ( PhB), Sawdust biochar (SDB)



96

4.5 Root volume of onion plant in soils under different types of

biochar Phragmites biochar ( PhB), Sawdust biochar (SDB)



97

5.1 Shoot and root dry weight of the maize plant in soil A 113

5.2 Shoot and root dry weight of the maize plant in soil B 114

5.3a Mycorrhizal fungi root colonization (%) in soil A 124

5.3b Mycorrhizal fungi root colonization (%) in soil B 125

6.1a Assimilation rate of CO2 in maize plant leaves in Cd-spiked

soil.Values are mean of three replicates.

138

6.1b Transpiration rate of the maize plant leaves in Cd-spiked

soil. Values are mean of three replicates.

139

6.1c Intercellular CO2 of maize plant leaves in Cd-spiked soil.

Values are mean of three replicates.

139

6.1d Stomatal conductance of the maize plant leaves in Cd-

spiked soil. Values are mean of three replicates

140

6.2 Dry weight of shoot and root of maize plant in Cd-spiked

soil. Values are mean of three replicates.

141

6.3 Root colonization of AMF in maize. Values are mean of

three replicates.

142

6.4 Uptake of Cd in maize. Values are mean of three replicates 147

6.5 Soil P concentration.Values are mean of three replicates 148

List of Appendices


1. Pictures 183

2. ANOVA tables 186

Abstract

Food security is a big challenge and it is getting more importance due to

economic growth, increase in population, and climatic changes. Biochar is a carbon-rich

pyrolyzed material widely used in agriculture as soil amendment for enhanced crop production,

soil quality improvement, C–sequestration, and mitigation of atmospheric C. Soil microbes are

the important component of soil ecosystem which influence ecological components and

processes including nitrogen cycling. The presence of the soil microbes establish a

symbiotic relationship with the plant roots to assist them in nutrients uptake; ultimately

enhancing the plant productivity in limited nutrients condition. Besides the bacterial

symbionts, there is another widespread group of symbiont termed ―mycorrhizal

association‖ in the plant roots which facilitate in the uptake of nutrients (N, P, K, Ca, Mg,

Fe, Cu, Mn, and Zn) from the soil and enhances the plant productivity under limited

nutrients condition. Studies in the present thesis were designed by considering a plant-

microbe-biochar system for plant growth promotion and heavy metal stress tolerance. In

this regard, onion and maize plants system was tested because of their economic

importance in the region of Pakistan and Turkey particularly and worldwide in general.

Onion and maize plant have an absolute requirement of nutrients (N, P, K) for growth

and development. The microbial application can facilitate in addressing limited access to

chemical fertilizer concern. Moreover, biochar and phosphorus solubilizing bacteria

(PSB) community can contribute together in nutrients availability. Both have the P-

supply potential to the soil, but their interaction has been tested less under semi-arid

climatic conditions. The purpose of the study was to evaluate the potential of

biochemically tested promising PSB strains and biochar for maize plant growth and

nutritional status in plant and soil. Therefore, two isolated PSB strains from maize

rhizosphere were biochemically tested in vitro and identified by 16S rDNA gene analysis.

The experiment was conducted in the greenhouse where the plant growth and nutrient

availability to the plants were observed. In this regard, all the treatments such as PSB

strains inoculated plants, biochar treated plants and combination of PSBs + biochar

treated plants were destructively sampled on day 45 (D45) and day 65 (D65) of sowing

with four replications at each time. PSB inoculation, biochar incorporation, and their

combinations have positive effects on maize plant height and nutrient concentration on

D45 and D65. In particular, plants treated with sawdust biochar + L. fusiformis strain

31MZR inoculation increased N (32.8%), P (72.5%) and K (42.1%) against control on

D65. Besides that, only L. fusiformis strain 31MZR inoculation enhanced N (23.1%) and P

(61.5%) than control which shows the significant interaction of PSB and biochar in

nutrient uptake. PSB and biochar have the potential to be used as a promising amendment

in improving plant growth and nutrient absorption besides the conventional approaches.

Multifunctionality of BC makes it valuable to use, however, the heterogeneity in its properties

raises questions on its suitability in a particular environment. The present study was designed to

explore the heterogenic properties of biochar in order to align its use for soil and environment.

Biochar was prepared from sludge (S), animal-waste (AW) and plant-derived feedstocks (FS)

originated from Mediterranean region. Physical and chemical characterization of BC was

performed to evaluate its suitability in the Mediterranean region regarding nutrient availability

concentrations to the plants. Considering that, pH, electrical conductivity (EC), proximate,

ultimate and nutrient analyses were done. Moreover, Scanning Electron Microscopy (SEM) was

performed, and C–stability trend was observed by thermogravimetric analysis. Plant-FS derived

biochar possess high moisture content, volatile matrix, fixed and total carbon (TC) as compared

to sludge biochar (SBC) and AW derived BC. Higher calcium carbonate (CaCO3) contents were

observed in AW derived BC. Furthermore, it is revealed from the porosity of BC that soil

microbes can sustain inside the porous structure when used as soil amendment. Different FS-

oriented biochar can be used as a soil amendment depending on the soil quality. The AW derived

BC and plant-FS derived biochar can be a good source of immediate nutrients release for plant

production in agriculture and C–sequestration respectively. Biochar can improve soil

properties, plant nutrient uptake by facilitating soil microbes and altering properties of

growth media. These studies were further designed to answer that how the biochar

interact with soil microbes in different soils for root colonization and plant nutrients

uptake. Moreover, to evaluate the incidence of biochar- and microbially induced changes

in the plant-soil system with P-application. Onion plant was grown in two soils amended

with two types of biochar with (or without) P2O5 application, having three microbially

inoculated treatments (and uninoculated control). Shoot and root biomass,macro, and

micronutrients concentration, N- and P-uptake and root colonization were analyzed.

Moreover, root attributes such as root surface area, root length, and root volume were

also evaluated by using WinRhizo. Biochar increased nutrient uptake and plant biomass

in the presence of P2O5 and microbial inoculation. Both soils were diversly responsive,

and the addition of biochar enhanced their responsiveness. Moreover, without-P addition,

soil microbial efficiency enhanced the nutrients uptake in shoot and root while

chlorophyll fluorescence was non-significant. Root colonization was also notably

increased in B+AM inoculated plants. Biochar types respond differently to varying soil

conditions. The P (with- or without-) application significantly influenced soil microbial

effectiveness in nutrient uptake and plant growth. Moreover, the root colonization was

also influenced by the biochar type and P application. Root attributes were significantly

influenced by the microbial inoculation. Cadmium (Cd) toxicity in agricultural crops is a

widespread problem. Little is known about the biochar and arbuscular mycorrhizal fungi

(AMF) effect, on Cd uptake and translocation in maize plant either applied separately or

combined. The current study was performed to demonstrate the effects of biochar and

AMF on growth, photosynthesis activity, nutrients and Cd uptake by maize is grown in

Cd-spiked soil. The alkaline soil was spiked by three various concentrations of Cd (0, 5,

and 10 mg Cd kg-1

) for each set of uninoculated control, biochar (Phragmites 1%), AMF

(Rhizophagus clarus) and biochar + AMF. Plants were harvested after 70 days, and

various morphological and physiological parameters, as well as elemental concentration

and root colonization, were recorded. Addition of biochar, AMF, and biochar + AMF

enhanced dry plant biomass in Cd-spiked soil. Root colonization decreased

proportionally by increasing Cd concentration. Besides that, addition of biochar either

separately or with AMF enhanced the root attributes in the Cd-spiked soil. However,

biochar + AMF neutralized Cd stress in maize plant for the gaseous attributes

(assimilation rate, transpiration rate, intercellular CO2, and stomatal conductance). The

AMF enhanced Cd uptake by plant while the addition of biochar phytostabilized the Cd

and reduced its uptake by plants. Phosphorus concentration was augmented in shoots and

roots of maize plant in biochar-amended soil than control plants. It is concluded that

biochar and AMF could ameliorate Cd toxicity effects in maize plant by changing the

morphological and physiological attributes along with elemental composition in the Cd-

spiked soil.

Chapter 1

General Introduction and

Review of Literature

1. General introduction and review of the literature

Food security is a big challenge and it is getting more importance due to economic

growth, increase in population, and climatic changes. According to an estimate, the

world‘s population will increase by 10 billion by 2050 (Khoshgoftarmanesh et al., 2010).

To meet the severe threat of food security in a long run, large-scale increase in food

production is need of the time. Besides that, agriculture is facing challenges which

include increasing demand and competition for natural resources (primarily phosphorus

availability as a key nutrient for plant growth) as well as biotic and abiotic

stresses.Increased fertilizer use has generally resulted in increased agricultural

productivity (Cameron et al., 2013). However, it has been noticed that long-term use of

chemical fertilizers, even at balanced application rates, can result in detrimental effects to

soil quality (Verma and Sharma, 2008), which subsequently decreases crop yield (Gilbert

et al., 2014). Generally, declining crop yields are countered by increasing fertilizer

application, perpetuating this cycle. Besides that, the mounting cost of agricultural inputs

and economic growth opened new frontiers of using chemical fertilizer alternatives.

Moreover, increasing awareness of the potential negative environmental consequences of

using chemical fertilizer have resulted in ever greater interest in beneficial crop–microbe

interactions and their application (Vance, 2001). Plants uptake P in the form of phosphate

(Pi) and phosphorus acquisition efficiency is naturally low in agricultural systems (plants

uptake only 15–20% of applied P) because Pi in the soil has low mobility and it forms Pi

depletion zone around the root (Syers et al., 2008).

1.1. Microbes

Soil microbes are the important component of soil ecosystem which influence

ecological components and processes including nitrogen cycling (Kowalchuk and

Stephen, 2001; Xu et al., 2018), nutrient acquisition (Sprent, 2001; Vimal et al.,

2017) and formation of soil (Miller and Jastrow, 2000). Furthermore, soil microbes

are the invisible majority in soil having huge genetic diversity (Fernandez-Gonzalez

et al., 2017; Mendes et al., 2017). According to an estimate, 1g of soil contains up to

200 m fungal hyphae, 10 10–10

11 bacteria and 6000–50,000 bacterial species

1.1.1. Plant Growth Promoting Rhizobacteria (PGPR)

The presence of the soil microbes establish a symbiotic relationship with

the plant roots to assist them in nutrients uptake; ultimately enhancing the plant

productivity in limited nutrients condition. Nitrogen (N)-fixing bacteria and plants

roots makes a symbiotic relationship, and this phenomenon is best studied where

bacteria convert atmospheric N2 into NH4+-N (Sprent, 2001). Besides that,

phosphorus (P)-solubilization is another important feature of the soil microbes

involved in the availability of P to the plants. Such microbes affect the soil P-

transformation which makes them a fundamental portion of the P-cycle.

Microorganisms act on organic and inorganic resources of the total soil P, where

they are solubilized and mineralized by a number of biological activities

odr guez and Fraga, 1999 . The P-solubilizing (PSB) and N-fixing bacteria

have additional characteristics of plant growth promotion (PGP) activity by

synthesizing plant hormones. In general, these bacteria are named as plant growth

promoting rhizobacteria (PGPR) which facilitate in uthe ptake of nutrients from

the soil too (Glick et al., 2007). These microbes produce different exudates

having organic acids and they solubilize various precipitated P forms. The PSBs,

consists of up to 40% of cthe ultivable soil bacterial population (Kucey, 1983;

Landeweert et al., 2001).

1.1.2. Phosphorus Solubilizing Bacteria (PSB)

There is a group of bacteria in the soil (rhizospheric) and within the roots

(endophytes) which facilitates plant growth promotion. They release certain types

of exudates containing organic acids which solubilize the nutrients in soil such as

P and K. Moreover, they help the plants in growth regulation by producing

various growth regulators (Rafique et al., 2017). The PSB, solubilize fixed-P

present in the soil and make it accessible for the plant roots uptake. Use of such

beneficial bacteria as biofertilizer in the agricultural industry is of key interest to

boost the agri-based economy. Usually the include Azospirillum, Arthrobacter,

Acinetobacter, Alcaligenes, Burkholderia, Bacillus, Erwinia, Enterobacter,

Serratia, Flavobacterium, Rhizobium, and Pseudomonas.

Stimulation of plant growth by the PGPR has been demonstrated on different

levels of trials in the laboratory and field. Different strains of Pseudomonas

putida, P. fluorescens, Bacillus subtilis, and Licinibacillus fusiformis have shown

increased shoot and root elongation in apple, beans, canola, citrus, lettuce, onion,

ornamental plants, potato, rice, radish, sugar beet, maize, tomato, and wheat

(Karakurt and Aslantas, 2010; Gupta et al., 2016; Rafique et al., 2017; Zuo et al.,

2018). The PGPR follow two types of the mechanism to enhance plant

productivity. First, an indirect mechanism where PGPR decrease the deleterious

effect of the pathogenic microorganisms due to the antibiotics synthesis (Fan et

al., 2017) siderophore production from bacteria (Ansari and Ahmad, 2018).

Second, the direct mechanism by fixation of N2 (Schutz et al., 2018), synthesis of

phytohormones (Tsukanova et al., 2017) and enzymes that modulate plant

hormones (Bjelic et al., 2018), moreover, solubilization of organic and inorganic

phosphate to make P available to the plants (Rao, 2016).

The primary mechanism of P-solubilization is the production of various

chemical compounds such as acid phosphatases, carbon dioxide, humic

substances, hydrogen sulfide, mineral acids, organic acids, protons, and

siderophores by PGPR (Ivanova et al., 2006; Ansari and Ahmad, 2018). The PSB

produce various organic acids such as 2-keto-gluconic, acetic, gluconic, glycolic,

oxalic, isobutyric, isovaleric, lactic, malonic, and succinic acid which result in the

acidification of surrounding soil resulting in the release of soluble orthophosphate

ions (HPO4-1

, HPO4-2,

and PO4-3

) (Vazquez et al., 2000). These organic acids are

commonly produced in all P-solubilizer bacterial strains, but they could be

specific to the bacterial genus too. Organic acids production induce acidic

conditions in the soil which solubilize orthophosphate ions from insoluble sources

and readily taken up by the plant roots (Kundu et al., 2009). Besides that, the

presence of chelating compounds in the bacterial strain such as siderophore also

plays a key role in insolubilization of insoluble phosphates (Han et al., 2017).

1.1.3. Mycorrhizal fungi

Besides the bacterial symbionts, there is another widespread group of

symbiont termed ―mycorrhizal association‖ in the plant roots which facilitate in

uptake of nutrients (N, P, K, Ca, Mg, Fe, Cu, Mn, and Zn) from the soil and

enhances the plant productivity under limited nutrients condition Ortaş et al.,

2017). Around 80% roots of the terrestrial plant species are getting benefits from

the mycorrhizal symbiotic association (Kamel et al., 2017). It involves the

bidirectional transfer of organic – C from plant to the arbuscular mycorrhizal

fungi (AMF), whereas soil-derived nutrients such as N, P, and Zn from AMF to

the plant (Jones and Smith, 2004; Smith and Read, 2008). It was first time

observed in 1885 by a German botanist Albert Bernhard Frank for the roots of

forest trees. Moreover, mycorrhizal fungi have the ability to provide resistance to

the plant in stress conditions, such as drought, disease, heat and under limited

nutrients condition in exchange for carbon. Mycorrhizal fungi ensure the resource

complementarity by the provision of macro and micronutrients which are

otherwise inaccessible to the plant roots (Figure 1.1). The abundantly present

mycorrhizal groups include the ectomycorrhizal (EM) fungi, endomycorrhizal

[arbuscular mycorrhizal (AM)] fungi, ectoendomycorrhizal fungi, ericoid

mycorrhizal (ERM) fungi, and the orchidaceous mycorrhizal fungi. Among them,

endomycorrhizae is of key importance and includes various genra such as

Aculospora, Entrophospora, Gigaspora, Glomus, Scelerocystis, and

Scutellospora. The AM fungi are rich in Savannah, grassland, tropical forests,

grasses, herbs, shrubs, fruit tree plants, cereals, and horticultural plants (Read and

Perez‐ Moreno, 2003; Ortaş et al., 2017). After certain modifications, genus

Scelerocystis is eliminated from endomycorrhizae, and its all members have been

added to the genus Glomus because of indifference in there morphological and

molecular characterization (Morton, 1988; Redecker et al., 2000). All the

members of genus Glomus are obligate like other gerna in the endomycorrhizae.

The AM fungi have the potential to enhance plant productivity by two folds

(Vogelsang et al., 2006). On the other hand, AM fungi can alter nutrients

distribution between co-existing grassland species without compromising on plant

output, where enhancement of P-uptake is one of the main contribution of AM

fungi in plant productivity. According to a study, AM fungi can contribute up to

90% P-uptake of the plant (Van Der Heijden et al., 2006). Enhancement of P-

uptake is significant in various plant species where high P-requirement is

mandatory for proper root development. Moreover, fixation of applied chemical-P

is a limiting factor in root development and plant productivity. Use of AM fungi,

enhance the P-uptake by extending the rhizospheric area which concludes as an

increase in plant productivity Ortaş and afique, 2017 . Besides that, AM fungi

enhance N-acquisition under certain conditions for plan productivity (Hodge et

al., 2001).

1.1.4. Co-inoculation of PSB and AM fungi

In a previous study, Khan and Zaidi (2007) performed individual

inoculation of a P-solubilizing bacteria (Bacillus spp.) and Glomus fasciculatum

on the wheat plant to determine dry matter content. Moreover, co-inoculation of

Bacillus spp. and Glomus fasciculatum was also performed. Results showed that

1.7-fold increase was observed in the root, 1.5-fold in shoot while in the whole

plant it was increased by 1.6-fold in comparison to the rest of treatments.

Similarly, Gamalero et al. (2004) studied the impact of G. mosseae with two

strains of Pseudomonas fluorescens, i.e. P190r and 92rk on Lycopersicon

esculentum Mill. Bacterial inoculation with G. mosseae increased mycorrhizal

colonization by 41% which was a significant increase. Moreover, a significant

increase in the shoot and root fresh weight was also observed. Similarly, in

another study, Khan and Zaidi (2006) inoculated green gram (Vigna radiata (L.)

Wilczek) with dual inoculation of G. fasciculatum and B. subtilis and observed a

significant increase in the root length and flowering stage than the control.

Similarly, maize plant was inoculated with G. deserticola¸and Azospirillum

brasilense where the results indicated a significant increase in shoot and root

weight (Vázquez et al., 2000). The production of IAA by Az. brasilense

stimulated mycorrhizal colonization which resulted in growth promotion of maize

plant. It is proposed that increased mycorrhizal colonization enhances the contact

chance between plant roots and fungal hyphae which indicates functional

compatibility between symbiotic and saprotrophic microorganisms. In the same

way, G. deserticola and B. pumillus were used for co-inoculation on alfalfa

(Medicago sativa). A significant increase in the root weight (190%) and shoot

weight (715%) was observed (Medina et al., 2003).

Figure 1.1: Schematic representation showing the impact of soil microbes on the

nutrient acquisition and plant productivity in natural ecosystems. Plant litter is

decomposed by a wide range of bacteria and fungi (1) making nutrients available

for uptake by mycorrhizal fungi (2) and plant roots or immobilizing nutrients into

microbial biomass and recalcitrant organic matter (4). Ecto-mycorrhizal fungi

and ericoid mycorrhizal fungi also have access to organic nutrients and deliver

these nutrients to their host plants (3). Some plants can also acquire organic

nutrients directly. Nutrients can also be lost from soil caused by denitrification of

ammonium into di-nitrogen gas or nitrogen oxides by denitrifying bacteria (5) or

when nitrifying bacteria and Archaea facilitate nitrogen leaching by transforming

ammonium into nitrate (6), which is much more mobile in soil. The contribution

of microbes to leaching losses of other nutrients (e.g., phosphorus) is still poorly

understood. Nitrogen-fixing bacteria (both free-living and symbiotic) transform

nitrogen gas into ammonium (7), thereby making it available to plants, enhancing

plant productivity. Finally, microbial pathogens attack plants and can reduce

plant productivity (8) (Leake et al., 2002).

1.2. Biochar

Biochar is a black material prepared from a number of organic feedstocks by

thermal degradation in the absence or limited presence of oxygen (pyrolysis) and used as

a soil amendment in distinction with charcoal (Joseph and Lehmann, 2009). It is gaining

attention as a soil amendment because of various soil improving factors such as positive

agronomic effects on plant and long-term carbon sequestration in soil (Sohi et al., 2010;

Verheijen et al., 2010). Besides that, biochar has some additional positive effects of

sorption due to characteristics of porosity, greater surface area and negative charge on the

surface (Mohan et al., 2006; Downie et al., 2009). Moreover, the biochar-amended soil is

further influenced in terms of porosity, soil structure, texture, particle size distribution,

cation exchange capacity and increase in soil water retention where it results in the plant

growth promotion (Atkinson et al., 2010). Further functions of biochar as soil

amendment include services to the soil ecosystem stability, improved soil fertility, and

climate change mitigation by sequestering carbon (Lehmann et al., 2006; Lehmann,

2007; Sohi et al., 2010). Such positive changes induced by the addition of biochar

contribute to nutrient cycling (Steiner et al., 2008). Rhizosphere bacteria and fungi may

also enhance plant growth directly (Compant et al., 2010).

1.3. Biochar and microbial interaction for plant growth in limited nutrients

condition

Phosphorus is a major nutrient required for the plant for the root development and

plant growth promotion. The soluble orthophosphate (Pi) is absorbed by the plants

mainly as P-source where it contributes <1% of the total ratio of P in soils because of

strong bonds (Sylvia et al., 2005). There are two forms of bound P in soil such as

organically and inorganically bound (Metcalf and Wanner, 1991). An immense range of

microorganisms have the ability to solubilize inorganic P (Bucher, 2007) by exudation of

gluconate, citrate, or oxalate to decrease the soil pH, dissociating Ca2+

bound P (Alikhani

et al., 2007).

Arbuscular mycorrhizal fungi, PGPR, and plant roots release various organic acids

such as oxalic, acetic, gluconic, and succinic acids. These acids solubilize organo-mineral

and secondary mineral surfaces to form orthophosphorus. Vassilev et al. (2013) reported

that nutrients which are present in biochar ash boosted the ability of microbes of

phosphate solubilization and inferred that animal bone biochar could be a sustainable

substitute for inorganic P fertilizer. He et al. (2014) amended the soil with ∼8 ton ha−1

rice husk biochar and inoculated with organic acid producing bacteria (Lysinibacillus

sphaericus and Lysinibacillus fusiformis). Results showed 47-54% more solubilization of

ortho-P. Almost three-fold higher ortho-P solubilization from rock phosphate (containing

13.97 P) was reported by de Oliveira Mendes et al. (2014) when mixed with holm oak

biochar (slow pyrolyzed at 480 °C) at a 1:1 phosphate rock:biochar ratio and applied to

the potato dextrose agar growth medium (pH 7) inoculated with Aspergillus niger.

Aspergillus niger increases production of citric acid by ∼2 fold and gluconic acid by

∼3.5 fold in comparison to control.

In nutrient-poor soil, there was a greater production of organic acids by nutrient-

loaded biochar to enhance ortho-P solubilizing activity than nutrient-rich soils (Deb et al.,

2016). Former findings from controlled conditions and environments show the potential

of biochar to stimulate and enhance the activity of phosphate solubilizing

microorganisms. However, advance studies in field environment conditions are needed to

confirm the prospects of biochar in this concern.

Association of mycorrhizal fungi and plant is very familiar to increase phosphorus

uptake by crops, and in this action, various mechanisms of fungi and plants are involved

in secreting extracellular phosphatases and phosphate solubilizing organic acids. Fungal

hyphae have a great ability to enter microsites that are unapproachable to plant roots;

mycorrhizal fungi efficiently acquire phosphate and transport to its host plant (Warnock

et al., 2007). Biochar is often known to promote mycorrhizal colonization of host crops

(Blackwell et al., 2010; Ortaş, 2010; Ortas, 2016). Hammer et al. (2014) by using

electron microscopy and 33

P tracer found that Rhizophagus irregularis was firmly

attached to the inner and outer surfaces of nutrient-loaded wood biochar (550 °C). It

brings about six times more 33

P translocation to the host plant Daucus carota in

comparison to the rest of the experimental units.

Warnock et al. (2007) and Atkinson et al. (2010) reported that biochar enhances the

association of mycorrhizal colonization and increases the growth of P solubilizing

bacteria. Although, two separate meta-analyses made by (Lehmann et al., 2011) and

Biederman and Harpole (2013) and empirical evidence (Warnock et al., 2007) comes to

the conclusion that very less association of mycorrhizal colonization will found in

nutrient-rich soil supplemented with biochar.

This recommendation is dependent on the idea that nutrient-rich soil decreases

plant dependence on mycorrhizal fungi for obtaining nutrients and leads to the hypothesis

that high mycorrhizal colonization will found in the soil with fewer nutrients

supplemented with wood-based biochar (Figure 1.2). This perception is supported by the

observation that greater mycorrhizal colonization of wheat roots (from 1.4 to 16%) and

higher wheat biomass (∼7–8 times more dry weight) occurred when a nutrient-poor

loamy sand soil was amended with 5 t ha−1

of various biochars, compared to the

unamended control (Blackwell et al., 2015).

Biochars in this experiment were obtained from Acacia saligna wood (slow

pyrolyzed at 380 °C) and Eucalyptus marginata wood (slow pyrolyzed at 550–650 °C)

mixed in a 10:1 (biochar:fertilizer) with advantageous microbe-inoculated inorganic

fertilizer (N, P, K, S, Ca, Mg with microbial inoculation at 750 g t−1

fertilizer). Inferring

results from (Blackwell et al., 2015) is challenging due to the complications of

interacting factors presented in the experiment, i.e., production temperatures and various

biochar feedstocks as well as supplemental nutrients and microbial inoculants. It does

demonstrate the need to reflect how biochar affects and change the soil physicochemical

and biological properties that are vital for the development of mycorrhizae.

Figure 1.2: The Hypothetical model is showing the mycorrhizal colonization of plants

grown in nutrient-poor and nutrient-rich soils. When the nutrient-poor soil is amended

with biochar containing the high surface area and low nutrient content (e.g., wood-based

biochars produced at high production temperatures), the reduction in bioavailable nutrient

concentration will stimulate mycorrhizal colonization. In nutrient-rich soil, such biochar

types enhance mycorrhizal colonization. Likewise, biochar with high nutrient content and

high surface area (e.g., biochars produced from manure and crop residue feedstocks)

result in high mycorrhizal colonization in nutrient-poor soil and low mycorrhizal

colonization in nutrient-rich soil. is biochar particles. The thin white lines

superimposed upon the plant roots indicate mycorrhizal hyphae (Gul and Whalen, 2016).

It was hypothesized by Warnock et al. (2007) that micropore in biochar decrease

grazing on bacteria and fungal hyphae. The greater abundance of microorganisms

comprising bacteria retrieving unapproachable plant source of phosphorus and sulfur

from the soil or the biochar directly influence nutrient supply and availability of plants

nutrients when these nutrients are in less quantity and help to enhance plant growth

(Figure 1.3).

Figure 1.3: The hypothetical relationship between nutrient availability and the

microbial contribution to plant productivity. Microbes are hypothesized to be

most important for the productivity of poor nutrient ecosystems. It is also

hypothesized that microbial diversity (–––) is negatively correlated with nutrient

availability (Van Der Heijden et al., 2008).

1.4. Biochar and microbial interaction for plant growth under heavy metal stress

Potentially toxic elements (PTEs) causes contamination of soils which is known

as the general problem of soil that may be due to excessive use of chemical fertilizers

and bio-solids that is co-mixed with waste materials containing heavy metals. Various

strategies have been established to ensure less bioavailability of PTE such as soil

washing, land excavation, phytoremediation (Kiikkilä et al., 2001; de Mora et al.,

2005; Fellet et al., 2011; Beesley et al., 2014). However, there is a dire need to find

effective and cheap practices in order to solve the problem of contaminated soils. One

such technique that is receiving attention is the application of biochars that have the

ability to adsorb PTEs and make less available to plants so inhibit uptake and transfer

of PTEs in the food chain (Puga et al., 2015).

Attention to the biochar potential for enhancing soil fertility and quality along

with remediation potential for soil contaminants is also attaining momentum. Biochar

could significantly lower the mobility of selected contaminants in the contaminated

soil, with mainly promising results for cadmium (Cd) (Beesley and Marmiroli, 2011).

Namgay et al. (2010) reported a biochar application causes a significant decrease in

the availability of lead (Pb) and Cd in a pot experiment. The main soil microbial

groups that affect metal uptake by plants and metal immobilization in soils

(Piotrowski and Rillig, 2008) is the AM that usually introduced into the soil for land

reclamation (Renker et al., 2004). It is also reported that AMF induces plant tolerance

to PTEs. This happened when fungal hyphae bind with metals (Gonzalez-Chavez et

al., 2002) and complexation of metals by glomalin (Gonzalez-Chavez et al., 2004),

which is a glycoprotein produced by all AMF that have been tested to date (Wright

and Upadhyaya, 1998; Nichols, 2003). AMF hyphal and glomalin production should,

therefore, be taken into account when phytostabilization technologies are used in

polluted soils. This capability of AMF to sequester and accumulate PTEs in a non-

toxic form may help to increase plant fitness and soil quality in polluted areas

(Gonzalez-Chavez et al., 2004).

Soil amendments with an abundance of AMF are very beneficial to plant hosts,

and by altering soil structure and affecting heavy metal immobilization, it improves

soil quality (Rillig et al., 2006). Inoculation of AMF on Cd spiked soil decreased

inorganically bound Cd fraction with an increase in residual Cd fraction (Aghababaei

et al., 2014). Recent research shows that supplementation of soil biochar can

stimulate AMF percent root colonization on plants in acidic soils (Ezawa et al., 2002;

Matsubara et al., 2002; Yamato et al., 2006).

1.5. Hypothesis

It is evident from the above discussion that phosphorus is a major nutrient

required by the plant for plant growth promotion and root development. To overcome

the P-losses in soil (present in the precipitated form), use of soil microbes is a viable

and eco-friendly approach. These soil microbes include PSB and mycorrhizae which

have been extensively and separately studied in different climatic conditions. Besides

that, use of biochar as a soil amendment in the agricultural field is another approach

to assist the plants for growth enhancement by altering soil physicochemical

properties. Combination of soil microbes (PSB and AMF) in biochar-amended soil

have not been studied before for plant growth promotion and bioremediation in

different environmental and soil conditions. Considering this major research gap in

advancing the scientific knowledge, various studies have been designed where PSB

and AMF were used in combination with biochar for plant growth promotion.

Varieties of biochar were produced to evaluate their qualitative flexibility with soil

microbes in P-limited condition and heavy metal stress tolerance.

1.6. Overall objectives

The overall objectives of the study were to evaluate the performance of soil microbes

and biochar to enhance plant growth under limited nutrient and heavy metal stress

conditions. Following objectives were targeted during the studies conducted:

a. Biochemical characterization and sequencing of bacteria to evaluate

potential of bacteria on plant growth.

b. Evaluation of plant-based biochar for plant nutrients uptake.

c. Quantification of plant and soil nutrients to identify combined effect of

PSB and biochar.

d. Quantitative evaluation of nutrients in different biochar prepared from

sewage sludge, animal waste and plant based feedstock.

e. Quantification of carbon content in various biochar.

f. Thermal stability estimation of biochar prepared from different

feedstocks.

g. Evaluation of different biochar influence on chlorophyll fluorecence in

PSB – mycorrhizae presence in onin plant.

h. Quantifiaction of macro and micronutrients in onion plant in biochar –

PSB – mycorrhizae system.

i. Evaluation of different biochar influence on chlorophyll fluorecence in

PSB – mycorrhizae presence in maize plant.

j. Quantifiaction of macro and micronutrients in maize plant in biochar –

PSB – mycorrhizae system.

k. Evaluation of gasous exchange in maize plant grown in biochar –

mycorrhizae syetem under cadmium stress.

Macro and micronutrients quantification in plants to evaluate cadmium influence on

plant growth.

Chapter 2

PSB and Biochar Interaction for


2.1. Introduction

In Pakistan, maize consumption is increasing on a regular basis. Exploiting the

potential of current maize plant varieties regarding profitability, the addition of chemical

fertilizers and other growth-promoting inputs are inevitable. Considering the low

phosphorus (P) status in Pakistani soils (80-90% soils are deficient) (Ahmad and Rashid,

2004), it is of basic importance to make P application essential in the rhizosphere. Hence

application of chemical fertilizer-P may be used efficiently to overcome yield gaps.

However, the application of fertilizer-P demands high cost as most of the P makes a

complex with calcium (Ca), aluminum (Al), and iron (Fe) which make it unavailable for

plants uptake (Herrera et al., 2016). In the undisturbed natural soil, a considerable

amount of P (400-1200 mg kg-1

) is present odr guez and Fraga, 1999 . Besides that, the

addition of chemical fertilizer accumulates a large proportion of insoluble P in the form

of a complex with Ca/Mg carbonates in alkaline soil while for acidic pH soil, Al/Fe

mineral complexes are formed. Organic forms of P may constitute 30–50% of the total

phosphorus in most soils odr guez and Fraga, 1999 .

Currently, shifting the insoluble proportion of P into the soluble pool is a key

objective in sustainable agriculture. It can be achieved by adopting various possible soil

management protocols and optimizing the P-availability for plants growth with minimum

losses from soil. For mining of P-minerals, phosphorus solubilizing bacteria (PSB) and

phosphorus solubilizing fungi (PSF) constitute about 1-50 % and 0.1-0.5 % of soil biota

respectively (Zaidi et al., 2009; Sharma et al., 2013). Soil biota contribution got the

attention of researchers for plant growth promotion (PGP) and yield enhancement due to

P solubilization potential (Fasim et al., 2002; Chen et al., 2006). The indigenous PSB in

combination with chemical fertilizer (superphosphate and rock phosphate) reduces the

dose requirement by 25-50% (Sundara et al., 2002). Some studies reported that

inoculation of PSB can solubilize the Ca/Mg/Al/Fe-bound P which becomes available to

the plant roots to enhance growth and proliferation (Liu et al., 2016). The release of

enzymes and other chemicals by PSB (organic acids, phosphorus solubilizing enzymes,

phytase, and siderophore) break down the bond between P and the fixing element to

make it available for the plant uptake (Hayes et al., 2000). Several bacteria such as

Azospirillum, Azotobacter, Bacillus, Burkholderia, Pseudomonas, Klebsiella, etc. have

been identified as phosphate solubilizers in soil and phytohormone producers in maize

plant (Sharma et al., 2013).

Conventional farming and intensive use of the chemical fertilizers developed a one-

way movement of the nutrients. When chemical-P fertilizer is applied to the rhizosphere,

most of the part is fixed in the soil as phosphate of Ca/Al/Fe and becomes unavailable for

plant uptake. Plant roots uptake the available proportion of chemical-P and fixes in

biomass. Phosphorus is one of the most limiting nutrients for plant productivity as its

reserves are depleting worldwide. Considering it, fixed P in the plant biomass can be

recycled from plant biomass is need of the time to enhance soil quality and plant

productivity (Cordell et al., 2009; Metson et al., 2014). To achieve maximum utilization

of P under minimum loss can be done through biochar application to the soil. Biochar

releases the P in presence of PSB and makes it available to the plant roots.

Biochar is a valuable by-product of pyrolytic biomass during generation of biofuel. It

is a potential source of P recycling from the agricultural wastes to enrich the soil quality.

As a new area of research, biological and chemical effects on the P release from biochar

still need further investigation (He et al., 2014). Biochar production and its application as

soil amendment achieved promising results for crop production (Dickinson et al., 2015;

Changxun et al., 2016; Ortas, 2016), soil quality improvement (Fang et al., 2016),

biochemical properties enhancement to facilitate soil biota (Puga et al., 2015; Hairani et

al., 2016), mitigation of climate change effects in a long run (Smith, 2016) and disposal

of large-scale waste biomass (Jeffery et al., 2015). Conventional scheme of using

agricultural waste as a soil amendment in the recycling of fixed P is supposed to be less

effective than biochar routed P cycling (Dai et al., 2016).

However, currently, available information on the use of PSB and biochar together in

association with maize plant for nutrients uptake is still limited. Here, the study aims to

clarify the effects of plant residues-based biochar and PSB inoculation on P recycling for

maize plant growth response. It was hypothesized that combined PSB and plant-based

biochar could increase nutrient availability to plants.

2.2. Objective

On the basis of foregoing discussion, objectives of the study were:

Biochemical characterization and sequencing of bacteria to evaluate

potential of bacteria on plant growth.

Evaluation of plant-based biochar for plant nutrients uptake.

Quantification of plant and soil nutrients to identify combined effect of

PSB and biochar.

2.3. Materials and Methods

2.3.1. Biochemical Characterization of Bacteria

Rhizospheric bacteria were isolated from 1g soil tightly adhering to the root by

serial dilution plating on Luria-Bertani (LB) agar plates (Somasegaran and Hoben, 1994).

The soil was mixed by shaking for 20 min to separate microorganisms completely from

the soil in autoclaved dispersion flasks. The plates were incubated at 28±2oC until the

appearance of bacterial colonies. Individual colonies were picked and streaked on LB

plates for further purification. Isolated bacterial strains were biochemically characterized

by respective methods described here.

2.3.1.1. Catalase activity

Catalase activity was determined by obtaining a bacterial

culture from a Luria-Bertani medium incubated for 24 h, and few drops of

H2O2 (30%) were added to a glass slide. Oxygen-bubble formation indicated

the catalase activity (Schaad et al., 2001).

2.3.1.2. Oxidase activity

To determine oxidase activity, Kovacs oxidase reagent was

employed (1-2 drops) in 24 h old culture on a small filter paper. Change in

color (dark purple) of filter paper in 60-90 minutes showed bacterial positivity

for oxidase.

2.3.1.3. Phosphate solubilization

Phosphate solubilization was examined on Pikovskaya‘s

medium (Pikovskaya, 1948) where the bacterial colony was spotted in the

center of plates which had tricalcium phosphate [Ca3(PO4)2] as the insoluble

phosphate source. After 7 days of incubation at 28±2oC, halos formation

confirmed the P-solubilization activity of bacteria.

2.3.1.4. N-fixation quality

N-fixation quality of the bacteria was determined by

incubation at 28 ± 2oC on nitrogen-free media for 3-4 days (Okon et al.,

1977). Growth exhibition on media confirmed nitrogen fixation quality of

bacteria.

2.3.1.5. Gelatinase activity

Nutrient gelatin stabs were inoculated and incubated at 25oC

for one week, liquefication of gelatin shows the positivity of gelatinase in

bacteria (Wood and Krieg, 1994). In the determination of gelatin hydrolysis,

milk agar was prepared and inoculated with bacteria. Clear zone formation

indicated the hydrolysis of casein (Smith et al., 1952).

2.3.1.6. Citrate utilization test

Simmon‘s citrate broth was used for the citrate utilization test

whereas Christensen‘s agar was used with incubation for 4 days (Christensen,

1946; Graham and Hodgkiss, 1967).

2.3.1.7. Indole Acetic Acid

Indole Acetic Acid (IAA) was measured through a

colorimetric method on a spectrophotometer using ferric chloride–perchloric

acid reagent (FeCl3–HClO4) by drawing a standard curve (Gordon and Paleg,

1957).

2.3.1.8. Hydrogen sulfide production

Sulfide Indole Motility agar medium tubes were inoculated

with both bacteria, incubated for 48 h at 37oC to determine the hydrogen

sulfide production (Clarke, 1953).

2.3.1.9. Urease Activity

On urea agar, bacterial isolate changed the color of the agar

from yellow to pink indicating presence of urease activity.

2.3.1.10. Antibiotic resistance test

Both bacterial strains were tested against 10 antibiotics for

their characterization. It was confirmed that wither the bacterial strains were

sensitive or resistant. Name of the antibiotics used were: ampicillin,

gentamicin, nitrofurantoin, tetracycline, norfloxacin, amoxicillin, ofloxacin,

erythromycin, ceftriaxone and clindamycin.

2.3.2. Bacterial strains Genetic Identification

Total genomic deoxyribonucleic acid (DNA) of bacterial strains was extracted using

Bacterial Genomic DNA Purification Kit (GM biolab Co, Ltd., Taichung, Taiwan)

according to the supplier‘s instructions and used as DNA template in polymerase chain

reaction (PCR) for amplification of the 16S rRNA gene following previously reported

protocol (Araújo et al., 2002). The DNA purity was quantified at 260 nm and 280 nm

using NanoDrop Spectrophotometer (ND 1000, Thermo Fisher Scientific Inc. Waltham,

MA, USA), 1.6-2.2, to detect protein contamination in the DNA (Calvo et al., 2001).

PC amplification was performed in a reaction mixture containing 3 μl of 10x buffer, 2.4

μl of dNTPs mixture 2.5mM of each dNTP , 0.6 μl 20pmol μl-1 of each primer 27F 5‘-

AGAGTTTGATCCTGGCTCAG-3‘ and 1492 5‘-TTCAGCATTGTTCCATCGGCA-

3‘ (Weisburg et al., 1991), 0.18 μl of One Taq DNA Polymerase Thermo Fisher

Scientific and 1.8 μl of template DNA. The PC program Bio-Rad DNA Engine,

Hercules, CA) started with an initial denaturation step for 5 min at 95oC followed by 40

amplification cycles of denaturation at 95oC for 30 sec., annealing at 59

oC for 30 s and 1

min extension at 72oC. Before cooling to 4

oC, final extension period of 5 min at 72

oC was

incorporated into the program (Branco et al., 2005). The suitability of DNA amplification

was visualized by electrophoresis of PCR products with 6X loading dye (Thermo

Scientific™ at 5:1 PC product: dye ratio and a marker 1kb DNA ladder, Fermentas

Gene uler™ in 1% w/v agarose gel in 1X Tris-acetate EDTA (TAE) buffer for 1 h at

80 V. The agarose gel was stained with Gel ed™ Biotium Inc., Hayward, CA, USA for

40 min and examined under UV light in a UV transilluminator (Bio-RadMolecular

Imager Gel Doc™ X + System, Bio-Rad, Hercules, CA, USA) (Figure 2.1). Gel image

was captured with Image Lab software (Version 4.1, Bio-Rad Laboratories, Segrate,

Italy). The sequences were analyzed using the Basic Local Alignment Search Tool

(BLAST) Sequence Similarity Search to identify the most closely related members in the

National Center for Biotechnology Information (NCBI) GenBank DNA database

(www.ncbi.nlm.nih.gov/geo). The partial 16S rDNA sequences of the phosphorus

solubilizing strains were submitted to the NCBI database under their respective accession

number as follows: Bacillus subtilis strain 18MZR (KX710213) and Lysinibacillus

fusiformis strain 31MZR (KX710214).

http://www.ncbi.nlm.nih.gov/geo

Figure 2.1: Agarose gel electrophoresis of PCR product 16S rRNA for strains 18 and 31.

2.3.3. Phylogenetic analysis

The partial 16S rDNA sequences of both strains were aligned with the closely

related bacterial sequences obtained from the NCBI database using Multiple Sequence

Comparison by Log-Expectation (MUSCLE) (Edgar, 2004). The neighbor-joining

phylogenetic tree was constructed after calculation of a maximum composite likelihood

method from distance matrix using the Molecular Evolutionary Genetic Analysis

(MEGA) 4.0 software by the method of Kimura two-parameter model with a discrete

Gamma distribution (Tamura et al., 2007).

2.3.4. Biochar preparation and analysis

The woody sawdust was collected from a local sawmill in Rawalpindi, Pakistan. The

wood chips were ground and sieved. Bagasse was collected from sugar mill, i.e., Frontier

Sugar Mill Thaktbhai Mardan. The obtained material was passed through a 50-mesh

screen to move large lumps. Particle size was reduced to 0.7-0.8 nm, and it was dried at

110oC for 24 hr and stored in a container before initial characterization. The samples

were pyrolyzed to the 350oC temperature using a heating rate of 10

oC min

-1. The process

was carried out in a closed muffle furnace with an outlet for the gases release (Sánchez et

al., 2009). Biochar samples were prepared at 350oC with a residence time of 1 hr and

obtained samples were properly labeled. The prepared biochar samples were

characterized for various chemical analysis where pH and EC of biochar were measured

according to Novak et al. (2009). To measure pH and EC, 2 g of biochar shaken with 40

mL of deionized water for 30 min and the sample was allowed to settle for 15 min before

recording pH and EC. The cation exchange capacity (CEC) of biochar was measured by

the ammonium acetate (NH4OAC) extraction method (Song and Guo, 2012; Melo et al.,

2013) (Table 2.1).

Table 2.1: Properties of biochar used in the study

N: nitrogen, P: phosphorus, K: potassium, EC: electrical conductivity, CEC: cation

exchange capacity

2.3.5. Setting pot experiment and plant-soil analyses

The experiment was conducted on maize plant. The experimental soil (Nabipur soil

series, Fine-loamy mixed hyperthermic Udic Ustochrept) was collected from 0–15 cm

soil depth at the National Agricultural Research Centre located (33° 43' 11.9784'' N, 73°

5' 45.7764'' E) at an altitude of 518 m above sea level in Islamabad. The soil collected

from the research field area was air-dried, sieved (2 mm mesh) and analyzed for its

Elements/Components Sawdust biochar Bagasse biochar

Yield (%) 56 49

N (%) 4.6 4.1

P (%) 1.6 1.1

K (%) 2.14 1.7

pH 8.1 7.9

EC (dS m-1

) 0.8 1.2

CEC cmolc kg-1

35 27

Ash (%) 2.9 3.2

physicochemical properties. The soil had 15% clay, 45% silt and texture was loamy with

3.1% CaCO3, whereas pH 8.34 (1:1, soil: water ratio; MeterLab® PHM210, Radiometer

Pacific Limited, Copenhagen, Denmark) (McLean, 1982); nitrate-N (Ammonium

bicarbonate-DTPA-extractable) (Soltanpour, 1985), 3 mg kg-1

; organic carbon (Walkley,

1947), 4.9 g kg-1

; available P (Soltanpour, 1985), 1.9 mg kg-1

and exchangeable K

(Soltanpour, 1985) were 120 mg kg-1

. The soil was autoclaved at 121oC for 20 min,

exactly 3 kg of the soil was weighed and placed in each pot (21 cm, D × 18 cm, H).

Uniform doses of Urea (46% N), Diammonium Phosphate (18% N and 46% P2O5) and

Muriate of Potash (MOP, 60% K2O) at the recommended rates of 160 kg N ha-1

, 80 kg

P2O5 ha−1

and 60 kg K2O ha−1

equivalents (NARC, 2017), were applied to each pot

respectively. The experiment was conducted in a randomized complete block design,

with two harvests at 45 days (D45) and 65 days (D65) after planting with four replications

at both stages (8 replications in total). The nine treatments were executed: (i) control (C)

(uninoculated and untreated), (ii) 1% bagasse biochar (BC-1) (equals to 30 g for 3 kg

soil), (iii) 1% sawdust biochar (BC-2), (iv) B. subtilis strain 18MZR (B1), (v) L.

fusiformis strain 31MZR (B2), (vi) B. subtilis strain 18MZR + 1% bagasse biochar (M1),

(vii) L. fusiformis strain 31MZR + 1% bagasse biochar (M2), (viii) B. subtilis strain

18MZR + 1% saw dust biochar (M3) and L. fusiformis strain 31MZR + 1% saw dust

biochar (M4). Biochar was thoroughly mixed with the soil before seed sowing for each

treatment.

The PSB inoculum was grown in LB broth media for 24 hr (200 rpm, 26±2°C) and

cell suspensions were adjusted to OD600 between 1.4 and 2.0 (Nandre et al., 2012), which

corresponded to the total plate counts of 109 cfu mL

-1, as determined on the LB media

agar. Five maize seeds of similar size and shape, var. Islamabad Gold was added in each

pot and thinned to two per pot at 10th

day of sowing. Each pot with PSB inoculation

treatment was injected with 15 mL of respective inoculum on seed sowing and 10th

day

(D10) in the rhizosphere after thinning. The pots were watered every second day to

maintain 80% field capacity throughout the experiment to avoid any possible loss of

applied fertilizer through denitrification in excessive moisture.

Leaves of the plant were harvested at D45 and D65. They were dried and ground to

determine N by Kjeldahl method (Van Schouwenburg and Walinga, 1975) using a UDK

142 Automatic Distillation Unit (VELP Scientifica, Milan, Italy), P by Ammonium

Molybdate-vanadate solution by reading on spectrophotometer (Hitachi U-1500, San

Jose, CA, USA) (Isaac and Johnson, 1975) and wet digested filtrate of the sample was

directly used to determine K by Flame Photometer (Jenway PFP7, Jenway, UK).

Representative soil samples of 200g from the rhizosphere were collected by removing all

the soil + root from a pot into a tray and shake gently. The soil surrounding the roots was

collected in a separate bag, air-dried and sieved (2 mm mesh). Soil NO3-N, P, K by

ammonium bicarbonate-diethylenetriaminepentaacetic acid (AB-DTPA) extraction were

analyzed, and pH was also determined (Soltanpour, 1985).

2.3.6. Recovery of inoculated bacteria

After harvesting, inoculated plants were sterilized and cut into small sections.

Samples were surface sterilized and homogenized in autoclaved distilled water. The

homogenized mixture was plated in nutrient agar plates. Emerged colonies were

identified by their morphological characteristics; gram staining and antibiotic resistance

of bacteria were detected by the disc diffusion method. A 0.1 mL bacterial culture [108

colony forming units (CFU) mL-1

] was spread on LB agar plates meanwhile, antibiotic

discs (gentamicin, tetracycline and erythromycin) were positioned on the surface of

media and plates were incubated for 24hrs at 27oC. (Arumugam et al., 2011) to check

sensitivity and resistance of bacteria on the basis of previous screening.

2.3.7. Statistical analysis

Data were analyzed using one-way analysis of variance procedure (ANOVA)

followed by Duncan Multiple Range Test (DMRT) at p≤0.05 using Statistical Analysis

System software (SAS version 9.0) (Robert et al., 1997). Pearson‘s correlation of

coefficient test was performed to estimate the relationships between measured parameters

of plant and soil at both stages of harvesting.

2.4. Results

2.4.1. PSB strains characterization

Phosphorus solubilizing ability of two selected strains and biochemical characteristics

are shown in Table 2.2. Bacterial strains gave positive reactions for phosphorus

solubilization and nitrogen fixation. The B. subtilis strain 18MZR and L. fusiformis strain

31MZR showed clear halo zone formation around their bacterial colonies when grown on

the Pikovskaya media, indicating phosphate solubilization ability.

Table 2.2: Biochemical characteristics of bacteria used in the study

Characteristics Properties

Bacillus subtilis

18MZR

Lysinibacillus fusiformis 31MZR

Gram staining + -

Cell shape Rod Rod

Catalse + +

Oxidase + +

Phosphate

solubilisation

+ +

Nitrogen fixation + +

Hydrolysis of gelatin + +

Hydrolysis of casein + +

Citrate utilization + +

Indole production + +

Hydrogen sulfide - -

Urease - +

Ampicillin R R

Gentamicin R S

Nitrofurantoin R R

Tetracycline R S

Norfloxacin R S

Amoxicillin R R

(+) Positive results, (-) Negative results, (S) sensitive, (R) resistant

2.4.2. Molecular characterization of PSB

The PSB strains were identified by sequencing of rRNA gene. The BLAST search

results showed that the strains 18MZR and 31MZR are more closely related to the species

of genus Bacillus and Lysinibacillus respectively (Figure 2.2) with 96% sequence

similarity in both strains. The sequence analysis showed that both strains showed fewer

similarity values (96%) with previously characterized validly published species. Strain

18MZR and strain 31MZR clustered together and belonged to the genus Bacillus and

Lysinibacillus respectively. The bacterial strains belonged to the same phylum, two

different genera of bacteria and they could be (i) Bacillus subtilis strain 18MZR (ii)

Lysinibacillus fusiformis strain 31MZR. Bacteria in phylum Firmicutes have a strong cell

wall which makes them resistant to desiccation and can survive extreme conditions.

Moreover, it is one the most abundant phylum present in the rhizosphere where they

promote plant growth and protect plants from pathogen attack by a range of mechanisms.

Ofloxacin R S

Erythromycin R S

Ceftriaxone R S

Clindamycin R S

Figure 2.2: Phylogenetic tree showing inter-relationship of Strain 18MZR (KX710213) and 31MZR

(KX710214) with closely related species of the genus Bacillus subtilus and Lysinibacillus fusiformis,

respectively inferred from aligned unambiguous sequences of 16S rRNA gene. Numbers at nodes indicate

percentages of occurrence in 500 bootstrapped trees. The analysis involved 16 nucleotide sequences. There

were a total of 1002 positions. Scale bar, 0.1 substitutions per nucleotide position. The tree was generated

by the maximum composite likelihood method and was rooted by Geobacillus stearothermophilus strain R-

35646 (FN428694). as an out group. Accession number of each strain is shown in parentheses.

2.4.3. Plant height

Biochar addition to the soil increased plant growth, a significant increase was

observed in sawdust biochar (BC-2) amended maize root (43.6 cm) and shoot (55 cm) at

D45 while at D65, it was 49.6 cm and 62 cm, respectively. Inoculation with PSB

significantly increased the growth of maize plants at both harvestings (D45 and D65),

particularly L. fusiformis strain 31MZR inoculated plant height was significantly

increased at D65 for root (44.4 cm) and shoot (63.1 cm) (Figure 2.3a, 2.3b.). The highest

increase in root and shoot length (54.2 cm, 92.4 cm) was observed for sawdust biochar-

amended the soil with L. fusiformis strain 31MZR inoculation (D65). Bagasse biochar also

increased root and shoot length on D45 (39.8cm, 40.3cm) and D65 (45.9cm, 52.1cm) in

comparison to the control treatment. Similarly, in sawdust biochar treated soil, the root

and shoot length increase was significant on D45 (43.6cm, 55cm) and D65 (49.6cm,

62cm). Comparatively, only PSB inoculation, such as B. subtilis 18MZR on D45 (32.5cm,

48cm) and D65 (36.7cm, 54.1cm), while L. fusiformis strain 31MZR on D45 (36.4cm,

42.9cm) and D65 (42.7cm, 59.2cm) increased plant growth which was less than the

biochar amended soils. The combination of L. fusiformis strain 31MZR with bagasse

biochar significantly increased plant height on D45 (38.5cm, 45cm) and D65 (44.5cm,

63.1cm). When L. fusiformis strain 31MZR was inoculated with sawdust biochar, plant

height increased on D45 (45.3cm, 78.8cm) and D65 (54.2cm, 92.4cm). Moreover, B.

subtilis strain 18MZR inoculation with bagasse biochar increased plant height on D45

(37.7cm, 39cm), D65 (41.4cm, 52.8cm), while inoculation with sawdust biochar increased

plant height on D45 (40cm, 60cm) and D65 (42.6cm, 68.1cm).

Figure 2.3a: Root and shoot length of maize plant after 45 days harvesting for all treatments: control

(uninoculated and untreated), BC-1 = bagasse biochar, BC-2 = sawdust biochar, B1 = B. subtilis

strain18MZR inoculation, B2 = L. fusiformis strain 31MZR inoculation, M1 = B. subtilis strain 18MZR +

bagasse biochar, M2 = L. fusiformis strain 31MZR + bagasse biochar, M3 = B. subtilis strain 18MZR +

sawdust biochar, and M4 = L. fusiformis strain 31MZR + sawdust biochar

Figure 2.3b: Root and shoot length of maize plant after 65 days harvesting for all treatments: control

(uninoculated and untreated), BC-1 = bagasse biochar, BC-2 = sawdust biochar, B1 = B. subtilis strain

18MZR inoculation, B2 = L. fusiformis strain 31MZR inoculation, M1 = B. subtilis strain 18MZR +

bagasse biochar, M2 = L. fusiformis strain 31MZR + bagasse biochar, M3 = B. subtilis strain 18MZR + saw

dust biochar, and M4 = L. fusiformis strain 31MZR + saw dust biochar

2.4.4. Plant Nutrient Concentration

Biochar amended soil significantly increased the N concentration in plants at both

harvesting stages on D45 and D65 (Table 2.3). Inoculation with B. subtilis strain 18MZR

and L. fusiformis strain 31MZR also significantly increased N concentration on D45

(3.7%, 23.1%) and D65 (7.7%, 20.1%) in comparison to control. Meanwhile, inoculation

with bacteria in biochar-amended soil increased N uptake by D45 and D65 as L. fusiformis

strain 31MZR + sawdust biochar (35.4%, 32.8%), followed by B. subtilis strain 18MZR

+ sawdust biochar (25.2%, 23.3%), L. fusiformis strain 31MZR + bagasse biochar

(21.8%, 19.8%) and B. subtilis strain 18MZR + bagasse biochar (19.9%, 17.5%).

Phosphorus concentration in the control was 0.23% (D45) and 0.25% (D65). In biochar

amended treatments, total P was significantly high against control, i.e., bagasse and

sawdust biochar at D45 (37.8%, 59.6%) and D65 (59%, 58.3%), respectively. Application

of PSB increases P concentration in the plant; it was significantly increased in B. subtilis

strain 18MZR on D45 (58.2%) and D65 (58.3%), also in L. fusiformis strain 31MZR

inoculation D45 (62.9%) and D65 (61.5%) than control plant. When PSB strains were

inoculated with biochar, P concentration was highest in all combinations on D45 and D65

than control, as B. subtilis strain 18MZR + bagasse biochar (63.5%, 62.1%), L. fusiformis

strain 31MZR + bagasse biochar (70.1%, 68.4%), B. subtilis strain 18MZR + sawdust

biochar (72%, 70.6%) and L. fusiformis strain 31MZR + sawdust biochar (73.6%,

72.5%), respectively.

The concentration of K was significantly increased in biochar-amended soil (D45 and

D65). Inoculation with B. subtilis strain 18MZR and L. fusifosrmis strain 31MZR also

significantly increased K concentration by D45 (1.3%, 17.3%) and D65 (1.3%, 17%) in

comparison to control. Meanwhile, inoculation with bacteria in biochar-amended soil also

increased K uptake by D45 and D65 as L. fusiformis strain 31MZR + sawdust biochar

(42.2%, 42.1%), followed by B. subtilis strain 18MZR + sawdust biochar (36.8%, 36%),

L. fusiformis strain 31MZR + bagasse biochar (28.8%, 30%) and B. subtilis strain

18MZR + bagasse biochar (23.3%, 23.4%), respectively.

Table 2.3: Plant nutrients concentration at D45 and D65 harvesting

a-e Means of different treatments for various parameters

Treatments: control (uninoculated and untreated), BC-1 = bagasse biochar, BC-2 = saw dust biochar, B1 = B. subtilis 18 inoculation, B2 = L. fusiformis 31

inoculation, M1 = B. subtilis 18 + bagasse biochar, M2 = L. fusiformis 31 + bagasse biochar, M3 = B. subtilis 18 + saw dust biochar, and M4 = L. fusiformis 31 +

saw dust biochar. N: nitrogen, P: phosphorus, K: potassium

N (%) P (%) K (%)

D45 D65 D45 D65 D45 D65

Control 2.37 ± 0.35 e 2.50 ± 0.29 e 0.23 ± 0.03 e 0.25 ± 0.05 e 1.48 ± 0.04 e 1.51 ± 0.05 d

BC-1 2.62 ± 0.51 de 2.72 ± 0.55 de 0.37 ± 0.05 d 0.38 ± 0.05 d 1.78 ± 0.12 d 1.82 ± 0.11 c

BC-2 3.34 ± 0.30 ab 3.56 ± 0.19 ab 0.57 ± 0.04 c 0.61 ± 0.03 c 1.89 ± 0.12 d 1.92 ± 0.11 c

B1 2.46 ± 0.52 de 2.71 ± 0.36 de 0.55 ± 0.07 c 0.60 ± 0.06 c 1.50 ± 0.04 e 1.53 ± 0.05 d

B2 3.08 ± 0.08 bc 3.13 ± 0.10 bcd 0.62 ± 0.06 c 0.65 ± 0.05 c 1.79 ± 0.05 d 1.82 ± 0.06 c

M1 2.96 ± 0.18 bcd 3.03 ± 0.17 cd 0.63 ± 0.13 c 0.66 ± 0.11 c 1.93 ± 0.04 cd 1.97 ± 0.04 c

M2 3.03 ± 0.15 bc 3.09 ± 0.15 cd 0.77 ± 0.07 b 0.79 ± 0.07 b 2.08 ± 0.11 c 2.18 ± 0.16 b

M3 3.17 ± 0.13 ab 3.26 ± 0.07 bc 0.82 ± 0.05 ab 0.85 ± 0.06 ab 2.34 ± 0.14 b 2.36 ± 0.13 b

M4 3.67 ± 0.43 a 3.72 ± 0.40 a 0.87 ± 0.06 a 0.91 ± 0.07 a 2.56 ± 0.26 a 2.61 ± 0.28 a

2.4.5. Soil Nutrient Concentration

Soil N concentration in bagasse biochar-amended soil was 12% (D45) and 8.5% (D65)

more than control, while N was observed higher in sawdust biochar-amended the soil as

29.9% (D45) and 27.8% (D65) more. The PSB strains inoculated soil increased N

concentration by 9.4% (D45) and 8.1% (D65), whereas 18.6% (D45) and 18% (D65) N was

increased in both treatments of B. subtilis strain 18MZR and L. fusiformis strain 31MZR

inoculated soil. Highest N concentration was observed in L. fusiformis strain 31MZR +

sawdust biochar-amended the soil with 37.2% (D45) and 36.2% (D65) increased than control.

Similarly, soil available P concentration was increased by 15.2% (D45) and 17.1% (D65)

in bagasse biochar-amended soil than control, while for sawdust amendment, it was 24.1%

(D45) and 26.6% (D65). PSB inoculation alone solubilized more P by 13.3% (D45), 22.7%

(D65) via B. subtilis strain 18MZR and 16.7% (D45), 25.9% (D65) by L. fusiformis strain

31MZR inoculation than control. The combination of L. fusiformis strain 31MZR + sawdust

biochar-amended soil showed 58.3% more P than control on D65, while in L. fusiformis strain

31MZR + bagasse biochar it was 47.9% (Table 2.4).

Biochar amended soil increased K concentration in both harvestings of D45 and D65. The

addition of biochar enhanced K concentration in soil, as 29% increase was observed in

bagasse amended soil on D45 and D65. Whereas for sawdust biochar amended the soil, it was

47% increase. Meanwhile, inoculation with B. subtilis strain 18MZR and L. fusiformis strain

31MZR increased K concentration on D45 (12.5%, 24%) and D65 (11.5%, 25%) in

comparison to control. Inoculation with bacteria in biochar-amended soil increased K by D45

and D65 as L. fusiformis strain 31MZR + sawdust biochar (58.3%, 59.5%), followed by B.

subtilis strain 18MZR + sawdust biochar (53.2%, 54%), L. fusiformis strain 31MZR +

bagasse biochar (48%, 49%) and B. subtilis strain 18MZR + bagasse biochar (37%, 38%).

In the present study, plant height and nutrients concentration were found positively

correlated in all treatments of biochar amendment, bacterial inoculation and their

combination (Table 2.5 and Table 2.6). Root and shoot height was significantly correlated

with phosphorus uptake. In general, the nutrient uptake induces an increase in plant growth.

Table 2.4: Soil nutrients concentration at D45 and D65 harvesting

N (mg kg-1

soil) P (mg kg-1

soil) K (mg kg-1

soil)

D45 D65 D45 D65 D45 D65

Control 7.51 ± 0.67 e 7.40 ± 0.68 e 13.14 ± 1.30 e 12.05 ± 0.63 e 86.63 ± 5.15 f 82.75 ± 2.06 f

BC-1 8.53 ± 1.16 de 8.09 ± 0.89 de 15.50 ± 0.49 de 14.54 ± 0.28 d 122.00 ± 5.77 e 116.400 ± 8.52 e

BC-2 10.71 ± 1.04 ab 10.25 ± 1.07 b 17.32 ± 0.76 c 16.41 ± 1.03 c 166.00 ± 14.35 c 157.50 ± 15.15 c

B1 8.29 ± 1.36 de 8.05 ± 1.37 de 15.16 ± 0.10 de 15.59 ± 0.83 cd 99.00 ± 2.58 f 93.55 ± 3.55 f

B2 9.23 ± 0.27 cd 9.03 ± 0.33 b-d 15.78 ± 0.66 d 16.27 ± 0.60 c 114.00 ± 10.2 e3 110.43 ± 10.37 e

M1 8.79 ± 0.40 de 8.59 ± 0.39 d-e 16.41 ± 0.65 cd 15.62 ± 0.60 cd 138.00 ± 6.48 d 133.90 ± 6.49 d

M2 10.24 ± 0.51 bc 9.78 ± 0.16 bc 17.19 ± 0.68 c 16.68 ± 0.43 c 166.25 ± 5.62 c 161.65 ± 5.55 c

M3 10.70 ± 0.70 ab 10.30 ± 0.71 b 18.73 ± 0.56 b 18.22 ± 0.72 b 185.00 ± 8.37 b 179.95 ± 7.58 b

M4 11.96 ± 1.01 a 11.60 ± 1.01 a 21.53 ± 1.68 a 22.13 ± 1.78 a 207.75 ± 10.78 a 204.54 ± 11.15 a

a-e Means of different treatments for various parameters

Treatments: control (uninoculated and untreated), BC-1 = bagasse biochar, BC-2 = sawdust biochar, B1 = B. subtilis 18 inoculation, B2 = L. fusiformis 31

inoculation, M1 = B. subtilis 18 + bagasse biochar, M2 = L. fusiformis 31 + bagasse biochar, M3 = B. subtilis 18 + sawdust biochar, and M4 = L. fusiformis 31 +

saw dust biochar. N: nitrogen, P: phosphorus, K: potassium

Table 2.5: Pearson‘s correlation coefficients among plant and soil parameters at D45

Parameters Root Shoot Plant N Plant P Plant K Soil N Soil P Soil K

Root 1.00

Shoot 0.74** 1.00

Plant N 0.59 0.63** 1.00

Plant P 0.62** 0.77** 0.57 1.00

Plant K 0.66** 0.77** 0.68** 0.76** 1.00

Soil N 0.65** 0.75** 0.94** 0.66** 0.76** 1.00

Soil P 0.72** 0.87** 0.59 0.81** 0.82** 0.70** 1.00

Soil K 0.75** 0.82** 0.69** 0.76** 0.90** 0.81** 0.85** 1.00

Significant at P≤0.01**, n = 36

Table 2.6: Pearson‘s correlation coefficients among plant and soil parameters at D65

Parameters Root Shoot Plant N Plant P Plant K Soil N Soil P Soil K

Root 1.00

Shoot 0.82** 1.00

Plant N 0.66** 0.72** 1.00

Plant P 0.58 0.82** 0.58 1.00

Plant K 0.70** 0.83** 0.63** 0.75** 1.00

Soil N 0.70** 0.82** 0.91** 0.67** 0.76** 1.00

Soil P 0.73** 0.94** 0.60** 0.87** 0.78** 0.70** 1.00

Soil K 0.77** 0.86** 0.70** 0.77* 0.91 0.80* 0.80** 1.00

Significant at P≤0.01**, n = 36

2.5. Discussion

Use of PSB for solubilization of P and plant growth promotion is previously tested for

various crops and biochar is used independently in altering soil properties. This study was

designed to evaluate the combined effect of indigenously isolated PSB and low-

temperature biochar to evaluate nutrients uptake in maize plant in the semi-arid region. In

the present study, biochar from two feedstocks (bagasse and sawdust) and two PSB

strains (B. subtilis strain 18MZR and L. fusiformis strain 31MZR) were used as

inoculants to analyze plant growth under greenhouse conditions with different

combinations of biochar and PSB application. According to 16S rDNA analysis, isolated

PSB strains belonged to Firmicutes: Bacillus sp. and Lysinibacillus sp. (Figure 2.2).

Similarly, in previous studies, an association of maize plant with specific bacterial genera

(Bacillus and Lysinibacillus) has been reported (Cavaglieri et al., 2005; Vigliotta et al.,

2016). Further studies also showed that B. subtilis and L. fusiformis are present in soil

ecosystem where they interact with plant roots and particularly in maize plant (Singh et

al., 2013; Posada et al., 2016; Zhang et al., 2016). Bacterial strains such as B. subtilis and

L. fusiformis have already been reported as effective maize plant growth promoting

rhizobacteria through P-solubilization, and this activity was confirmed by the

biochemical test in the present study (Sgroy et al., 2009; Chauhan et al., 2016).

Inoculation with Bacillus and Lysinibacillus for various crops significantly promoted

plant growth leading to increasing in plant height and biomass. In the current study,

inoculation of PSB enhanced plant growth regarding root and shoot length which were

also observed in studies of Sgroy et al. (2009) and Chauhan et al. (2016).

Similarly, biochar addition to the soil may increase soil inorganic nitrogen which

assists the plant to increase its biomass regarding plant height. Moreover, it improves

moisture content in the soil for enhanced nutrient availability (Chen et al., 2010), and the

current study showed that application of bagasse biochar and sawdust biochar improved

soil condition in comparison to the control (Nguyen et al., 2017). Biochar made from

sawdust feedstock has been reported to assist plant growth by improving soil

physicochemical properties such as enhancing nutrient retention by up to 59%, and

nutrients content of plant, which was also observed in the sawdust amended soil of the

current study which enhanced nutrients concentration in soil and plant than control

(Laghari et al., 2016). Biochar addition widely enhances the potential of soil to boost

plant growth as observed in the bagasse and sawdust biochar-amended soil where the

plant length was notably increased than control by increasing soil porosity (De Tender et

al., 2016; Mollinedo et al., 2016). According to an estimate, more than 80% of

rhizospheric bacteria can produce growth promoting chemicals and increase in plant

height which is endorsed in PSB inoculated plants in the current study by enhancement of

root and shoot length, particularly the L. fusiformis inoculated maize plant than the

control (Arruda et al., 2013). The combination of bacteria and biochar for plant growth

has been reported as a promising approach against various crops. Similar results were

monitored in the study where the combination of PSB and biochar is more effective to

enhance the plant growth than a single application of either PSB or biochar. The addition

of biochar recruits the microbiome which produces certain compounds in the rhizosphere

to make nutrients available for plant growth and biomass production (De Tender et al.,

2016; Shanta et al., 2016) and in this study, a combination of PSB and biochar increased

in plant growth. Plant height increase can be attributed to nutrient availability such as N

and P due to biochar amendment and PSB inoculation where they may produce various

organic compounds in the rhizosphere such as indole acetic acid (IAA), gibberellin, and

cytokinin.

The selected strains showed a positive response for P-solubilization on Pikovskaya‘s

agar media, a selective media for the screening of P-solubilizing organisms by halo zone

formation (Kaundal et al., 2016). This medium contains tricalcium phosphate which is

broken down by the activity of PSB into various acids (malic acid, formic acid, citric

acid, succinic acid, lactic acid, and tartaric acid) and their chelation capacity implicates

major mechanism in the solubilization of inorganic phosphates by micro-organisms (Park

et al., 2009). During the screening procedure, clear halo zone formation due to organic

acids production was observed for both isolated bacterial strains. In addition, some PSB

(B. subtilis) also reported for Acetylene Reduction Activity where they fix N from the

atmosphere (82.9 mg L-1

) and converted to ammonium (Xie et al., 1998; Suksabye et al.,

2016). The N derived from biological nitrogen fixation (BNF) is fixed as ammonia with

minimum losses to the environment. According to an estimate, about 50-70% of chemical

N-fertilizer in soil losses through denitrification, leaching, and volatilization (Hodge et

al., 2000). Besides that, both bacterial strains had plant growth promotion abilities such

as IAA, chitinase production which make the plant stress tolerant. The L. fusiformis had

produce IAA (32.1 µg ml-1

), chitinase (3.2 mU ml-1

) and solubilize P (198.2 µg ml-1

)

(Trivedi et al., 2011). B. subtilis inoculation on Artemisia annua L. yielded plant height

as 93.3cm and assisted in N (1.52%), P (0.2%) and K (2.1%) uptake (Awasthi et al.,

2011). Rhizospheric bacteria and plant roots release specific exudates in the rhizosphere

which induce the activity of phosphatase and enhance P uptake (Geneva et al., 2006).

Besides that, Bacillus sp. helped the plant to uptake K (Sheng and He, 2006).

Application of biochar to the soil improves the microbial community and bacterial

population affiliated with Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria

phyla are activated by making 92-95% of the community which benefits the plant growth

(Kolton et al., 2011). In this study, isolated bacteria belong to the phylum Firmicutes and

further results showed a significant correlation between biochar and bacterial

combination on plant growth and nutrient uptake. Moreover, biochar treated with L.

fusiformis released P up to 54% (He et al., 2014). However, positive growth effects from

bacteria and biochar were less apparent at D45. Biochar as a porous material enhances the

soil porosity, allows water and air to infiltrate which facilitated extension of the root

system. Some rhizospheric factors along with photosynthetically assimilated carbon

compounds such as the root exudates (vitamins, amino acids, amides, and carbohydrates)

influence the rhizospheric microbial community (Lugtenberg and Kamilova, 2009).

Moreover, the addition of low pyrogenic biochar in the soil can do positive priming of the

carbon which facilitates microbial community to grow in the rhizosphere (Zimmerman et

al., 2011). PSB inoculation (B. subtilis strain 18MZR and L. fusiformis strain 31MZR)

significantly increased total N and P concentration in the maize plant on D45 and D65.

These increments were attributed to the inherent bacterial growth promoting abilities.

2.6. Conclusion

The conducted greenhouse study demonstrated that biochar (bagasse and sawdust)

addition to the soil, inoculation with indigenously isolated PSB strains (Bacillus subtilis

strain 18MZR and Lysinibacillus fusiformis strain 31MZR) and their various

combinations significantly increased plant growth by enhancing nutrients uptake (N, P,

K) in maize plant. The increments in plant growth are mainly attributed to the P-

solubilization by bacterial strains in the soil, P from the biochar and other PGP abilities

such as IAA, cytokinin and gibberellic acid production. Biochar and bacterial interaction

may form soluble-P which resulted in 0.87% uptake to the plant leave in L. fusiformis

strain 31 MZR + sawdust biochar amended maize on D65. Plant height and available

nutrient concentration are strongly correlated among all treatments. Maize plants

inoculated with bacteria and biochar together exhibited maximum growth and nutrient

concentration than biochar and bacterial treatments separately. Thus, this study shows

that PSB and biochar have the potential to use as a promising approach in improving

plant growth and nutrient absorption besides the conventional approaches under semiarid

soil conditions. Further studies are necessary to evaluate (i) the suitability of B. subtilis

strain 18MZR and L. fusiformis strain 31MZR on maize yield under field conditions and

(ii) the suitability of PSB in combination with biochar for nutrient availability in the field

and carbon sequestration potential. Moreover, the use of molecular approaches related to

maize plant and isolated bacterial strains can further uncover their interactive relation to

boost plant growth. Key findings of the study are:

Combination of biochar and isolated PSB strains enhanced plant growth and

nutrients uptake.

The bacterial ability of P-solubilization and it‘s PGP traits enhanced plant

growth.

Bacteria can solubilize P connected to biochar and make it available to plant.

Further compatibility of PSB and biochar can be tested at the molecular level.

Chapter 3

Quality Assesment of Biochar

3.1. Introduction

Biochar is produced by pyrolyzing various organic wastes and feedstocks under

anoxic conditions at 250-900 oC which enrich the carbon concentration (Cao et al., 2011;

Xie et al., 2015). A wide set of different feedstocks is suitable for biochar production

derived from sludge, animal-waste (chicken manure, cow manure, cow manure, poultry

litter) and plant-derived feedstock (wood biomass, agricultural residues, forestry

residues). The application of biochar to soil has various agricultural and environmental

benefits, including carbon sequestration, water resource protection, reduction of synthetic

fertilizers, crop production and soil improvement (Rehrah et al., 2016). In agriculture,

biochar application to soil has gained much attention due to long-term improvement in

soil biochemical and physical properties in nutrient retention, influencing soil biota and

soil biogeochemistry (Ayodele et al., 2009; Lehmann et al., 2011). Sludge, animal-waste

derived biochar, and plant-feedstock derived biochar have different chemical and

physical properties which make them suitable for soil in various perspectives such as

carbon sequestration, fertilization and liming, etc. (Joseph and Lehmann, 2009).

Biochar has real potential to be utilized in the absorption of environmental

contaminants due to its various properties such as easy to make from broadly cost-

effective feedstocks, chemical and physical surface characteristics, microporous

structuring, high pH and active functional groups (Do and Lee, 2013; Tan et al., 2016).

Biochar varies in nutrient contents, carbon and also surface structure (Joseph et al., 2010;

Spokas, 2010). Pyrolysis process alters constituent carbon compound by increasing

aromatic carbon in contrast to biomass feedstock which makes it chemically recalcitrant

and resistant against biochemical decomposition which makes biochar as carbon

sequestration tool (Abbott and Robson, 1982; Lehmann et al., 2006). During the pyrolytic

process, 20-60% of the carbon in biomass is carbonized into aromatic carbon

(Zimmerman, 2010; Lanza et al., 2015; Enders and Lehmann, 2017). Thermochemical

property of biochar is proposed to classify the long-term carbon stay in soil (Harvey et

al., 2012; Mańek et al., 2013).

Application of biochar in agriculture could be beneficial to design a

characterization framework which would influence the plant growth by improving soil

health in the short and long run. According to agronomic standpoint, biochar quality

assessment is an important task which may affect plant growth by attributing soil

physical properties and enhancing nutrient availability. Since the biochar has a large

surface and structure which also can be a shelter for soil microorganisms such as

mycorrhizal fungi and bacteria to grow in soil (Ortas, 2016). Therefore, the aim of this

study was to assess the quality of feedstock and biochar prepared from the Mediterranean

region based sludge, animal-waste and plant-feedstock derived material. It is important to

understand feedstock availability, type, suitability to the soil properties, appropriation for

plant growth and soil microbiota for further use in agricultural production and carbon

sequestration.

3.2. Objective

Quantitative evaluation of nutrients in different biochar prepared from

sewage sludge, animal waste and plant based feedstock.

Quantification of carbon content in various biochar.

Thermal stability estimation of biochar prepared from different

feedstocks.


3.3.1. Biochar preparation

The feedstocks of municipal sewage sludge, animal-waste and plant origin were

collected from local suppliers. Feedstocks of chicken manure (ChM), cow manure (CM),

municipal sewage sludge (S), sheep manure (SM), eucalyptus (Eu), Phragmites (Ph), and

sawdust (SD) were collected for biochar production. Biochar was prepared at two

pyrolytic temperatures considering their characterization, easy accessibility to the

feedstock and their application into the soils for plant growth and carbon sequestration.

Considering it, ChM, CM, S, SM, Eu, and Ph were pyrolyzed at 550°C with a residence

time of 1 hr in a closed container under oxygen-limited conditions in a muffle furnace

(RD50, REF-SAN, Turkey) for characterization only. Moreover, Ph and SD were

pyrolyzed at 350°C with a residence time of 1 hr for characterization and further

exploration of plant-biochar interaction in soil (discussed in chapter 4, and 5). Biochar

was milled to pass through 2 mm sieve for further analyses.

3.3.2. Electrical conductivity and pH

Biochar pH values were obtained in triplicate using a ratio of 1.0 g of biochar in

20 mL deionized water with the modification that the time on the shaker was increased to

1.5 hr to ensure sufficient equilibration between the solution and biochar surfaces.

Electric conductivity (EC) was then determined with an Orion model 115A plus

conductivity meter (Thermo Fisher Scientific, Waltham, MA) (Rajkovich et al., 2012).

3.3.3. Moisture, volatile matrix and ash contents

The moisture, volatile matrix (VM) and ash content of biochar measured

following the standard ASTM D1762 – 84 methods (ASTM, 2007). Briefly, 5.0 g of

oven-dry sample was weighed into a pre-ignited crucible and heated at 500 oC overnight

(>8 hr). The crucible was then cooled to room temperature in a desiccator and weighed

again. The ash content then calculated:

( ) ( )

( )

3.3.4. Total nutrient analysis

Total P, K, Ca, Mg, Fe, Mn, and B obtained after dry combustion by heating to

500 oC over 2 hr and holding at 500°C for 8 hr. Five-milliliter HNO3 was added to each

vessel and digested at 120 oC until dried. Tubes were removed from the block and

allowed to cool before adding 1.0 mL HNO3 and 4.0 mL H2O2. Samples were placed

back into a preheated block and processed at 120 oC to dryness, then dissolved with 1.43

mL HNO3, made up with 18.57 mL deionized water and filtered (Enders and Lehmann,

2012). Nutrient concentration in digested plant samples was analyzed by inductively

coupled plasma optical emission spectrometry (ICP-OES), Perkin Elmer, USA.

3.3.5. Carbon analyses

Different fractions of carbon in biochar and its stability were analyzed. Total

carbon (TC) and nitrogen content of the biochar samples determined by combustion using

a Thermo Fisher Scientific FLASH 2000 Series CN Elemental Analyzer (Thermo Fisher

Scientific, Waltham, U.S.A.). About 3.5 – 5.5 mg of biochar samples were analyzed and

compared to calibration with the reference material as Aspartic acid (2-

aminobubutanedioic acid). Fixed carbon (FC) was derived from mass balance equation:

Calcium carbonate (CaCO3) was estimated by calcimeter (Loeppert and Suarez,

1996). Moreover, the stability of biochar carbon against mineralization was evaluated

using the dichromate oxidation method (Schumacher, 2002). Briefly, 0.1 g of biochar

(<0.15 mm) was weighed into a 500 mL conical flask, followed by addition of 10 mL

0.167 M K2Cr2O7 and 20 mL concentrated H2SO4. The mixture was settled on a bench

until the room temperature reached. Deionized water was then added to bring the mixture

to 60.0 mL. This solution was pipetted into a 250 mL conical flask and titrated with

freshly standardized 0.5 M FeSO4 to the endpoint using 0.2% N-phenoantranilic aqueous

solution as an indicator. Blanks without biochar addition included as a control. Unstable

organic carbon (OC) in biochar calculated as:

( ) ( )

( )

where V0 is the volume (mL) of FeSO4 consumed in titrating the control, V is the volume

(mL) of FeSO4 consumed in titrating the biochar sample, and carbon is the concentration

(mol L-1

) of the freshly standardized FeSO4 solution. Stable/recalcitrant OC calculated as

the difference between total OC and unstable OC contents of the biochar.

3.3.6. Scanning Electron Microscopy

Samples were analyzed by environmental scanning electron microscope (ESEM)

model Quanta FEI 650 (FEI, Netherlands). Before observation, the surface of the sample

was coated with a thin, electric conductive gold film.

3.3.7. Fourier transform infrared spectroscopy (FTIR)

Fourier transform infrared spectroscopy analysis of feedstock and biochar

samples were carried out to characterize the surface functional groups. Both feedstock

and biochar were ground and mixed with KBr to 0.1% and pressed into pellets. Spectra

recorded in the range of 4000 – 500 cm–1

with a resolution of 4 cm–1

using a PerkinElmer

Spectrum One spectrometer (Du et al., 2009).

3.3.8. Thermal gravimetric analysis (TGA)

The TGA and differential thermal analysis (DTA) were used to measure thermal

decomposition of biomass and the respective biochar under air atmosphere by a thermal

analyzer (Perkin-Elmer STA 6000, simultaneous Thermal analyzer); moreover, the

devolatilization rate determined. Samples of 5 – 9 mg were heated from 50 to 105 °C at a

rate of 20 °C min-1

and kept at 105 °C for 10 min to remove the moisture. Samples were

then heated from 40 to 800 °C at 20 °C min-1

(air flow rate of 90 mL min-1

) to collect

thermal decomposition curves (Enders and Lehmann, 2012). The treatment of thermal

curves involved the analysis of TG and DT combustion thermograms. The sample weight

loss at a particular temperature and the rate of weight loss determined following the TG

and DT combustion curve, respectively.

3.3.9. Statistical analysis

All the samples were analyzed in triplicate, and Pearson‘s correlation was used to

evaluate the correlation among biochar properties for quality assessment. The correlation

was considered to be statistically significant at a 95% confidence interval (p≤0.05). All

data analysis and plotting were performed with OriginPro 9.0 data analysis software

(OriginLab Corporation, Northampton, MA).

3.4. Results

3.4.1. pH and electrical conductivity

The results of the pH and EC analysis of biochars produced from municipal

sewage sludge, animal-waste and plant-derived feedstocks at different pyrolysis

temperatures are given in Table 3.1. The rise in pyrolysis temperature increased biochar

pH, showing that the biochar produced at 500oC are more alkaline in nature than 350

oC.

Biochars (ChMB) had the highest EC among all studied biochars. The EC values (0.33 –

3.37 mS cm-1

) showed that biochar materials are not saline and suitable as the soil

amendment.

3.4.2. Moisture, volatile matrix and ash contents

Moisture contents are variable in all the biochar, and they ranged from 1.65 -

5.47%. Biochar from the plant feedstock had comparatively more moisture content than

rest of biochars. The VM of the biochars also varied from 25.56 - 66.99% at both

pyrolysis temperature where the low temperature had more VM in PhB350 and SDB. Ash

content ranged from 3.11 - 66.62% in biochars depending on pyrolysis temperature and

feedstock types. Plant-derived biochar had significantly lower ash content than rest of

biochars.

3.4.3. Mean nutrient analysis

In this study, the total nutrient analysis was done for sustainable high crop yields

by application of biochar. Nutrients concentration in biochar (N, P, K, Ca, Mg, Fe, Mn,

and B) was abundant in municipal sewage sludge, and animal-waste derived biochar in

comparison to the plant-feedstock derived biochar. Maximum N was found in ChMB as

1.62%, while the minimum was 0.51% in PhB350 (Table 3.1). Similarly, the highest P

concentration was found in SB as 1.33%, while it was 0.03% in SDB as the lowest.

Similarly, micronutrients showed more concentration in municipal sewage sludge, and

animal-waste derived biochar than plant-feedstock derived biochar. The Fe concentration

ranged from 0.003 to 1.56% with an increasing order as

PhB350<PhB550<EuB<SDB<SMB<CMB<ChMB<SB. These results suggested that the

municipal sewage sludge and animal-waste derived biochar may be used in crop

improvement by the provision of more concentrated nutrients in the soil environment.

Table 3.1: pH, EC, proximate, ultimate analyses and nutrient elements of biochars

Element

Biochar

ChMB CMB SB SMB EuB PhB550 PhB350 SDB

pH 9.90±0.02 8.11±0.24 9.24±0.12 10.26±0.01 9.15±0.06 8.98±0.06 7.49±0.08 6.29±0.09

EC (mS cm-1

) 3.37±0.29 0.84±0.06 0.61±0.04 2.82±0.18 1.31±0.97 2.38±0.32 2.23±0.16 0.33±0.00

Proximate analysis

Moisture (%) 2.69±0.14 1.65±0.12 2.58±0.12 3.89±0.22 3.69±0.07 5.47±0.30 3.91±0.11 2.56±0.99

Asha (%) 64.82±0.56 64.35±0.11 66.62±0.06 44.34±1.33 7.70±0.20 11.96±0.15 8.18±0.02 3.11±0.01

VMb

(%) 25.21±0.34 20.56±1.38 20.61±0.11 32.27±1.28 31.26±1.00 30.23±1.13 56.05±1.50 66.99±0.58

FCc (%) 7.27±0.55 13.45±1.25 10.19±0.25 19.49±0.97 57.35±0.89 52.35±1.54 31.86±1.59 27.34±1.35

Ultimate/Nutrient analysis

C (%) 20.67±1.18 29.92±1.47 17.65±1.67 34.07±1.83 73.63±12.63 68.31±1.29 67.89±8.76 61.96±3.96

N (%) 1.62±0.23 1.43±0.80 1.21±0.14 1.52±0.32 0.63±0.13 0.51±0.03 0.57±0.13 0.56±0.05

P (%) 1.12±0.11 1.00±0.03 1.33±0.06 0.56±0.00 0.06±0.00 0.23±0.01 0.09±0.00 0.03±0.00

K (%) 2.17±0.11 0.29±0.00 1.18±0.03 2.95±0.03 0.44±0.02 2.56±0.11 2.07±0.01 0.22±0.01

Ca (%) 5.65±0.62 10.15±0.00 4.24±0.07 3.27±0.11 3.41±0.25 0.37±0.02 0.12±0.00 0.79±0.03

Mg (%) 5.55±0.32 1.34±0.02 2.64±0.00 4.17±0.12 0.20±0.01 0.22±0.01 0.09±0.00 0.08±0.00

Fe (%) 1.199± 0.03 0.768± 0.02 1.526± 0.02 0.633± 0.03 0.017± 0.00 0.014±0.00 0.003±0.00 0.023± 0.00

Mn (mg kg-1

) 123.80±15.40 78.05±1.95 177.00±2.80 59.25±1.85 11.20±0.50 6.95±0.45 4.40±0.00 28.60±22.40

B (mg kg-1

) 19.15±0.45 4.30±0.60 12.65±2.05 20.45±0.85 4.85±0.15 1.10±0.00 0.19±0.01 2.00±0.70

a: ash on dry basis, b:volatile matrix on dry basis, c: fixed carbon on dry basis, Biochar; ChMB: chicken manure biochar, CMB: cow manure biochar, SB: sludge

biochar, SMB: sheep manure biochar, EuB: eucalyptus biochar, PhB550: phragmites biochar, PhB350:phragmites biochar, SDB sawdust biochar

3.4.4. Carbon analyses

This study showed that plant-feedstock derived biochar has shown a higher amount of

total carbon in comparison to the municipal sewage sludge and animal-waste derived

biochar. Total carbon concentration ranged from 17.65 to 34.07% in municipal sewage

sludge, and animal-waste derived biochar while, in plant-feedstock derived biochar, it was

61.96 to 73.63%. Maximum total carbon was noted in EuB which was then the SB having

17.65%. There was no significant difference observed in PhB550 and PhB350 as the

difference was only a 0.6% increase. Similarly, the amount of FC in municipal sewage

sludge and animal-waste derived biochar ranged 7.27 to 19.49% whereas, in plant-derived

biochar, it was 27.35 to 57.35% as the same pattern was observed in the TC of plant-derived

biochar. Observed stable carbon was not much different than the TC and the difference range

was found from 0.0001 to 0.0014 (Table 3.2). Hence, it can be concluded from the data that

the total carbon of the biochar is also stable and recalcitrant to the environment. Results of

the study suggested that alkalinity and acidity of biochar are dependants on carbon fractions

and the presence of various functional groups on biochar surface.

Table 3.2: Stable, unstable and calcium carbonate content in biochar

Biochar type Stable C (%) Unstable carbon (%) CaCO3 content (%)

ChMB 20.67±1.2 0.0002±0.0000 6.28±0.09

CMB 29.92±10.5 0.0010±0.0001 15.74±0.37

SB 17.65±1.7 0.0014±0.0002 3.20±0.07

SMB 34.07±12.8 0.0006±0.0000 3.98±0.09

EuB 73.63±12.6 0.0001±0.0000 3.46±0.34

PhB550 68.31±1.3 0.0014±0.0000 0.19±0.04

PhB350 67.89±8.8 0.0004±0.0001 0.30±0.04

SDB 61.96±4.0 0.0006±0.0001 0.95±0.10

Biochar; ChMB: chicken manure biochar, CMB: cow manure biochar, SB: sludge biochar,

SMB: sheep manure biochar, EuB: eucalyptus biochar, PhB350: phragmites biochar,

PhB350:phragmites biochar, SDB sawdust biochar

3.4.5. Scanning Electron Microscopy

The SEM images of municipal sewage sludges, animal-waste and plant feedstock

derived biochars had a great difference as illustrated in Figure 3.1. Municipal sewage sludge

and animal-waste derived biochar surfaces, particularly ChMB and CMB are amorphous in

comparison to the plant-feedstock derived biochar. A variety of porous structure and biochar

shapes can be seen in the SEM photographs which show the diversity of porous structure.

Some of the biochars had spherical cavities along with smooth surface.

Figure 3.1 SEM images of biochar obtained at different pyrolysis temperatures and feedstock sources: at 550oC; A)

chicken manure biochar (ChMB), B) cow manure biochar (CMB), C) sludge biochar (SB), D) sheep manure biochar

(SMB), E) eucalyptus biochar (EuB), F) phragmites biochar (PhB550), at 350oC; G) phragmites biochar (PhB350), H)

sawdust biochar at (SDB)

3.4.6. Fourier transform infrared spectroscopy

Environmental interaction with the biochar is dependent on the surface chemistry of

biochar. The FTIR spectroscopy peaks showed that the aliphatic and aromatic functional

groups were predominant in municipal sewage sludge, animal-waste and plant-feedstock

derived biochar while most of the functional groups were alike (Figure 3.2). Dominant peaks

(3600-3000 cm-1

) were observed for O-H and N-H group. Details of the biochar to the

particular peak range and functional groups are presented in Table 3.3. The C-H, CH3, CH2

stretches were also dominant in all the feedstocks and biochar at 3000-2600 cm-1

except for

Eu, EuB.

In municipal sewage sludge, NH and organic OH groups are very unstable at

pyrolytic temperature. Thermodecomposition of C-OH and aliphatic C-H groups increases

C=O peak in ChMB. After pyrolysis, C=O groups remain intact in the peptide structures.

Increased stability with decreasing aliphatic C-H, O-H groups concurrently increasing the

aromatic C-H, C=C groups presence, which appears as pyrolytic temperature increases.

Plant-derived biochar had cellulose and hemicellulose which was confirmed by the presence

of oxygenated hydrocarbons. Results showed that presence of carboxyl and hydroxyl groups

in the biochar make them suitable soil amendment for acidic soils in raising pH and cation

exchange capacity improvement (Yuan et al., 2011; Oh et al., 2012).

Figure 3.2 Fourier transform infrared (FTIR) spectra of feedstock (A) as chicken manure (ChM), cow manure (CM),

sludge (S), sheep manure (SM), phragmites (Ph), and sawdust (SD),and biochar (B) as chicken manure biochar (ChMB),

cow manure biochar (CMB), sludge biochar (SB), sheep manure biochar (SMB), eucalyptus biochar (EuB), phragmites

biochar (PhB550), phragmites biochar (PhB350), sawdust biochar at (SDB)

Table 3.3: FTIR spectroscopy wave number (cm-1

) for feedstocks and their respective biochar. A

Feedstock Biochar Wave number (cm-1

) Assignments

ChM, SD ChMB, SB, SDB 3600-3000, O–H (hydroxyl), phenols, organic acids, H-bonded N-H

groups

Ph PhB350, PhB550, SMB 3620-2500 O–H (stretching), alcohol

ChM, SD, Ph ChMB, SB, SMB, SDB,

PhB350, PhB550, CMB

3000-2600 C–H (stretching), CH3-stretch, CH2-stretch, NH2

- SMB, EuB 2360-2364 P-H (Organophosphorus group), C–O vibrations of the

CO2 molecule

- SMB 1882-1797 C–O (stretch)

ChM, Ph SMB 1800-1514 Carboxylic acids C=O (stretching) and C–C

- ChMB, EuB 1685-1461 Cyclic amides, Out-of-plane bending of carbonates

- SB 1600-1580 Quinones

Ph, SD PhB550, PhB350 1636-1600 O-H bending (of H2O), C=O, C=C

- ChMB 1574-1330 Nitro groups

- SB, EuB 1570-1550 Ketones (C=O stretching), Amides II, aromatic C=C

ChM - 1543 Amide

- EuB 1510 Substituted benzene carbon skeleton vibration

ChM - 1457 CH2 deformations

SD - 1422 C6 ring

ChM SB, EuB 1450-1380 CH2 and CH3 bending, C-OH deformation of CO2H,

COO– symmetric stretch, OH of phenol,

Ph - 1371 O-H bending

ChMB 1314 The C-N stretch of primary amines

ChM, Ph ChMB, SB, SMB 1400-1000 Phenolic groups C–OH (stretching), C-O stretching

Ph PhB550, PhB350 1400-1000 Ethers (C–O stretching, bending), C-O-H (OH

association), –CH2–

ChM - 1314-1190 C–N stretch

EuB, PhB550, PhB550 1120-1117 C=O stretching and bending of ketones

Ph - 1061 C-OH bending

CM - 1100-1035 angular deformation of O-H

Ph, SD SB, SDB 1070-1030 C-O-C stretching (pyranose ring skeletal)

- SMB 775 C-H and O-H

- ChMB 757-714 β -ring of pyridines

SD EuB, SDB 700-500 OH (bending), N-H bending of amides, CH– of alkenes

and alkanes

Feedstocks; ChM: chicken manure, CM; cow manure, S: sludge, SM: sheep manure, Eu: eucalyptus, Ph; phragmites, SD: sawdust,

Biochar; ChMB: chicken manure biochar, CMB: cow manure biochar, SB: sludge biochar, SMB: sheep manure biochar, EuB:

eucalyptus biochar, PhB350: phragmites biochar, PhB350:phragmites biochar, SDB sawdust biochar

3.4.7. Thermal gravimetric analysis

The thermogravimetric analysis was performed for the municipal sewage sludge,

animal-waste and plant-derived feedstocks and their biochar which are presented in Figure

3.3. By analyzing the TG and DTA curves generated for municipal sewage sludge and its

biochar, three main areas of the peak can be recognized. In the initial area (20–250°C),

physically adsorbed water to the biochar was lost. In the second peak area (250 to 600°C),

VM in municipal sewage sludge was lost by degasification which resulted in 37.7% weight

loss. Third peak area continued up to 800oC where inorganic matter decomposition, for

example, CaCO3 took place. In chicken manure, adsorbed water was removed up to 220oC

while in the second phase (220-600oC), 15% weight was lost during pyrolysis. At the

beginning of pyrolysis, CM had weak endothermic peak followed by an exothermic peak. In

the manures, endothermic decomposition at 220-250oC lead to the formation of SO2 and CO2

just like nitrate in manures. In Eu and EuB, weight loss started due to evaporation of

adsorbed moisture at 90oC. Additionally, thermal degradation of cellulose, lignin, and

hemicellulose at 160oC and 500

oC are considered major weight loss areas by 31% and 51%

weight reduction, respectively. The first peak in PhB550 was located at 100oC, the second

peak at 330oC and third peak was ranging from 330-540

oC which resulted in the loss of

weight as 2% (moisture), 4.5% (cellulose and hemicellulose) and 22% (cellulose

decomposition) respectively. In PhB350, 25% weight loss was observed up to 152oC, while

until 470oC it was 45% loss. Similarly, in SDB 4.8% weight loss happened in the form of

moisture, while 13% was observed up to 412oC which further continued to decrease up to

800oC by 34% weight loss.

Figure 3.3-a TGA-DTA curves of various animal feedstock and their biochar at 550oC; A) sludge (S) B) cow manure (CM),

C) sheep manure (SM), D) chicken manure (ChM), E) sludge biochar (SB), F) cow manure biochar (CMB), G) sheep manure

biochar (SMB), and H) chicken manure biochar (ChMB)

Figure 3.3-b TGA-DTA curves of plant-derived biochar at 550oC; A) phragmites biochar (PhB550) and B) eucalyptus biochar (EuB)

Figure 3.3-c TGA-DTA curves of plant-derived biochar at 350

oC; A) phragmites (Ph), B) sawdust (SD), C) phragmites biochar

(PhB350), D) sawdust biochar (SDB)

3.4.8. Correlation

Pearson‘s correlation showed a positive interaction between pH and EC (Table 3.4)

which are key factors in the selection of biochar suitability as a soil amendment (acidic or

alkaline soil). Similarly, positive correlation between pH and ash showed availability of

nutrients in alkaline pH biochar which was further confirmed by N availability depending on

biochar pH. Similarly, EC and VM were positively correlated in biochar. Moreover, TC was

enhanced in high VM produced biochar.

Table 3.4: Pearson‘s correlation values among biochar properties for quality assesment

pH EC Moisture

(%)

Ash (%) VM (%) FC (%) TC (%) N (%) C:N

pH 1

EC 0.59* 1

Moisture

(%)

0.22 0.45* 1

Ash (%) 0.53* 0.10 -0.55* 1

VM (%) -0.77* -0.18 0.11 -0.75* 1

FC (%) -0.13 -0.027 0.67 -0.82* 0.23 1

TC (%) -0.43* -0.08 0.51* -0.91* 0.57* 0.85* 1

N (%) 0.50* 0.26 -0.42* 0.74* -0.50* -0.67* -0.69* 1

C:N -0.54* -0.17 0.49* -0.86* 0.54* 0.80* 0.89* -0.90* 1

*shows the significant correlation, - shows negative correlation

3.5.Discussion

Biochar is an emerging soil amendment rich in carbon followed by essential

nutrients required for plant growth. Moreover, it‘s presence in soil alters soil properties

and influence the soil microbial activity. Beneficial effects of biochar are dependant on

it‘s physicochemical and biological properties. Biochar having high pH range shows

alkalinity which could be due to the separation of feedstock minerals from the organic

matrix at a high pyrolysis temperature (Cao and Harris, 2010). Biochar is alkaline in

nature, and pH of biochar increases with increasing temperature of pyrolysis (Yao et al.,

2011; Zhang et al., 2015) owing to proceeding removal of acidic functional groups on

biochar surface (Mukherjee et al., 2011) leaving behind the hydrophobic aromatic carbon

surface. Alkaline biochar could be used in acidic soils whereas the PhB350 and SDB with

neutral pH can also be used in neutral to slightly alkaline pH soils to absorb positive ions.

Moreover, the variation in biochars EC could be due to the loss of VM leaving behind the

ash content (Cantrell et al., 2012). Pyrolysis process and release of VM concentrate the

elements responsible for EC enhancement that could be considered to select suitability of

large-scale biochar application (3-30 ton ha-1

) as a soil amendment (Johannes and

Stephen, 2009).

Ash content in the biochar is enriched with P, S, K, Ca, and Mg (Gaskin et al.,

2008). The desirable moisture content in biochar is supposed to be 10% by weight

(Bridgwater and Peacocke, 2000). In this study, moisture content was in a desirable range

which may be useful in biochar handling and reduction in loss of soil nutrients (Ahmad et

al., 2012). The VM is well correlated with elemental ratios, and it could be a suitable

predictor of biochar carbon stability (Spokas, 2010). The ash contents and carbon are

strongly correlated with each other. In biochar with low ash contents, high amount of

carbon was reported. Agricultural residues and manures produce biochar with high ash

contents, in comparison to that from woody feedstock‘s (Demirbas, 2004).

Some of the elements such as P are relatively stable at low pyrolytic temperature

biochar, and it may release slowly into the soil (Knoepp et al., 2005). However,

increasing total P concentration did not always affect P-available fraction, which has an

agronomical value but using P-solubilizing organisms can make it available. In the case

of K, 2.95% was found in SMB whereas 0.22% as the lowest in the SDB. Pyrolysis

temperature variation effect on biochar nutrient concentration was non-significant in

PhB350 and PhB550. In the pyrolysis process, VM escape feedstock (DeLuca et al.,

2015) leaving behind the high concentration of elements in biochar.

Pyrolysis temperature changes the nutrient concentration, as the temperature rises,

volatilization of nutrients starts, and they leave the feedstock in a sequence. For example,

N volatilizes at low temperature (200°C), P and K volatilize between 700-800 °C and rest

of the nutrients such as Ca need higher temperature (>1000°C) to volatilize (Kookana et

al., 2011; DeLuca et al., 2015). Although the biochar produced at low temperature have

less amount of N, they are more efficient in absorbing NH4 (Chan and Xu, 2009). Iron

plays a significant role in both the stabilization of organic carbon and uptake of nutrients

by plants. The Fe oxide can cause either precipitate or adsorb onto the surface of

microorganisms forming an amorphous phase (Gilbert and Banfield, 2005).

Using SEM micrographs, it is likely to observe the structural modification under

pyrolysis conditions (Uzun et al., 2010). Formation of amorphous surface and porous

structure shows vesicles established due to the release of VM (Keiluweit et al., 2010;

Uzun et al., 2010). The biochar produced at a higher temperature usually has

homogeneous and well porous structures in comparison to the slow pyrolyzed biochar as

discussed by Uzun et al. (2010). The large pores in plant-derived biochar might be

originated from the vascular bundles of the starting wood biomass (Hernandez-Mena et

al., 2014). The porous texture of biochar is beneficial in soil quality improvement by

providing habitats for symbiotic microorganisms such as bacteria and mycorrhizal fungi

(Thies and Rilling, 2009).

Fixed carbon increases as the pyrolysis temperature increases which can be seen

in EuB, PhB550, PhB350 and SDB (Song and Guo, 2012). They concluded that

recalcitrant carbon increased with the increase in pyrolytic temperature. Similar results

were observed by Enders et al. (2012) where the difference in chicken manure and its

biochar was 60%. Besides that, CaCO3 content in the biochar was found interesting as

they ranged from 0.19 to 15.74%. Plant-feedstock derived biochars had shown less

CaCO3 than municipal sewage sludge and animal-waste derived biochars. CMB had

significantly more content than SB whereas, in plant-derived biochar, EuB had more

CaCO3 than PhB550. Similar results for cow manure (3.5-4.8% CaCO3) were found in

biochar prepared at 400-500oC respectively by Singh et al. (2010). Moreover, it could be

suggested that biochars produced at slow pyrolysis temperature are easily prone to

biological decay instead of biochars produced at high pyrolysis temperature which can

stay for a longer time in the terrestrial system with the slow release of CO2 (Al-Wabel et

al., 2013). The content of VM in biochar has also been observed to be related to biochar

stability (Enders et al., 2012). The carbon-nitrogen ratio of biochar was 12.8 – 40.5

which is important in agriculture. Municipal sewage sludge and animal-waste derived

biochar had a narrow C/N ratio which could be useful for the soil fertility. Regarding C/N

ratio, plant-feedstock derived biochar had wide C/N ratio than animal-waste derived

biochar which mineralizes quickly in soil (Keiluweit et al., 2010).

Biochar being carbonaceous material is of key importance in carbon sequestration

and climate change. Presence of different types of functional groups which are aromatic

in nature strengthens the recalcitrance property of biochar. Nature of functional groups

presence varies depending on feedstock source and quality. The plant-derived biochars

were dominated with O-H bending, carboxyl C=O stretch, and aliphatic C-H stretch.

During the pyrolysis process, aliphatic CH peaks shifts to aromatic. The interpretations of

the FTIR spectra are based on data presented in numerous studies (Chefetz et al., 1998;

Lichtfouse et al., 1998; Silverstein et al., 2014). The peak area continued up to 800oC

where inorganic matter decomposition, for example, CaCO3 took place Zielińska et al.,

2015). The increase in the peak of SB showed the stability of aromatic compounds, and it

may be cyclization (Hossain et al., 2011). During the pyrolysis process, peaks at 2900-

2800 cm-1

disappeared due to loss of aliphatic compounds which enhanced the

recalcitrance property of biochar (Wang et al., 2013). Moreover, loss of C-H and -OH

groups during pyrolysis resulted into formation of porous biochar which was observed in

SEM images (Bagreev et al., 2001).

Biochar is prepared at high pyrolytic temperatures which induce various

biochemical and structural changes. Thermal stability of biochar determines it‘s quality

depending on the presence of various biological molecules present in the feedstock which

are transformed during pyrolysis. Along the way, the reaction becomes exothermic

(Gabbott, 2008). Specifically, thermal degradation of cellulose and hemicellulose

continue between 250-350oC (Jung et al., 2008). Besides that, lignin degradation

continues throughout the temperature range (Uzun et al., 2007; Kumar et al., 2010). The

structure of lignin is aromatic, and it is found in excess at spaces between cellulose

crystals which makes the lignin resistant against thermal degradation (Braga et al., 2014).

These results were consistent with the data on different types of biomasses (Okoroigwe

and Saffron, 2012; Salaheldeen et al., 2014). For lignin decomposition, there was no peak

for any of the plant studied, which may be because of its wide range of decomposition

temperatures from 150 to 800 °C without a sharp weight-loss peak (Abed et al., 2012;

Huang et al., 2015). The analysis of the TGA curve patterns showed that plant-derived

biochars had higher thermal stability than municipal sewage sludge, and animal-waste

derived biochar. All plant-derived biochar were thermally stable up to 400oC which could

be due to high contents of VM (Conti et al., 2016). On the other hand, biochars with high

organic matter content are more suitable as the soil amendment.

3.6.Conclusion

Preparation and characterization of biochar from municipal sewage sludge,

animal-waste, and plant-derived feedstock are of vital importance for sustainable

agriculture and environment. Eight different biochars were prepared at various

temperatures, and their physical and chemical characterization was determined. Based on

the analysis, plant-derived biochars performed better and found suitable in long run

sequestration of carbon and slow release of nutrients for plant production. Besides that,

municipal sewage sludge and animal-waste derived biochar had a low amount of carbon,

suitable for the agriculture as they had a high amount of nutrients required for the plants

and liming effect for the soils. At low pyrolysis temperature, biochar of neutral pH can be

made. Porus structure of biochar is suitable for the soil as it will provide habitat to the

soil microbiota in facilitating plant growth. It seems that plant-derived biochar is

significantly different than municipal sewage sludge and animal-waste derived biochar.

Key points of the findings of the study are:

Biochar either prepared from animal derived feedstock or plant-based

feedstock is important for sustainable agriculture and environment.

Plant-derived biochars are suitable for long-term carbon sequestration.

Animal-derived biochar is suitable for sustainable agriculture.

Porous surface structure of biochar could provide habitat to soil microbes

Animal-derived biochar is rich in the essential nutrients required for plant growth.

73

Chapter 4

Microbe-Biochar Sytem for

Onion Plant Growth

4.1. Introduction

The pivotal roles of vegetables are predominantly important for health due to

availability of minerals, vitamins, phytochemicals and antioxidants. Onion importance is

greatly increasing and now it has become second most medicinal and horticultural crop

after tomatoes (Arshad et al., 2017). Moreover, rhizosphere is a rich niche of microbes

and should be explored for potential plant growth promoting rhizobacteria (PGPR) which

can be useful in developing bio-inoculants for enhancement of growth and yield of crop

plant. Bacteria predominates the rhizosphere and take nutritional substances (amino acid,

vitamins and other nutrients) released from plant tissues for growth (Reetha et al., 2014).

Additionaly, arbuscular mycorrhizal fungi (AMF) form association with plants and its

hyphae penetrates into the cortical cells to excange nutrients/sugar (Taylor et al., 2015).

Besides that, the physical structure, e.g., porous and high surface area of biochar, is

beneficial to increasing air (oxygen) content, enhancing water storage capacity and

improving the living condition of microorganisms in soil (Atkinson et al., 2010). The

inorganic compounds, e.g., the compounds of N, P, and K in biochar could provide

nutrient elements to the plants (Tan and Lagerkvist, 2011). Biochar increases plant

growth under specific soil conditions (Crane-Droesch et al., 2013) ascribed as more N-

and P- uptake by the plant, owing to nutrients input from biochar or a larger amount of

plant-available N and P in biochar-amended soils (Jones et al., 2012; He et al., 2014).

Biochars formed from woody feedstock material such as sawdust, reed, eucalyptus or

bamboo can serve as a slow release of nutrients where the plant-available N is negligible

(Mukherjee and Zimmerman, 2013; Yao et al., 2013). Biochar can modify N cycling in

soils by exerting effects on the soil microbial community and is proposed as a mechanism

for increased or decreased plant available N depending on whether biochar exerts effects

on N mineralization or volatilization (Anderson et al., 2011). Moreover, Anderson et al.

(2011) further concluded from the controlled greenhouse study that, that biochar

promotes phosphorus solubilizing bacteria which was further explained by He et al.

(2014) that specific bacteria such as different strains of Lysinibacillus fusiformis,

solubilize the P of biochar by 47-54%. Most of the biochar usually contained 0.2–0.8% of

P. Studies show that environment has a strong influence on the release of P from biochar.

According to an estimate <50% P in biochar releases under natural environmental

conditions (Qian et al., 2013). As the P is a non-renewable and irreplaceable resource in

plant growth and development (Seyhan, 2009), and the reserves of phosphorus-rock will

become depleted in 30–100 years with the severe loss of P in agricultural fields and the

very slow recycle rate of P by natural cycle (Weikard and Seyhan, 2009).

Besides the role of biochar in P availability, management of soil microbes such as

bacteria and mycorrhizal fungi interaction with the plant roots possibly will augment P

availability from biochar in different soils. In comparison to further nutrients, P has little

mobility in soil particularly in the soils having P-fixation ability. The P (available) from

chemical fertilizer can be considerably reduced over its exchanges with mineral oxides

that chemisorb PO4-2

from the soil solution (Parfitt et al., 1975). Increased P uptake

kinetics and mycorrhizal symbiosis are strategies that favor P acquisition (Lynch, 2011).

Moreover, the introduction of PSB in this root-mycorrhizae-biochar system could

possibly enhance the P utilization. The root mass function of the plant shows whether the

plant is favoring investment in shoot functions (chlorophyll fluorescence) or root

functions (nutrient uptake); an increase directs sophisticated investment in root functions

(Smit et al., 2013). Biochar containing P can be a potential P source to mitigate the

coming ‗‗P crisis depletion ‘‘, moreover, the use of PSB and mycorrhizal fungi can lead

to the sustainable production of plant growth. Depending of micronutrients role within

the metabolism, trace elements are generally classified in essential and non-essential. In

particular, three trace elements, i.e. copper (Cu), manganese (Mn) and zinc (Zn) were

selected due to their intriguing relation with human health showing either nutritional and

toxicogical effects (Michalke and Fernsebner, 2014; Bost et al., 2016; Gibson et al.,

2016).

Onion tend to be more dependent on AMF than other cultivated plants due to their

thick, sparsely branched roots and lack of root hairs and genetic evidence has shown that

modern onion breeding has not selected against response to AMF , (Brewster, 2008;

Galván et al., 2011). To date, few studies have examined the nutrient acquisition

strategies on plant P-uptake under soil amendments in the presence of PSB and

mycorrhizal fungi under controlled conditions. Especially in the presence (also absence)

of P fertilizers and different biochars, managing the foraging capacity of plants, bacterial

and mycorrhizal associations may improve plant P acquisition. Yet we are not aware of

studies examining the combined effects of plant root-bacteria-mycorrhizal fungi, and

uptake of P in different biochar amended soils.

We grew onion in two different soils, under greenhouse conditions and measured

macro- and micronutrient concentration, nutrient uptake, chlorophyll fluorescence, and

root colonization in response to different biochar amended soils with (and without) P-

application. Therefore, the objectives of this study were: (1) how the biochar interact with

soil microbes in different soils for plant nutrient uptake and (2) whether the biochar- and

microbially- induced changes in the plant-soil system coincide with P-application.

4.2. Objective

Evaluation of different biochar influence on chlorophyll fluorecence in PSB –

mycorrhizae presence in onin plant.

Quantifiaction of macro and micronutrients in onion plant in biochar – PSB –

mycorrhizae system.


4.3.1. Nursery development and plant growth in greenhouse

An experiment was conducted in greenhouses of the Cukurova University, Adana,

Turkey, from 28 February 2016 to 15 May 2016. For the onion seedlings production,

Andesitic tuff (local substrate with 0.5-1 mm granulometry) + peat moss (Potgrond P,

Geeste, Germany) (1:1 v:v) mixture was prepared in the plastic trays where the onion

seeds (Balkan Hybrid, Adana, Turkey) were broadcasted. The seeds were covered by

spreading peat moss and irrigated with distilled water. Tays were further covered with the

polyethylene plastic sheet of 0.25mm thickness and put in the greenhouse to increase the

temperature and humidity. Environmental conditions of greenhouse were 25 ± 3°C, 80 ±

3% relative humidity and 16:8 h day:night cycle. After three weeks of seed sowing,

vegetatively grown uniform seedlings were transferred to the pots according to respective

treatments. Treatments were factorial combinations of two soil types, two biochar, two

phosphorus levels and three biological inoculants in addition to the control were applied

to onion plant, resulting in four treatments. For each soil type, three replicates per

treatment were prepared for a total of 96 pots. Pots were arranged in a randomized

complete block design with one pot per treatment per block. Two types of soil were used

in experiment where Soil A was designated as Menekse (Typic xerorthent Ortehnt

Entisol) and Soil B as Kiziltapir (Lithic Rpodoxeralf Xeralf Alfisol) soil series by USDA

classification located at the research farms of Cukurova University, Adana (Table 4.1

shows initial soil properties).

Table 4.1: Soil properties before addition of chemical fertilizer and biochar

amendment

Soil A Soil B

Texture analysis (%)

Sand 6.0 25.0

Silt 67.0 17.0

Clay 27.0 58.0

Characteristics

SOM (%) 1.10 1.6

pHwater 7.84 6.68

CaCO3 (%) 22.16 1.33

Bulk Density (g cm-3

) 1.0 1.3

CECest (meq [100g]-1

) 21.23 20.65

Nutrient content (mg kg-1

)

NO3-N 8.0 8.0

P2O5 (mg kg-1

) 1.45 1.87

K2O (mg kg-1

) 82.05 75.91

SOM soil organic matter, CECest estimated cation exchange capacity, NO3-N nitrate nitrogen, P2O5

phosphorus pentaoxide, K2O potassium oxide (means and standard deviation; n = 3)

4.3.2. Biochar preparation

Two types of biochar were prepared from feedstock of common reed (Phragmites

australis) and sawdust were collected from the vicinity of the research area of Cukurova

University, Adana, Turkey. Before making the biochar, feedstock was ground and sieved.

The obtained material was passed through a 50-mesh screen away large lumps. Particle

size was reduced to 0.7-0.8 nm, and it was dried at 110oC for 24 h. The feedstock was

charred at 350°C for 2 h using a heating rate of 10oC min

-1 in a closed container under

oxygen-limited conditions in a muffle furnace (RD50, REF-SAN, Turkey) (Sánchez et

al., 2009). The residence time of preparing biochar was 1 hr. Biochar was milled to pass a

2 mm sieve and labeled properly for further analysis and utilization. Preliminary

properties of biochar used in the study are given in Table 4.2.

Table 4.2: Properties of biochar used as soil amendment

PhB SDB

pH 7.49±0.08 6.29±0.09

EC (mS cm-1

) 2.23±0.16 0.33±0.00

Proximate analysis (%)

Moisture 3.91±0.11 2.56±0.99

Asha 8.18±0.02 3.11±0.01

VMb 56.05±1.50 66.99±0.58

FCc 31.86±1.59 27.34±1.35

Ultimate/Nutrient analysis (%)

Total C 67.89±8.76 61.96±3.96

Stable C 67.89±8.74 61.96±4.0

Unstable C 0.0004±0.0001 0.0006±0.0001

CaCO3 0.30±0.04 0.95±0.10

N 0.57±0.13 0.56±0.05

P 0.09±0.00 0.03±0.00

K 2.07±0.01 0.22±0.01

Ca 0.12±0.00 0.79±0.03

Mg 0.09±0.00 0.08±0.00

Fe 0.003±0.00 0.023± 0.00

Mn (mg kg-1

) 4.40±0.00 28.60±22.40

B (mg kg-1

) 0.19±0.01 2.00±0.70

PhB Phragmites biochar, SDB Sawdust biochar (means and standard deviation; n = 3)

4.3.3. Pot study setup

Nursery pots (3 L, 21 cm diameter, 18 cm height) were each filled with 3 kg of soil

amended with 30 g of biochar. Two levels of diphosphorus penta-oxide (P2O5) were

established as 0 mg kg-1

(without-P) and 38 mg kg-1

(with-P). The dose of P was exactly

half of the locally recommended P2O5 dose for onion plant. All pots received equal doses

of N and K fertilizer according to farmer practice based on soil test results of nitrate

nitrogen and potassium oxide after soil collection. Pots filled with Soil A and Soil B

received 0.13g of urea (equivalent to 160 kg N ha-1

) in all pots and 7.02g potassium

dihydrogen phosphate (equivalent to 38 kg P2O5 ha-1

) in with-P pots. To balance the K in

rest of the pots (without-P), 3.84g of potassium chloride was added. Pots then received

1.0% w/w dry biochar and biological inoculants based on experimental treatments. The

treatments were; uninoculated control (C), Lysinibacillus fusiformis 31MZR (B) as 108-9

cfu, Rhizophagus clarus (M), L. fusiformis 31MZR + R. clarus (B + M). Soil, biochar,

and fertilizer were thoroughly mixed by hand, to ensure uniform distribution of

amendments and then placed with germination paper to prevent soil loss through holes at

the bottom of the pot. Pots were watered to field capacity every two to four days,

necessary for the duration of experiment. In each pot, five healthy and uniform seedlings

germination based on vegetative growth were transplanted. Treatments labeled with

bacterial inoculation were inoculated with L. fusiformis, isolated from corn (Rafique et

al., 2017). For the mycorrhizal fungi inoculation, R. clarus (BEG248) was originally

isolated from Tephrosia purpurea located in South Amerrica and submitted to The

International Bank for the Glomeromycota in 1997 by donor P. Lovato. It was obtained

and propagated using sorghum (Sorghum bicolor) as the host plant, and the infected

roots, hyphae, spores, and substrates were collected. Purity of the R. clarus was ensured

by properly managing the propogation and spore collection. Each mycorrhizal fungi

inoculated pot was filled with 50 g (equivalent to ~ 700 spores) inoculum (Ortas, 2012).

4.3.4. Plant harvesting and sample preparation

Every plant in the pot was harvested after attaining vegetative maturity, 65 days after

transplanting. Aboveground biomass was harvested by cutting the stem at the soil

surface, dried to a constant weight at 60 °C and weighed to determine dry biomass

production. Moreover, belowground biomass was also harvested, rinsed with tap water,

deionized water and then roots were dried to a constant weight at 60 °C and weighed to

determine dry biomass production. All dried plant tissues (above- and belowground) were

ground with a Tema mill, RM100 (Retsch Solutions in Miling and Sieving, Haan,

Germany) to pass through a 0.5 mm mesh sieve, samples were and stored in sealed

containers for analyses.

4.3.5. Chlorophyll Fluorescence Measurement

Chlorophyll fluorescence parameters of the uppermost leaves of onion were measured at

room temperature using a FluorPen FP 100 (Photons Systems Instruments, Drasov,

Czech Republic) following the protocols (Ritchie and Bunthawin, 2010). Plants were

kept in dark for a minimum of 30 min prior to measurement after which minimal

fluorescence in the dark-adapted state (F0) was recorded. A saturating pulse of irradiation

(2 mmol m-2

s-1

) for 3 s was then administered to measure the maximal fluorescence in

the dark-adapted state (Fm) (Gong et al., 2013). The leaves were then placed under actinic

light (300 mmol m-2

s-1

) to determine the maximal fluorescence (Fm 0), the minimal

fluorescence in the light-adapted state (F0 0) and the steady-state fluorescence (Fs). We

calculated chlorophyll fluorescence parameters (Fv/Fm) following Zai et al. (2012).

4.3.6. Tissue nutrient analyses and AMF root colonization

Total N concentration (%) was determined for above- and belowground biomass

tissue using an elemental analyzer (Thermo Fisher Scientific FLASH 2000 Series CN

Elemental Analyzer, Thermo Fisher Scientific, Waltham, U.S.A.). Nutrient concentration

(P, K, Cu, Mn, and Zn) in digested biomass was analyzed by inductively coupled plasma

optical emission spectrometry (ICP-OES), Perkin Elmer, USA (Wheal et al., 2011). The

roots of onion were cut into small pieces (about 1 cm) and stained with Trypan Blue

following a modification of the procedure described by Phillips and Hayman (Koske and

Gemma, 1989) to determine AMF colonization. There were thirty root pieces of 1 cm

length from every plant root sample to visually ensure colonization which makes thirty

microscopic views for each treatment. The AMF colonization in onion root was

determined using the method described by (Giovannetti and Mosse, 1980).

( )

4.3.7. Recovery of inoculated bacteria

After harvesting, inoculated plants were sterilized and cut into small sections.

Samples were surface sterilized and homogenized in autoclaved distilled water.

Homogenized mixture was plated in nutrient agar plates. Emerged colonies were

identified by their morphological characteristics; gram staining and antibiotic resistance

of bacteria was detected by disc diffusion method. A 0.1 mL bacterial culture [108 colony

forming units (CFU) mL-1

] was spread on LB agar plates meanwhile, antibiotic discs

(gentamicin, tetracycline and erythromycin) were positioned on the surface of media and

plates were incubated for 24hrs at 27 oC. (Arumugam et al., 2011) to check sensitivity

and resistance of bacteria on the basis of previous screening..

4.3.8. Calculations and statistical analyses

The N uptake per plant (mg plant−1

) was calculated by multiplying the shoot tissue N

concentration (%) with dry shoot biomass (mg) and then dividing by 100 for plants

harvested. Similarly, the P uptake per plant (mg plant-1

) was calculated. Data were

analyzed using Statistix software (Statistix, 2008). The application of P (0 and 38 kg ha-

1), effect of biochar type [phragmites biochar (PhB) and sawdust biochar (SDB)] and

their interaction were treated as fixed effects in the model; replicate nested within soil

type was treated as a random factor. Dependent variables were soil N, P, K, Cu, Mn, and

Zn concentration, shoot and root dry biomass, N and P uptake per plant, additional

dependent variables were root colonization and chlorophyll fluorescence. Data were

pooled to include both soils for all dependent variables after performing a Fisher F-test to

verify the assumption of homogeneity of variances among sample populations. Statistical

significance was postulated at p ≤ 0.05; biologically interesting differences with 0.05 < p

≤ 0.10 are also presented. Pearson‘s correlation of coefficient test was performed to

estimate the relationships among different factors and the observed nutrients

concentration.

4.4. Results

4.4.1. Chlorophyll Fluorescence

Soil A and Soil B amended with SDB showed high values of Fv/Fm in comparison

to the PhB (Table 4.3). There was a significant difference between the same treatments of

both soils. Moreover, the treatments without-P had more value than with-P applied

treatments. In Soil A, B+M combination had highest Fv/Fm value than rest of the

treatments. Besides that, a notable difference was observed between B and M treatments

of both soils amended with PhB.

Table 4.3: Chlorophyll fluoresence (Fv/Fm) of the onion plant under different soil

conditions

Soil A

Phragmites biochar Sawdust biochar

Treatments without-P with-P without-P with-P

C 0.64±0.01 c-g 0.65±0.02 b-g 0.72±0.02 ab 0.69±0.04 a-e

B 0.62±0.07 f-h 0.65±0.07 b-f 0.70±0.01 a-d 0.69±0.02 a-f

M 0.67±0.03 a-f 0.68±0.03 a-f 0.69±0.02 a-e 0.70±0.04 a-e

B + M 0.67±0.02 a-f 0.49±0.07 i 0.73±0.02 a 0.68±0.01 a-f

Soil B

C 0.64±0.02 e-g 0.68±0.02 a-f 0.71±0.01 a-c 0.68±0.02 a-f

B 0.65±0.03 b-g 0.68±0.01 a-f 0.69±0.02 a-e 0.67±0.02 a-f

M 0.58±0.09 gh 0.55±0.02 hi 0.65±0.00 b-f 0.65±0.02 c-g

B + M 0.67±0.01 a-f 0.66±0.03 a-f 0.65±0.03 c-g 0.64±0.01 d-g

C control, B L. fusiformis, M R. clarus, B+M L. fusiformis + R. clarus (means and standard deviation; n =

3)

4.4.2. Tissue macronutrient analyses

The Soil A, amended with PhB increased 2% shoot N without-P in B-treatment

whereas with-P, it decreased by 15% (Table 4.4). Maximum reduction of 60% was

observed in the M-treatment, without-P whereas with-P it was only 2%. In shoot, B+M-

treatment with-P, 8% increase was observed. Similarly, when SDB was applied, B+M-

treatment with-P had a similar increase of 8% while the maximum increase of 31% was

observed in B-treatment without-P. In Soil B, amended with PhB, 10 and 3% increase

was noted in B-treatment of without-P and with-P respectively. Whereas, only B+M-

treatment had 8% increase with-P in SDB amended Soil B. The root N was observed on

the higher side in Soil A amended with PhB (with-P) than without-P, whereas on the

addition of SDB, this change was nonsignificant. In Soil B, SDB application without-P

enhanced root N in comparison to other combinations.

In Soil A, M- and B+M-treatment significantly enhanced the shoot P by 76 and 17%

respectively without-P for PhB, while only B-treatment enhanced it by 25% in with-P

(Table 4.5). When SDB was applied in same soil (without-P), M-treatment enhanced P

concentration in a shoot by 10% and with-P it was 15% in B-treatment. Soil B response

was quite different and 61% increase was observed in M-treatment of PhB amended soil

(without-P), while it was only 8% in with-P. On addition of SDB in Soil B, 61% shoot P

was depicted in B-treatment whereas 11% increase in B+M-treatment (without-P). Only

12% was observed for the B-treatment with-P. Soil microbes significantly increased the

root P for Soil A amended with PhB without-P, whereas it decreased in with-P except for

the B-treatment as 33%. A similar trend was observed for the Soil B amended with PhB.

Besides that, on the application of SDB, root P was enhanced in M- and B+M-treatments

which shows the contribution of mycorrhizal fungi in both combinations of P applicat

Table 4.4: Concenteration of N (%) in plant shoot under different soil conditions and P application

C control, B L. fusiformis, M R. clarus, B+M L. fusiformis + R. clarus (means and standard deviation; n = 3)

Soil A


without-P with-P without-P with-P

Treatments Shoot N Root N Shoot N Root N Shoot N Root N Shoot N Root N

C 3.2±0.2 ab 1.9±0.0 lm 1.8±0.2 h-j 2.0±0.1 kl 2.0±0.2 gh 1.7±0.2 m 1.9±0.1 hi 1.9±0.0 lm

B 3.3±0.0 ab 2.0±0.1 j-l 1.6±0.1 k 2.5±0.1 e-g 2.9±0.0 c 2.0±0.2 kl 1.5±0.0 k 2.2±0.0 h-j

M 2.0±0.1 gh 2.2±0.2 h-j 1.6±0.0 k 1.9±0.1 k-m 2.0±0.1 gh 2.0±0.0 j-l 1.5±0.1 k 2.1±0.1 j-l

B + M 1.9±0.2 hi 2.4±0.1 f-h 1.7±0.2 i-k 2.4±0.1 f-h 2.0±0.0 gh 2.1±0.1 i-k 1.6±0.0 jk 2.1±0.1 j-l

Soil B

C 3.4±0.3 a 2.6±0.2 c-e 2.7±0.1 d 3.2±0.1 a 3.4±0.1 a 2.3±0.1 g-i 2.6±0.1 d 2.8±0.0 bc

B 2.4±0.1 ef 2.8±0.1 b-d 2.5±0.1 de 3.1±0.1 a 3.1±0.1 bc 2.6±0.3 d-f 2.5±0.1 de 2.8±0.2 bc

M 2.6±0.2 d 2.7±0.1 b-d 2.6±0.1 de 2.8±0.1 b-d 3.1±0.2 bc 2.9±0.1 b 2.2±0.1 fg 2.5±0.1 e-g

B + M 2.6±0.1 de 2.5±0.1 e-g 2.5±0.1 de 2.6±0.1 d-f 2.6±0.2 de 2.9±0.1 b 2.3±0.1 ef 2.7±0.1 c-e

Table 4.5: Concenteration of P (%) in plant shoot under different soil conditions and P application


Soil A



Treatments Shoot P Root P Shoot P Root P Shoot P Root P Shoot P Root P

C 0.1±0.0 op 0.1±0.0 m 0.5±0.0 b 0.4±0.1 ij 0.1±0.0 l-o 0.1±0.0 k-m 0.4±0.1 d-f 0.6±0.1 g-i

B 0.0±0.0 p 0.1±0.0 lm 0.7±0.1 a 0.7±0.1 e-g 0.1±0.0 m-p 0.1±0.1 k-m 0.5±0.1 b-d 0.5±0.1 h-j

M 0.1±0.0 m-p 0.2±0.0 k-m 0.5±0.0 b-d 0.5±0.1 h-j 0.1±0.0 k-o 0.1±0.1 k-m 0.4±0.2 d-f 0.6±0.2 f-h

B + M 0.2±0.0 k-n 0.2±0.0 k 0.4±0.0 e-g 0.5±0.0 h-j 0.1±0.0 k-o 0.2±0.0 k-m 0.3±0.1 f-i 0.4±0.1 j

Soil B

C 0.2±0.1 j-m 0.1±0.0 k-m 0.4±0.0 e-g 0.9±0.1 c 0.1±0.0 m-p 0.1±0.0 k-m 0.4±0.1 c-e 1.5±0.1 a

B 0.9±0.0 n-p 0.1±0.0 k-m 0.4±0.0 e-g 1.4±0.1 a 0.1±0.0 m-p 0.1±0.0 k-m 0.5±0.1 bc 1.3±0.1 a

M 0.2±0.0 i-k 0.2±0.0 k-m 0.4±0.0 d-f 1.0±0.0 b 0.3±0.0 h-j 0.2±0.0 kl 0.4±0.0 d-f 0.7±0.1 ef

B + M 0.2±0.0 i-l 0.2±0.0 k-m 0.3±0.0 f-h 0.8±0.0 cd 0.3±0.0 g-j 0.2±0.0 k 0.3±0.0 f-h 0.7±0.2 de

In Soil A, amended with PhB, a significant increase in shoot K was observed than

for M- and B+M-treatments (without-P) (Table 4.6). A similar trend was noted for the

soil amended with SDB (with-P) while, change in the shoot K was minute in Soil B

against both amendments and P application. On application of SDB, this change was

negligible. Root K in Soil A amended with PhB (without-P) increased in comparison to

without-P. In rest of the combinations of Soil A and Soil B, the root K was negligible to

mention.

4.4.3. N- and P- uptake

Nutrients uptake in onion plant was measured, and results showed that in Soil A

amended with SDB showed highest N-uptake for B+M-treatment followed by PhB in

without-P (Table 4.7). Similar trend with-P application was observed by the sequence of

B+M > B > C > M while for PhB, it was B+M > C > B > M. When these treatments were

applied to the Soil B, response was quite different in terms of the N-uptake amount, and

that was M > C > B+M > B for the PhB (without-P) and similar trend was observed in

SDB amended soil (without-P). When P was applied to the PhB amended soil, N-uptake

increased significantly. Bacteria and mycorrhizal fungi worked synergistically in PhB,

and SDB amended (without-P) Soil A. Whereas, with-P soil, B-treatment had highest P-

uptake in both biochar for Soil A. Moreover, in Soil A, this trend was different but no

continuous pattern was observed. Besides that, P-uptake was more than the Soil A.

Table 4.6: Concenteration of K (%) in plant shoot under different soil conditions and P application


Soil A



Treatments Shoot K Root K Shoot K Root K Shoot K Root K Shoot K Root K

C 3.9±0.1 n 1.7±0.2 p 4.3±0.2 l-n 7.5±0.8 a 4.9±0.1 e-i 2.7±0.5 op 4.4±0.4 j-m 7.4±0.4 ab

B 2.9±0.2 o 2.6±0.2 p 4.4±0.2 k-n 5.6±0.5 f-l 5.1±0.2 d-g 4.1±0.9 mn 4.4±0.3 i-m 6.0±0.3 d-i

M 4.6±0.1 g-m 4.8±0.3 k-n 4.1±0.1 mn 6.8±1.1 a-e 5.7±0.1 ab 3.9±0.3 mn 4.9±0.4 e-j 5.4±0.3 g-l

B + M 5.0±0.2 e-h 5.0±0.4 i-m 4.1±0.0 mn 6.9±0.6 a-d 4.3±0.1 l-n 3.7±0.0 no 4.7±0.3 f-l 5.4±0.8 g-l

Soil B

C 4.8±0.3 e-k 6.3±0.6 b-g 5.5±0.2 bc 6.3±0.2 b-g 5.1±0.4 c-f 6.8±0.4 a-e 6.2±0.2 a 7.1±0.4 a-c

B 4.7±0.3 f-l 6.2±0.5 c-h 5.2±0.1 c-e 6.5±0.8 a-f 4.8±0.3 e-k 5.6±1.0 f-l 5.5±0.3 b-d 5.8±0.3 e-k

M 4.8±0.2 e-l 4.8±0.6 k-m 4.5±0.16 h-m 4.8±0.4 j-m 5.1±0.4 c-f 5.5±0.1 f-l 5.3±0.2 b-e 5.6±0.7 f-l

B + M 5.0±0.1 e-h 4.6±0.3 l-n 4.9±0.5 e-k 5.5±0.5 f-l 5.2±0.1 c-e 5.2±0.3 h-l 4.9±0.16 e-k 5.9±.0.6 d-j

Table 4.7: Nitrogen and Phosphorus uptake (%) under different soil conditions and P application

Soil A



Treatments N-uptake

P-uptake

N-uptake

P-uptake

N-uptake

P-uptake

N-uptake

P-uptake

C 11.2±2.5 kl 0.2±0.1 l 42.9±14.6 f-l 12.4±3.9 d-i 16.1±2.2 j-l 1.1±0.2 kl 67.3±23.0 d-i 15.2±8.6 c-h

B 8.6±3.2 l 0.1±0.0 l 35.7±4.7 h-l 15.9±1.7 c-f 19.3±8.9 j-l 0.8±0.4 kl 78.2±11.6 c-g 23.9±5.7 ab

M 30.6±8.4 i-l 1.9±0.5 kl 32.2 ±3.5 h-l 9.8±0.7 f-j 29.4±2.6 i-l 2.0±0.1 kl 50.6±8.9 e-k 14.1±7.6 c-h

B + M 40.5±7.2 g-l 3.2±0.6 j-l 51.6±7.4 e-j 10.6±0.8 f-i 43.1±1.9 f-l 3.2±0.7 j-l 81.2±5.3 c-f 15.4±4.1 c-g

Soil B

C 92.1±50.9 cd 5.7±4.8 i-l 145.9±13.0 ab 20.4±3.2 bc 42.8±6.1 f-l 1.3±0.2 kl 145.7±14.0 ab 23.9±3.2 ab

B 35.4 ±3.0 h-l 1.3±0.1 kl 135.4±44.6 ab 19.7±7.0 b-d 50.8±9.5 e-k 1.6±0.3 kl 141.1±16.0 ab 28.2±5.0 a

M 94.6±23.7 cd 8.1±1.8 g-k 141.2±8.7 ab 21.2±1.9 a-c 141.5±25.2 ab 11.3±1.2 e-i 162.1±14.1 a 28.4±1.5 a

B + M 90.0±23.9 c-e 7.8±2.2 h-k 136.7±6.2 ab 18.4±1.3b-e 113.3±13.9 bc 12.4±1.2 d-i 71.2±63.4 d-h 10.9±10.3 f-i


4.4.4. Tissue micronutrient analysis

The presence of Cu in onion shoot for M- and B+M treatments was >5ppm in

PhB amended Soil A without-P, whereas, SDB amended soil it was 6.5 and 6.2ppm

respectively (Table 4.8). Inconsistent results were observed in treatments with-P for both

biochar type amended Soil A. Generally, in both soils, without-P, Cu concentration

decreased in order of B+M > M > C > B and in treatments of with-P, the order is C > B >

(M and B+M alternate) independent of the biochar type used. The concentration of Cu in

plant root showed highest concentration in SDB amended Soil A with-P.In case of Mn,

soil type had a significant effect on shoot Mn concentration (Table 4.9). Soil B provided

manifold more Mn then Soil A, moreover, its concentration is prominent in with-P

treatments then the without-P. Soil B strongly influenced the Mn concentration in plant

roots in comparison to the Soil A. The similar trend was shown by the Zn concentration

in shoot and plants grown in Soil B had more Zn concentration in their shoots (Table

4.10).

Table 4.8: Concenteration of Cu (ppm) in plant shoot under different soil conditions and P application


Soil A



Treatments Shoot Cu Root Cu Shoot Cu Root Cu Shoot Cu Root Cu Shoot Cu Root Cu

C 3.5±0.0 i-m 14.3±1.6 d-h 4.2±0.8 e-h 12.6±0.7 f-j 6.1±0.1 a 28.5±2.9 a 3.5±0.1 i-l 15.0±2.2 d-g

B 2.7±0.0 n 12.5±1.7 f-j 4.5±0.0 d-f 14.2±0.5 d-h 6.2±0.0 a 28.3±3.3 a 3.4±0.1 j-m 11.6±0.5 h-k

M 5.4±0.0 b 16.2±1.3 de 3.2±0.2 lm 15.5±2.7 d-f 6.2±0.1 a 23.4±4.3 b 4.4±0.8 d-g 13.6±0.1 d-h

B + M 5.1±0.2 bc 24.9±1.2 b 3.5±0.3 i-l 12.8±0.9 f-i 6.5±0.0 a 23.9±3.1 b 3.0±0.5 mn 11.2±0.0 h-k

Soil B

C 3.8±0.5 h-k 9.0±0.1 k 4.0±0.3 f-i 11.8±0.9 g-k 4.5±0.0 d-f 11.3±0.1 h-k 4.6±0.3 c-e 13.5±0.9 d-h

B 3.3±0.0 k-m 9.4±0.1 jk 4.0±0.1 f-j 13.0±0.7 e-i 3.6±0.1 i-l 10.0±0.0 i-k 3.9±0.1 g-j 9.7±0.8 i-k

M 4.9±0.2 b-d 12.5±1.8 f-j 3.9±0.3 g-j 11.0±0.4 h-k 5.1±0.8 bc 16.7±1.2 cd 3.6±0.2 i-l 12.0±0.6 g-k

B + M 4.3±0.1 e-g 11.1±0.8 h-k 3.2±0.2 lm 11.2±0.3 h-k 6.5±0.2 a 19.5±0.3 c 3.8±0.2 h-k 13.9±2.7 d-h

Table 4.9: Concenteration of Mn (ppm) in plant shoot under different soil conditions and P application


Soil A



Treatments Shoot Mn Root Mn Shoot Mn Root Mn Shoot Mn Root Mn Shoot Mn Root Mn

C 17.3±0.2 i 25.7±3.5 n 24.8±2.0 i 37.2±0.5 mn 25.4±1.9 i 65.5±1.9 l 24.7±3.6 i 46.6±8.0 l-n

B 22.5±2.6 i 54.3±9.8 lm 24.7±2.1 i 46.2±6.0 l-n 23.6±3.1 i 56.9±0.0 lm 23.9±4.3 i 48.0±14.6 lm

M 23.8±3.3 i 57.0±4.0 lm 25.7±2.6 i 35.8±5.9 mn 29.6±1.6 i 56.7±5.7 lm 20.6±1.6 i 61.4±7.7 l

B + M 21.2±1.0 i 53.4±9.3 lm 20.9±1.9 i 44.3±1.4 l-n 25.7±1.2 i 57.4±10.3 lm 23.8±4.1 i 53.5±2.9 lm

Soil B

C 78.8±9.0 h 144.2±0.7 ij 257.2±36.0 b 279.5±11.2 c 78.9±2.9 h 190.8±3.8 fg 117.6±11.8 f 125.6±4.7 j

B 205.5±1.5 c 370.7±4.1 b 186.9±9.4 c 170.8±3.7 gh 106.7±15.8 fg 167.0±9.4 h 372.2±46.3 a 442.5±23.4 a

M 159.1±2.0 d 246.7±43.4 de 148.1±9.6 de 268.3±9.6 cd 89.1±4.0 gh 149.5±2.2 hi 126.3±10.9 ef 124.3±11.9 j

B + M 158.9±4.7 d 204.7±4.6 f 154.4±8.8 d 228.2±4.3 e 110.6±4.1 fg 158.2±11.2 hi 90.2±3.3 gh 87.9±6.6 k

Table 4.10: Concenteration of Zn (ppm) in plant shoot under different soil conditions and P application


Soil A



Treatments Shoot Zn Root Zn Shoot Zn Root Zn Shoot Zn Root Zn Shoot Zn Root Zn

C 4.3±1.0 p 19.2±5.2 d 5.3±1.4 n-p 20.0±3.3 d 25.2±2.3 bc 87.0±11.3 ab 6.5±0.3 m-p 21.5±3 d

B 5.3±0.9 n-p 24.9±10.4 d 3.6±0.5 p 96.8±14.8 a 24.3±3.6 cd 52.9±21.4 b-d 7.3±1.5 l-p 18.9±1.7 d

M 9.3±0.1 k-m 34.1±3.9 cd 4.1±0.2 p 19.3±3.0 d 36.6±2.7 a 51.2±2.6 b-d 21.0±3.7 de 24.2±4.1 d

B + M 12.1±2.0 h-k 56.5±10.6 a-d 4.9±1.0 op 18.7±2.0 d 26.3±6.7 bc 56.1±20.5 a-d 9.0±0.2 k-o 25.1±3.1 d

Soil B

C 10.2±0.7 j-m 31.7±3.3 d 11.2±0.5 i-l 19.9±0.7 d 19.4±1.9 ef 42.4±3.9 cd 15.1±1.5 g-i 25.1±2.7 d

B 14.3±1.1 h-j 42.4±2.3 cd 10.9±0.8 j-l 16.7±1.9 d 15.7±0.8 f-h 41.0±1.9 cd 13.9±2.3 h-j 23.5±2.8 d

M 18.9±0.9 e-g 55.2±4.1 a-d 9.6±0.2 k-m 19.5±1.5 d 27.0±2.8 bc 75.7±0.5 a-c 12.2±0.2 h-k 21.9±2.1 d

B + M 19.3±0.6 ef 55.4±4.5 a-d 11.6±2.0 h-k 21.1±1.3 d 28.8±3.4 b 89.9±1.6 ab 11.0±1.7 i-l 20.3±1.7 d

4.4.5. Root colonization and root traits

The root colonization in both soils; Soil A and Soil B showed similar behavior. In

all soils, C- and B- treatments had <10% colonization that could be due to the transfer of

mycorrhizal spores by air (Ortas et al., 2017) (Figure 4.1 and Figure 4.2). Soil A had

more colonization without-P treatments in comparison to the with-P treatments.

Treatment of B+M had the highest colonization as 73% and 70% for without-P and with-

P application in PhB respectively, with SDB it was 58% and 68% respectively. In Soil B,

again B+M-treatment had the highest colonization of 82% and 85% for without-P and

with-P application, respectively under PhB amendment. A similar trend was observed in

the SDB amended Soil B, as 80% and 82% for without-P and with-P respectively. In all

the combinations, bacteria and mycorrhizal fungi together ensured more colonization

than mycorrhizal fungi alone.

Figure 4.1: Root colonization in onion plants in soils under different types of biochar

(phragmites biochar (PhB), sawdust biochar (SDB)) and treatments as control (C),

bacteria (B), mycorrhizal fungi (M), bacteria + mycorrhizal fungi (B+M).

Figure 4.2: Microscopic picture of root colonization in only mycorrhizal fungi (M)

inoculated plant and bacteria + mycorrhizal fungi (B+M) inoculated the plant

Root length seems predominant in the B+M treatment of Soil A in both conditions

of P2O5 irrespective of the biochar type (Figure 4.3). Besides that, root length for B+M

treatment was predominant in Soil B amended with PhB (without- and with-P condition).

While in SDB amended Soil B, the B+M treatment followed M in root length. In a

similar way, surface is of the plant roots was modified. Roots in the Soil B had

significantly more root volume than Soil A which shows that soil properties altered the

root characteristics (Figure 4.4). Addition of P2O5 enhanced root formation which was

M

B+M

endorsed in the increase of root length, surface area, and root volume (Figure 4.5).

Biochar amendment had a generally beneficial impact on plant resource allocation, but

this was not observed in all the parameters of roots. Type of biochar addition in soil did

not have a significant impact on the root characteristics.

Figure 4.3: Root length of onion plant in in soils under different types of biochar



Figure 4.4: Root surface area of onion plant in soils under different types of biochar



Figure 4.5: Root volume of onion plants in soils under different types of biochar



Pearson‘s correlation analysis was done for the macro- and micronutrients in relation to

different sources of variance (Table 4.11) and (Table 4.12). Results showed that soil and

biochar types positively correlate with observed parameters.

Table 4.11 p-values (probability) from analysis of variance for macronutrients of shoot

and root

Sources DF Shoot N Shoot P Shoot K Root N Root P Root K

Soil 1 <0.0001 0.8605 <0.0001 <0.0001 <0.0001 <0.0001

Biochar 1 0.0120 0.4614 <0.0001 <0.0001 0.6603 0.9404

Soil x Biochar 1 <0.0001 0.0070 0.0183 0.5878 0.4011 0.0305

Phosphorus 1 <0.0001 <0.0001 0.2001 0.0002 <0.0001 <0.0001

Soil x

Phosphorus

1 <0.0001 <0.0001 0.0011 0.2572 <0.0001 <0.0001

Biochar x

Phosphorus

1 0.4421 0.0112 0.1308 0.0606 0.8702 0.1961

Soil x Biochar

x Phosphorus

1 <0.0001 <0.0001 0.0033 0.0083 0.9299 0.1401

Treatments 3 <0.0001 0.1489 0.0052 <0.0001 <0.0001 0.0411

Soil x

Treatmentss

3 <0.0001 0.0020 <0.0001 <0.0001 <0.0001 <0.0001

Biochar x

Treatments

3 0.0003 0.9294 0.0005 0.0073 <0.0001 0.0760

Soil x Biochar

x Treatments

3 0.0003 0.2197 0.0374 <0.0001 0.0016 <0.0001

Phosphorus x

Treatments

3 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001

Soil x

Phosphorus x

Treatments

3 <0.0001 0.5737 0.0532 0.0048 <0.0001 <0.0001

Biochar x

Phosphorus x

Treatments

3 <0.0001 0.7084 0.0011 0.4359 <0.0001 0.9358

Soil x Biochar

x Phosphorus x

Treatments

3 0.0015 0.0012 <0.0001 0.0460 <0.0001 0.8230

Df: Degree of freedom; p indicates significant differences (p < 0.05)

Table 4.12 p-values (probability) from analysis of variance for micronutrients of shoot

and root

Sources DF Shoot

Cu

Shoot

Mn

Shoot

Zn

Root

Cu

Root

Mn

Root Zn

Soil 1 0.0002 <0.0001 <0.0001 <0.0001 <0.0001 0.7718

Biochar 1 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 0.1466

Soil x Biochar 1 0.0103 <0.0001 <0.0001 0.0272 <0.0001 0.7209

Phosphorus 1 <0.0001 <0.0001 <0.0001 <0.0001 0.3519 <0.0001

Soil x

Phosphorus

1 <0.0001 <0.0001 0.0041 <0.0001 0.0013 0.1390

Biochar x

Phosphorus

1 <0.0001 0.0018 <0.0001 <0.0001 0.0035 0.0096

Soil x Biochar

x Phosphorus

1 <0.0001 <0.0001 <0.0001 0.0001 0.0035 0.1424

Treatment 3 <0.0001 <0.0001 <0.0001 0.0007 <0.0001 0.6464

Soil x

Treatments

3 0.0515 <0.0001 0.0022 0.1378 <0.0001 0.2172

Biochar x

Treatment

3 0.1377 <0.0001 <0.0001 0.0479 <0.0001 0.1924

Soil x Biochar

x Treatment

3 <0.0001 <0.0001 0.0002 <0.0001 <0.0001 0.3262

Phosphorus x

Treatment

3 <0.0001 <0.0001 <0.0001 0.0001 0.000 0.0478

Soil x

Phosphorus x

Treatment

3 0.5752 <0.0001 0.0036 0.0029 0.0004 0.2748

Biochar x

Phosphorus x

Treatment

3 <0.0001 <0.0001 0.2457 0.0108 <0.0001 0.6038

Soil x Biochar

x Phosphorus

x Treatment

3 <0.0001 <0.0001 0.0135 0.0005 <0.0001 0.0567

Df: Degree of freedom; p indicates significant differences (p < 0.05)

4.5. Discussion

In this study, different soils, biochar types, and P-application levels indicate that

biochar may have different effects on the plant under the same environmental conditions.

It may influence the soil microbes too which effectively participate in the plant growth

promotion by nutrients uptake. In case of the healthy plants, usually the Fv/Fm values are

over 0.8, but in our study, it ranged from 0.5 – 0.73 which is slightly lower. This

indicates that plants during the mycorrhization system were under suboptimal conditions.

The Fv/Fm value below 0.7 in C- and B- treatments show that the photosynthesis in plants

was suboptimal even to the end of the experiment which is in agreement with a previous

study (Herrmann et al., 2004). It can also lead to the point that plants may have lower

Chla a + b content which is consistent with the decreased quantum efficiency of

photosystem II (ΦPSII). Our data highlight an important aspect that, treatments with

mycorrhizal colonization had high (Fv/Fm) data close to the 0.8 which correlate to the

high photosynthetic activity of the plant resulting in greater photosynthetic carbon

assimilation (Seaton and Walker, 1990). These results are in accordance with the high

demand of carbon by the fungi (Hutchison and Piché, 1995). This data further highlights

the importance of pre-mycorrhizal phase with plant roots which need high carbon

assimilation rate and should not be interrupted by forced reduction which may include

growth regulators. They imbalance the system and reduces plant growth.

Biochar and mycorrhizal fungi effect on plant growth and nutrients uptake are

limited in the crops fertilized with the recommended dose of chemical fertilizer. Such

plants already have sufficient amount of nutrients which suppress the mycorrhization and

biochar effects; therefore in such conditions, mycorrhizal fungi cannot harvest the

benefits as exploited in the P-deficient environment (Gazey et al., 2004; Solaiman et al.,

2010). Mycorrhizal fungi have the capability to significantly create a pathway for P-

uptake into the roots (Smith et al., 2004). Nutrient availability to the plants is strongly

dependant on the soil properties and water use efficiency (Theodose and Bowman, 1997),

mycorrhizal fungi use in the Soil A improved the P and water supply even in unfavorable

conditions. Neumann and George (2004) also studied the role of mycorrhizal fungi in P

supply, and water uptake which are inconsistent with this study. When biochar interacts

with the bacteria, particularly PSB such as L. fusiformis 31MZR, it increases N-

concentration by 23% more than the control, and P-concentration as 63% more than

control (Rafique et al., 2017). In this study, bacterial strain enhanced the plant growth

and nutrient uptake because of its ability to interact with plant roots positively. Such

bacteria present in the soil ecosystem and they interact with plant roots in terms of

nutrients uptake (Zhang et al., 2016). L. fusiformis 31MZR is already reported as P-

solubilizer which promotes the plant growth confirmed by various biochemical tests

(Chauhan et al., 2016). It may solubilize the P in biochar-amended soil and make it

available for the plant roots and mycorrhizal fungi for transportation into the root.

In our studies, biochar-amended soil increased the root colonization and improved

plants growth. Literature shows, the increase, and decrease of mycorrhizal abundance

depending on biochar type and soil properties. Besides, increase in mycorrhizal

abundance in biochar-amended soil is common as observed in our studies with different

types of soil and biochar (Lehmann et al., 2011). The commonly known mechanism

behind reduced utility of symbiosis in the presence of nutrients is explained by (Lehmann

et al., 2011). Our studies also showed the same response and mycorrhizal colonization

was comparatively less in with-P soils. The root colonization by R. clarus in onion is

shown Figure 4.1. As the biochar is considered soil conditioner, it improves soil physical

and chemical properties. Thus, mycorrhizal inoculation has a linear trend with increasing

dose of the biochar (Elmer and Pignatello, 2011).

4.6. Conclusion

This study showed that onion plant showed physiological and nutritional

improvement due to use of biochar and soil microbes in absence/limited supply of P-

fertilizer. Chlorophyll fluorescence was on the higher side in plants with a mycorrhizal

association manifesting rapid photosynthetic carbon assimilation. Macro and

micronutrients presence in the shoot and root of the plant showed that soil type is the

main contributor in association with the biochar type. Moreover, use of soil organisms

further enhanced the nutrient uptake based on their suitability with biochar. The onion

plants inoculated with mycorrhizal fungi and combination of bacteria and mycorrhizal

fungi enhanced the nutrients uptake by many folds in comparison to the plants inoculated

with bacteria only. Improved crop growth provides the evidence that the interaction of

biochar with soil microbes is diverse. Biochar and microbially induced changes are

specified in the plant-soil ecosystem for the sustainable plant growth promotion nutrients

harvesting. Some of the key findings include:

Presence of biochar and microbes induced positives changes on physiologial

and nutritional properties of onion plant.

Mycorrhizal association enhanced chlorophyll fluorosence which indicates

rapid photosynthetic carbon assimilation.

Soil type influenced nutrient uptake by plant.

Chapter 5

Microbe-Biochar Sytem for

Maize Plant Growth

5.1. Introduction

Cereal crops (particularly maize) are cultivated under vast areas for their role as

staple food and energy source on a global scale, especially in developing countries

(Farhad et al., 2009). However, the agriculture productivity is consistently on the decline

due to a reduction in soil quality and poor nutrient use efficiencies in developing

countries (Jones et al., 2013). Recycling of nutrients from organic sources into the soil is

a sustainable approach for improving soil physical, chemical and biological properties

(Girmay et al., 2008). Such practices are, in particular, very important to enhance soil

fertility and crop productivity in soils with intrinsically low soil fertility (Anjum et al.,

2011). Biochar, charcoal-like material, is produced from pyrolysis of biomass under

limited or no-supply of oxygen and have high surface area and highly porous structure

(Lehmann and Rondón, 2006; Atkinson et al., 2010). Use of biochar is gaining

considerable global interest for its potential of improving soil nutrient retention, water

holding capacity and sequestering carbon (C) in largely recalcitrant form (Downie et al.,

2009). The high porosity of biochar is generally linked with enhanced water retention in

soils (Singh et al., 2010). Biochar acts as a soil conditioner, enhances plant growth by

supplying nutrients efficiently and increases crop yields (Atkinson et al., 2010; Spokas et

al., 2012). Biochar application has been shown to have positive effects on soil C stability,

especially in soil with low native organic matter contents (Sohi et al., 2009; Riaz et al.,

2017). Biochar is emerging as an attractive option to improve fertilizer use efficiency

(Zhang et al., 2010). A few recent field-based studies have found increased fertilizer use

efficiencies of chemical (Agegnehu et al., 2016)) and chemical-organic (Zhang et al.,

2016) fertilizers in biochar amended low fertility soils. Biochar can even act as a source

of soluble P after application to the soil (Parvage et al., 2013). The release of P from

biochar has also been approved by a considerable amount of desorption of P from biochar

at zero P level in P sorption experiments (Morales et al., 2013). Addition of biochar along

with microbial inoculation, have positive effects on maize plant height and nutrient

concentration. Moreover, plants treated with sawdust biochar + L. fusiformis strain

31MZR inoculation increased N, P and K (Rafique et al., 2017).

Low P availability to plants is a global problem limiting crop production

(Richardson and Simpson, 2011). As a result, a number of agronomic practices have been

proposed to enhance P availability and use by crops under diverse climatic conditions

(Simpson et al., 2011). However, very limited number of studies have focussed on the

use of biochar to increase P utilization from organic and inorganic P fertilizers (Shen et

al., 2016; Rafique et al., 2017). Very recently, (Gul and Whalen, 2016) also indicated

about lack of research on P use efficiency despite availability of data regarding P uptake

by various crops under biochar amendments. Total P content in biochar generally

increases with increasing pyrolysis temperature whereas the bioavailable P greatly

decreased with rising temperature (Cantrell et al., 2012; Iqbal et al., 2015).

5.2. Objectives

Evaluation of different biochar influence on chlorophyll fluorecence in PSB

– mycorrhizal fungi presence in maize plant.

Quantification of macro and micronutrients in maize plant in biochar – PSB

– mycorrhizal fungi system.


5.3.1. Experimental design and pot study setup

An experiment was conducted in greenhouses of the Cukurova University, Adana,

Turkey, from 01 June 2016 to 04 August 2016. Already treated soils with different

treatments of biochar, bacteria and mycorrhizal fungi were used in the present study after

harvesting of the onion to evaluate the influence of aged biochar on growth and nutrient

parameters of a maize plant. The biochar used in the study was prepared from the

feedstock of common reed (Phragmites australis) and sawdust. Five maize seeds (LG

37.10, Anadolu, Hybrid, Turkey) were added to every pot (3 L, 21 cm diameter, 18 cm

height) filled with 3 kg of soil amended with 30 g of biochar in respective treatments and

irrigated with distilled water. Pots were placed in the greenhouse whose environmental

conditions were 25 ± 3°C, 80 ± 3% relative humidity and 16:8 h day:night cycle. Two

levels of P were set, where no P was applied to half of the pots while rest of the pots

received a half dose of P of the local recommendations (i.e. 40 kg P2O5 ha−1

). Each of the

pot was provided with a basal dose of ammonium nitrate (34% N), Potassium

Dihydrogen Phosphate (34% K2O and 52% P2O5) and Muriate of Potash (MOP, 60%

K2O) at the recommended rates of 160 kg N ha-1

, and 60 kg K2O ha−1

equivalents based

on the soil test results. Pots were already having 1.0% w/w dry biochar biological

inoculants based on experimental treatments at the time of onion sowing. The treatments

were; uninoculated control (C), Lysinibacillus fusiformis 31MZR (B), Rhizophagus

clarus (M), and L. fusiformis 31MZR + R. clarus (B + M). The bacterial counts were 108-

9 cfu/ml. Pots were watered to field capacity before seeding and then watered to field

capacity every two to four days, necessary for the duration of the experiment. For the

mycorrhizal inoculation, R. clarus was propagated using sorghum (Sorghum bicolor) as

the host, and the infected roots, hyphae, spores, and substrates were collected. Each

mycorrhizal fungi inoculated pot was filled with 50 g (equivalent to ~ 700 spores)

inoculum (Ortas, 2012).

After the germination, 3 uniform heightened plants were kept in each pot for

further growth after thinning. Treatments were factorial combinations of two soil types,

two biochar, two phosphorus levels and three biological inoculants in addition to the

control were applied to maize plant, resulting in four treatments. For each soil type, three

replicates per treatment were prepared for a total of 96 pots. They were arranged in a

randomized complete block design with one pot per treatment per block. The experiment

was conducted on two soils, where Soil A was designated as Menekse (Typic xerorthent

Ortehnt Entisol) and Soil B as Kiziltapir (Lithic Rpodoxeralf Xeralf Alfisol) soil series by

USDA classification located at the research farms of Cukurova University, Adana,

Turkey. The study was conducted just after harvesting the onion plants (Chapter 4) and

maize seeds were incorporated in the same soil. Soil P is mentioned in Chapter 4.

5.3.2. Harvest and sample preparation

Every plant in the pot was harvested after attaining vegetative maturity of 65 days

after sowing. Aboveground biomass was harvested by cutting the stem at the soil surface,

dried to a constant weight at 60 °C and weighed to determine dry biomass production.

Moreover, belowground biomass was also harvested, rinsed with tap water, deionized

water and then roots were dried to a constant weight at 60 °C and weighed to determine

dry biomass production. All dried plant tissues (above- and belowground) were ground

with a Tema mill, RM100 (Retsch Solutions in Miling and Sieving, Haan, Germany) to

pass through a 0.5 mm mesh sieve, samples were and stored in sealed containers for

analyses.

5.3.3. Root characterization

The harvested roots were thoroughly washed using deionized water (Kachenko

and Singh, 2006). Then, the root system of the plants was placed on a scanner (Epson

Perfection V700, Photo Long Beach, CA, USA), in a transparent plastic tray filled with

water. Root length, root surface area, and root volume were analyzed using WinRHIZO

Pro 3.10 (Regent Instruments Inc.) (Himmelbauer, 2004).

5.3.4. Tissue nutrient analyses and microbial root colonization





optical emission spectrometry (ICP-OES), Perkin Elmer, USA (Wheal et al., 2011).

To determine the extent of AMF colonization, the roots of onion were cut into

small pieces (about 1 cm3 in dimension) and stained with Trypan Blue following a

modification of the procedure described by Phillips and Hayman (Koske and Gemma,

1989). AMF colonization in the onion root was determined using the method described

by (Giovannetti and Mosse, 1980).

( )


The N uptake per plant (mg plant−1

) was calculated by multiplying the shoot

tissue N concentration (%) with dry shoot biomass (mg) and then dividing by 100 for

plants harvested. Similarly, the P uptake per plant (mg plant-1

) was calculated. Data were

analyzed using Statistix software (Statistix, 2008). The application of P (0 and 38 kg ha-

1), and biochar type [phragmites biochar (PhB) and sawdust biochar (SDB)] effects and

their interaction were treated as fixed effects in the model; replicate nested within soil

type was treated as a random factor. Dependent variables were soil N, P, K, Cu, Mn, and

Zn concentration, shoot and root dry biomass, N and P uptake per plant, additional

dependent variables were root colonization and chlorophyll fluorescence. Data were

pooled to include both soils for all dependent variables after performing a Fisher F-test to

verify the assumption of homogeneity of variances among sample populations. Statistical



estimate the relationships among different factors and the observed nutrients

concentration.

5.4. Results

5.4.1. Root characteristics

Addition of P2O5 in soil has a significant impact on root length enhancement

which was endorsed in the Soil B amended with SDB (with-P) having a 98% increase in

root length (Table 5.1). Besides that, biochar type also influenced the root parameters,

and up to 43% increase in root length have been noticed for the PhB amended Soil A in

B+M treatment (without-P). Similarly, Soil B amended with SDB (with-P) enhanced root

length by 22% in B+M treatment. The root surface area was also influenced by biochar

type and it was more in PhB amended Soil A (without-P) for the B+M treatment by 46%

while it increased only 20% for with-P (Table 5.2). In contrary to this, enhancement in

the Soil B was less for the surface area and maximum 25% increase was noticed for B+M

treatment in SDB amended Soil B (with-P). Root volume enhancement was also noticed

for the treatments used in PhB amended soil up to 49% in B+M treatment (without-P)

whereas only 22% root volume increased in SDB amended R. clarus inoculated plants

(with-P) (Table 5.3).

Table 5.1: Root length of the maize plant influenced by the biochar-microbial interaction

Soil A



Treatments Root length (cm)

C 8178.6±1444.2 l 26625.4±1778.7 c-h 8244.5±1390.5 l 24774.3±2718.8 e-h

B 6743.8±1156.5 l 27437.8±2636.8 c-h 8817.4±1803.9 l 24964.7±4044.6 e-h

M 12914.9±1047.8 j-l 28131.7±4600.0 c-h 12199.3±3098.7 kl 31037.2±3788.1 b-g

B + M 20237.8±5993.3 h-k 28174.2±2963.9 c-h 14079.6±4628.4 i-l 23209.9±5138.9 f-i

Soil B

C 22773.7±5395.2 g-j 20051.3±1176.7 h-k 35105.3±6525.0 a-d 32826.71±12238.83 b-g

B 23286.7±890.2 f-i 31827.6±5701.5 b-g 25846.2±8971.3 d-h 34824.59±7354.78 a-e

M 39214.5±6101.2 ab 33134.4±2022.2 a-f 32577.9±729.2 b-g 34553.00±1720.10 a-e

B + M 28520.0±4217.6 c-h 33637.8±10333.2 a-e 36264.8±711.4 a-c 43105.04±8757.85 a

Table 5.2: Root surface area of the maize plant influenced by the biochar-microbial interaction

Soil A



Treatments Root surface area (cm2)

C 758.8±79.9 h 2661.8±431.0 b-e 786.7±82.0 h 2037.0±116.2 ef

B 694.3±118.0 h 2431.8±230.4 b-f 852.6±102.5 h 2190.1±444.6 c-f

M 1246.5±76.1 gh 2414.1±375.2 b-f 1067.3±297.9 gh 2885.8±338.1 b-d

B + M 1788.1±361.0 fg 2612.3±343.2 b-e 1222.4±254.1 gh 2175.4±312.3 c-f

Soil B

C 2029.0±270.9 ef 1749.2±186.2 fg 2375.0±268.87 b-f 2644.81±691.33 b-e

B 2120.5±72.2 d-f 3013.3±527.7 ab 2161.0±552.21 c-f 2932.76±639.56 bc

M 3104.5±342.4 ab 3043.3±321.9 ab 2615.3±140.49 b-e 2697.50±99.64 b-e

B + M 2509.8±409.5 b-f 2771.0±789.3 b-e 2892.63±187.46 b-d 3720.86±935.72 a

Table 5.3: Root volume of the maize plant influenced by the biochar-microbial interaction

Soil A



Treatments Root Volume (cm3)

C 0.5±0.0 l 2.0±0.8 ab 0.6±0.0 kl 1.2±0.1 d-i

B 0.5±0.1 l 1.6±0.3 b-f 0.6±0.1 j-l 1.4±0.4 c-g

M 0.9±0.0 g-l 1.5±0.3 b-f 0.7±0.2 i-l 1.9±0.2 a-c

B + M 1.2±0.2 e-j 1.8±0.3 a-e 0.8±0.1 h-l 1.5±0.2 b-f

Soil B

C 1.3±0.1 d-h 1.1±0.2 f-k 1.17±0.05 e-j 1.59±0.29 b-f

B 1.4±0.1 c-g 2.1±0.4 ab 1.32±0.22 d-h 1.79±0.42 a-d

M 1.8±0.2 a-d 2.0±0.3 ab 1.52±0.14 b-f 1.53±0.18 b-f

B + M 1.6±0.5 b-f 1.7±0.4 b-f 1.67±0.18 b-f 2.33±0.71 a

5.4.2. Shoot and root dry weight

Dry shoot weight in without-P was noticed maximum by 50% increase in B+M

treatment of Soil A (PhB) whereas only 33% increase was observed in SDB amended

Soil A with respect to control (Figure 5.1). In contrary, only 6% increase was noticed in

the L. fusimormis 18MZR inoculated plant of PhB while 3% reduction in SDB amended

Soil A (without-P) respectively. Besides that, with-P, R. clarus inoculated plant had only

7% increase in PhB amended Soil A and 14% increase in the SDB amended Soil A. In

Soil B, PhB enhanced the dry shoot weight by 34% in M-treatment of the plant followed

by 14% increase in B+M (without-P). Addition of the P2O5 didn‘t significantly enhance

the dry shoot weight. Data showed that PhB was more responsive in dry shoot weight

enhancement than SDB.

Figure 5.1: Shoot and root dry weight of the maize plant in Soil A

0

2

4

6

8

10

C B M

B+

M C B M

B+

M C B M

B+

M C B M

B+

M



Wei

ght

(g 3

kg

-1 s

oil

)

DSW DRW

Figure 5.2 Shoot and root dry weight of the maize plant in Soil B

Root dry weight also followed a similar trend and a maximum increase of 55% was

noticed for the B+M treatment of Soil A amended with PhB (without-P) whereas, in SDB

amended soil it contributed for 47% (Figure 5.2). When P2O5 was added to the soi A, only

15% enhancement was noticed for the R. clarus inoculated plants. Besides that, trend

deviated in Soil B where mycorrhizal fungi addition increased 35% dry root weight in

PhB amended Soil B (without-P) and 32% in SDB amended Soil B (without-P). Addition

of P2O5 had a positive influence on root weight and SDB amended Soil B had 32% and

34% more dry root weight in M- and B+M – treatment respectively. While, PhB amended

Soil A (with-P) had 27% and 28% enhancement for M- and B+M – treatment.

5.4.3. Root and shoot tissue nutrients analyses

In Soil A, M- and B+M-treatment significantly enhanced the shoot P by 33% and

20% respectively without-P for PhB, while in with-P, only B-treatment enhanced it by

52% while M-treatment enhanced 24%. When SDB was applied in the same soil

(without-P), B+M-treatment enhanced P concentration in a shoot by 12% (Table 5.4).

Soil B response was different as 27% increase was observed in M-treatment of PhB

amended soil (without-P). On addition of SDB in Soil B, 13% shoot P was observed in B-

0

4

8

12

16

20

C B M

B+

M C B M

B+

M C B M

B+

M C B M

B+

M



Wei

ght

(g 3

kg

-1 s

oil

)

DSW DRW

treatment whereas 32% increase in M-treatment (without-P). Only 2% increase was

observed for the B-treatment with-P. Soil microbes significantly increased the root P for

Soil A amended with PhB without-P. The P uptake in the roots was also influenced by

the addition of P2O5 in the soil. Maximum 31% more P was observed in the R. clarus

inoculated plant in comparison to the control for Soil B amended with SDB (without-P).

In Soil A amended with PhB, a significant increase in shoot K was observed for B- (20%)

and B+M- (11%) treatments (without-P) (Table 5.5). Change in the shoot K was

significant in Soil B against both amendments and P application as M- treatment (16%) in

without-P, while with-P it was 9% (B-treatment), and 8% (M-treatment) in PhB amended

the soil. On application of SDB, this change was negligible while only 15% increase was

observed in the B-treatment. Root K in Soil A amended with PhB (with-P) increased

2.9% in comparison to without-P. In plant roots, R. clarus enhanced the uptake of K in

both soils and the maximum 69% increase in K was observed for the plants inoculated

with R. clarus in Soil A amended with SDB (without-P). In rest of the combinations for

Soil A and Soil B, the root K was negligible.

The 41% increase in Ca was noted in the root inoculated with B+M treatment in

Soil A amended with SDB (with-P) (Table 5.6). While in the Soil B, maximum of 36%

increase in Ca was noticed for PhB amended soil and inoculated with B+M (with-P).

Besides that, addition of P2O5 in soil enhanced the Mg uptake to the roots irrespective to

the soil type (Table 5.7).

The Mn concentration in the shoot of Soil A was influenced by the biochar type

used and data showed that use of SDB reduced the Mn uptake in plant shoot (Table 5.8).

Similar behavior of Mn was observed in Soil B. L.fusiformis significantly contributed to

Mn uptake. The Mn uptake was non-significant and was not affected by the addition of

biochar. The presence of Cu in different treatments of PhB amended Soil A without-P

ranged as 5.27-8.07ppm whereas in SDB amended soil it ranged as 4.37-22.33ppm

(Table 5.9). In the case of Cu, biochar had a nominal impact on the uptake, SDB

amended soil had more Cu in comparison to the PhB amended the soil. Maximum

increase of 67% was noted in B-treatment of with-P soil amended with SDB. In the case

of Mn, soil type had a significant effect on shoot Mn concentration. Soil B provided

manifold Mn than Soil A, moreover, its concentration is prominent with-P treatments

than without-P. Similar trend was shown by the Zn concentration in the shoot. Plants

grown in Soil B had more Zn concentration in their shoots and the soils with-P have less

concentration of Zn than without-P (Table 5.10). For the Zn, 36% increase in the uptake

was noticed in the B+M treatment of Soil A amended with PhB (without-P).

5.4.4. Root colonization

The root colonization in both soils; Soil A and Soil B showed similar behavior. In

all soils, C- and B- treatments had <10% colonization instead of C-treatment in Soil-A

amended with PhB (without-P) that could be due to the transfer of mycorrhizal fungi

spores by air (Ortas et al., 2017) (Figure 5.3a and 5.3b). Soil A had more colonization

without-P treatments in comparison to the with-P treatments. Treatment of M had the

colonization of 78% while 77% in B+M-treatment in without-P PhB amended Soil A.

The combination of B+M had more colonization in both soils and biochar which ranged

from 73-82%. In Soil B, PhB amended soil had 73-76% colonization whereas SDB

amended soil had 80-82% colonization which shows that biochar type influences the root

colonization. In all the combinations, bacteria and mycorrhizal fungi together ensured

more colonization than mycorrhizal fungi alone.

Table 5.4: Concentration of P (%) in plant shoot under different soil conditions and P application


Soil A



Treatments Shoot P Root P Shoot P Root P Shoot P Root P Shoot P Root P

C 0.08±0.0 n 0.08±0.0 m-o 0.27±0.0 i 0.15±0.0 gh 0.15±0.0 kl 0.10±0.0 k-n 0.66±0.0 f 0.30±0.0 e

B 0.09±0.0 mn 0.08±0.0 m-o 0.55±0.0 g 0.21±0.0 f 0.13±0.0 l-n 0.08±0.0 m-o 0.63±0.0 f 0.30±0.0 e

M 0.13±0.0 l-n 0.08±0.0 l-o 0.35±0.0 h 0.22±0.0 f 0.13±0.0 l-n 0.08±0.0 m-o 0.61±0.0 f 0.32±0.0 e

B + M 0.16±0.0 kl 0.09±0.0 k-o 0.21±0.0 jk 0.13±0.0 h-j 0.14±0.0 lm 0.08±0.0 l-o 0.33±0.0 h 0.17±0.0 g

Soil B

C 0.16±0.0 kl 0.08±0.0 l-o 1.30±0.0 b 0.67±0.0 ab 0.14±0.0 lm 0.07±0.0 o 1.07±0.0 d 0.67±0.0 ab

B 0.16±0.0 kl 0.08±0.0 l-o 1.42±0.0 a 0.59±0.0 c 0.16±0.0 kl 0.08±0.0 no 1.10±0.0 cd 0.65±0.0 b

M 0.22±0.0 ij 0.10±0.0 k-m 1.15±0.0 c 0.46±0.0 d 0.24±0.0 ij 0.11±0.0 i-k 1.02±0.1 e 0.58±0.0 c

B + M 0.26±0.0 ij 0.11±0.0 j-l 1.01±0.1 e 0.48±0.0 d 0.26±0.0 iS 0.13±0.0 hi 0.97±0.0 e 0.68±0.0 a

Table 5.5: Concentration of K (%) in plant shoot under different soil conditions and P application


Soil A



Treatments Shoot K Root K Shoot K Root K Shoot K Root K Shoot K Root K

C 5.33±0.4 p 0.28±0.1 p 8.22±0.3 bc 1.48±0.1 g-i 8.00±0.1 cd 0.25±0.0 p 7.04±0.2 h-j 2.35±0.2 ab

B 6.67±0.1 k-m 0.18±0.0 p 9.23±0.4 a 1.72±0.2 e-g 6.30±0.1 no 0.21±0.0 p 6.84±0.1 i-k 2.16±0.1 bc

M 6.44±0.1 l-o 0.68±0.1 n 8.14±0.2 bc 1.90±0.1 de 6.63±0.1 k-n 0.69±0.1 mn 6.47±0.3 l-o 2.56±0.2 a

B + M 7.20±0.0 gh 0.40±0.0 op 8.03±0.1 cd 1.56±0.1 f-h 6.43±0.3 l-o 0.94±0.2 k-m 7.11±0.3 hi 2.03±0.1 cd

Soil B

C 6.65±0.2 k-m 0.96±0.2 kl 7.70±0.1 de 2.29±0.1 b 6.35±0.3 m-o 0.74±0.0 l-n 6.55±0.3 k-n 2.01±0.0 cd

B 6.15±0.2 o 0.94±0.2 kl 8.48±0.2 b 1.78±0.1 d-f 7.49±0.1 e-g 0.63±0.0 no 6.35±0.3 m-o 1.05±0.2 jk

M 7.29±0.2 f-h 1.44±0.2 hi 8.33±0.1 bc 1.75±0.3 ef 7.17±0.1 g-i 1.27±0.1 ij 6.43±0.3 l-o 1.31±0.1 hi

B + M 7.31±0.1 f-h 0.74±0.0 l-n 7.61±0.2 ef 1.46±0.2 hi 6.71±0.0 j-l 1.36±0.1 hi 6.41±0.1 l-o 1.31±0.0 hi

Table 5.6: Concentration of Ca (%) in plant shoot under different soil conditions and P application


Soil A



Treatments Shoot Ca Root Ca Shoot Ca Root Ca Shoot Ca Root Ca Shoot Ca Root Ca

C 1.58±0.0 d-f 1.95±0.0 f-i 1.27±0.1 i-k 2.35±0.1 bc 1.84±0.0 c 1.99±0.1 e-h 0.77±0.0 q 2.35±0.1 bc

B 2.69±0.1 a 2.59±0.1 a 0.96±0.1 no 1.37±0.0 o-q 1.45±0.1 f-h 2.06±0.1 ef 0.79±0.1 pq 1.73±0.0 jk

M 1.48±0.1 e-g 2.03±0.0 e-g 0.92±0.0 op 2.17±0.1 c-e 1.38±0.0 g-i 2.43±0.2 ab 0.73±0.0 q 1.05±0.1 s

B + M 1.65±0.0 d 2.02±0.0 e-g 1.11±0.0 2.59±0.1 a lm 1.24±0.0 i-l 1.67±0.0 j-m 0.87±0.0 o-q 1.76±0.1 i-k

Soil B

C 1.62±0.2 de 1.33±0.1 o-q 1.23±0.1 j-l 1.71±0.1 j-l 1.27±0.0 ij 1.37±0.1 o-q 1.08±0.0 mn 1.16±0.0 q-s

B 2.07±0.0 b 1.51±0.2 l-o 1.32±0.1 h-j 1.84±0.1 g-j 1.60±0.0 de 1.23±0.1 p-s 0.97±0.0 no 1.61±0.3 k-n

M 1.63±0.1 d 1.47±0.1 m-o 1.13±0.1 k-m 1.30±0.0 o-r 1.12±0.1 lm 1.10±0.0 rs 1.01±0.0 m-o 1.79±0.1 h-k

B + M 1.82±0.1 c 2.09±0.1 d-f 1.31±0.0 h-j 2.01±0.1 e-g 1.29±0.2 ij 1.43±0.1 n-p 1.00±0.0 m-o 2.27±0.2 b-d

Table 5.7: Concentration of Mg (%) in plant shoot under different soil conditions and P application


Soil A



Treatments Shoot Mg Root Mg Shoot Mg Root Mg Shoot Mg Root Mg Shoot Mg Root Mg

C 0.85±0.0 c 0.87±0.0 cd 0.65±0.0 fg 1.18±0.1 a 1.09±0.1 a 0.78±0.0 fg 0.51±0.0 hi 1.02±0.1 b

B 1.07±0.0 a 0.72±0.1 gh 0.71±0.1 ef 0.86±0.0 de 0.94±0.0 b 0.73±0.1 gh 0.56±0.0 h 0.94±0.0 c

M 0.75±0.0 de 0.79±0.1 e-g 0.63±0.0 g 1.15±0.0 a 0.73±0.1 e 0.89±0.0 cd 0.49±0.0ij 0.69±0.0 h

B + M 0.80±0.0 cd 0.85±0.1 d-f 0.62±0.0 g 1.14±0.0 a 0.74±0.1 de 0.75±0.1 gh 0.53±0.1 hi 0.72±0.1 gh

Soil B

C 0.36±0.0 l-o 0.35±0.0 i-l 0.31±0.0 n-q 0.28±0.0 lm 0.31±0.0 n-q 0.31±0.0 k-m 0.26±0.0 q 0.27±0.0 m

B 0.42±0.0 j-l 0.40±0.0 ij 0.38±0.0 k-m 0.33±0.0 j-m 0.38±0.0 k-m 0.28±0.0 lm 0.28±0.0 pq 0.31±0.0 k-m

M 0.37±0.0 k-o 0.37±0.0 i-k 0.34±0.0 m-p 0.31±0.0 k-m 0.30±0.0 n-q 0.37±0.0 i-k 0.27±0.0 q 0.39±0.0 ij

B + M 0.44±0.0 jk 0.42±0.1 i 0.37±0.0 k-o 0.39±0.0 ij 0.34±0.1 m-p 0.34±0.0 j-m 0.31±0.0 n-q 0.30±0.0 k-m

Table 5.8: Concentration of Cu (ppm) in plant shoot under different soil conditions and P application


Soil A



Treatments Shoot Cu Root Cu Shoot Cu Root Cu Shoot Cu Root Cu Shoot Cu Root Cu

C 5.60±0.2 h-j 6.30±2.50 k 7.67±0.5 a-c 10.23±1.09 e-j 7.80±0.1 ab 11.07±1.39 d-i 7.37±1.5 a-d 9.00±0.79 g-k

B 6.60±0.5 c-h 8.60±0.49 h-k 6.90±0.6 a-f 9.07±1.32 g-k 6.80±0.2 b-g 10.07±1.23 e-j 5..33±0.9 g-j 8.30±0.45 i-k

M 6.60±0.7 c-h 9.87±0.66 f-j 6.03±0.0 e-i 9.70±0.45 f-j 6.17±0.0 e-i 10.70±2.24 d-i 4.67±0.9 jk 7.53±0.97 jk

B + M 8.07±1.1 a 11.23±1.16 d-h 5.27±0.5 i-k 9.23±0.45 g-j 6.47.33±5.4 d-h 11.47±0.76 d-g 4.37±0.2 k 7.73±1.29 jk

Soil B

C 6.77±0.5 b-h 16.07±1.39 a 7.47±0.3 a-d 10.77±0.45 d-i 5.70±0.4 g-j 13.23±0.58 b-d 6.33±0.5 d-i 11.57±1.84 d-g

B 6.47±0.6 d-h 12.50±1.47 c-f 8.07±1.4 a 12.70±2.01 c-e 6.73±0.1 b-h 13.43±1.62 a-d 6.10±0.1 e-i 11.00±1.79 d-i

M 6.13±0.2 e-i 11.27±c1.48 d-h 6.53±0.2 c-h 12.23±1.70 c-f 5.77±0.5 f-j 15.67±1.10 ab 6.00±0.3 e-i 12.13±2.16 c-f

B + M 7.17±0.7 a-e 13.27±2.32 a-d 7.67±0.5 a-c 14.90±1.71 a-c 5.90±0.8 f-i 14.77±0.63 a-c 5.73±0.7 f-j 12.37±1.49 c-f

Table 5.9: Concentration of Mn (ppm) in plant shoot under different soil conditions and P application


Soil A



Treatments Shoot Mn Root Mn Shoot Mn Root Mn Shoot Mn Root Mn Shoot Mn Root Mn

C 56.80±8.4 m-q 83.17±3.4 i-k 59.63±5.1 k-p 135.25±12.3 gh 52.00±6.7 n-q 66.90±1.3 j-m 45.70±4.1 pq 82.95±5.6 i-k

B 67.47±6.8 j-o 88.80±2.1 i-k 71.87±6.4 i-n 56.90±1.5 k-m 61.40±4.8 k-p 65.10±9.9 j-m 45.07±5.2 pq 75.83±7.2 i-l

M 70.77±13.1 i-n 61.80±1.2 j-m 52.90±3.9 m-q 83.00±10.6 i-k 48.57±2.6 o-q 79.47±8.5 i-k 41.03±2.1 pq 36.60±1.8 m

B + M 78.50±10.3 h-l 72.50±4.3 j-l 57.47±4.3 l-q 92.30±4.7 ij 53.73±4.6 m-q 45.25±1.5 lm 38.43±3.4 q 44.10±8.4 lm

Soil B

C 130.27±18.2 b-d 247.57±36.3 a 175.67±13.8 a 177.60±20.4 c-e 102.73±10.4 e-g 140.93±7.8 fg 66.90±3.8 j-o 107.30±0.9 hi

B 174.37±22.0 a 231.10±14.4 ab 144.07±22.1 b 220.05±12.1 ab 140.73±3.8 bc 163.93±4.0 d-g 148.27±5.4 b 172.75±32.2 d-f

M 114.43±8.3 d-f 186.05±11.9 cd 95.73±9.7 f-h 173.73±14.3 de 87.30±8.8 g-j 206.77±6.4 bc 73.60±9.1 i-m 137.95±49.0 gh

B + M 128.20±20.8 b-d 237.00±14.8 ab 120.47±3.7 c-e 249.73±34.5 a 89.57±7.5 g-i 172.33±11.2 d-f 79.17±21.1 h-k 147.93±9.4 e-g

Table 5.10: Concentration of Zn (ppm) in plant shoot under different soil conditions and P application


Soil A



Treatments Shoot Zn Root Zn Shoot Zn Root Zn Shoot Zn Root Zn Shoot Zn Root Zn

C 12.13±2.2 kl 26.60±0.01 g-j 5.37±0.9 lm 29.60±0.01 d-j 61.93±10.2 a 33.47±0.03 b-f 3.20±1.0 m 25.63±0.05 ij

B 17.07±2.3 i-k 24.53±0.01 j 4.43±1.2 lm 28.77±0.02 e-j 45.50±2.0 bc 36.10±0.05 a-c 3.50±0.4 m 27.87±0.01 e-j

M 27.13±4.3 e-h 26.50±0.03 g-j 4.97±0.8 lm 35.53±0.03 a-d 48.70±3.8 b 32.93±0.02 b-f 3.87±1.0 lm 26.13±0.02 h-j

B + M 41.13±8.0 b-d 41.65±0.02 a 5.80±1.1 lm 32.50±0.02 b-g 46.87±11.0 bc 37.47±0.01 ab 6.00±0.5 lm 24.70±0.01 j

Soil B

C 30.47±4.9 e-g 33.13±0.01 b-f 22.87±1.6 g-j 36.90±0.03 ab 25.27±2.4 f-i 29.33±0.00 e-j 21.63±1.5 h-j 31.33±0.04 b-i

B 33.30±6.0 d-f 27.83±0.02 e-j 22.77±3.1 g-j 31.60±0.05 b-i 30.20±0.4 e-g 30.10±0.03 c-j 18.67±2.1 h-k 29.95±0.02 c-j

M 33.90±3.4 de 27.53±0.02 f-j 17.93±0.5 i-k 29.37±0.03 d-j 40.33±7.2 b-d 33.97±0.02 b-e 19.80±1.4 h-k 32.65±0.00 b-g

B + M 39.97±0.7 cd 32.23±0.04 b-h 19.53±1.4 h-k 33.33±0.09 b-f 39.50±9.5 cd 36.00±0.06 a-c 16.67±0.4 jk 33.25±0.02 b-f

Figure 5.3a Mycorrhizal fungi root colonization (%) in Soil A

0

20

40

60

80

100

C B M B+M C B M B+M C B M B+M C B M B+M


Phragmaites biochar Sawdust biochar

Colo

niz

atio

n (

%)

Figure 5.3b Mycorrhizal fungi root colonization (%) in Soil B

0

20

40

60

80

100

C B M B+M C B M B+M C B M B+M C B M B+M


Phragmaites biochar Sawdust biochar

Colo

niz

atio

n (

%)

5.5. Discussion

In the present study, two soils of different physiochemical properties were

amended with PhB and SDB separately under two levels of P2O5. Moreover, they were

inoculated with the L. fusiformis 18MZR, R. clarus and combination of both. Generally,

poor, dry, compacted soils impair total root length, increase root diameter and alter root

hair length and density. We found that mycorrhizal fungi symbiosis affected root

architecture and phosphorus acquisition efficiency of maize, but the effect of AM was

dependant upon phosphorus treatment. Higher phosphorus availability reduced the

mycorrhizal colonization perhaps because the carbon costs associated with maintaining

the fungal tissue outweighed the benefit of obtaining additional phosphorus. Mycorrhizal

fungi symbiosis did alter the root architecture and increased phosphorus acquisition of

plants under low phosphorus conditions.

The higher root diameter found in the alkaline soil may imply a contribution of

more developed aerenchyma to the higher PAE under P-limited conditions in the alkaline

soil as suggested previously (Lynch, 2007; Fernandez and Rubio, 2015). Biochar and

mycorrhizal fungi effect on plant growth and nutrients uptake are limited in the crops

fertilized with the recommended dose of chemical fertilizer. Such plants already have

sufficient amount of nutrients which suppress the mycorrhization and biochar effects;

therefore in such conditions, mycorrhizal fungi cannot harvest the benefits as exploited in

the P-deficient environment (Gazey et al., 2004; Solaiman et al., 2010). Mycorrhizal

fungi have the capability to significantly create a pathway for P-uptake into the roots

(Smith et al., 2004). Nutrient availability to the plants is strongly dependant on the soil

properties and water use efficiency (Theodose and Bowman, 1997), mycorrhizal fungi

use in the Soil A improved the P even in unfavorable conditions. Neumann and George

(2004) also studied the role of mycorrhizal fungi in P supply and water uptake which is

inconsistent with this study. When biochar interacts with the bacteria, particularly PSB

such as L. fusiformis 31MZR, it increases P-concentration more than control (Rafique et

al., 2017). In this study, bacterial strain enhanced the plant growth and nutrient uptake

because of its ability to interact with plant roots positively. Such bacteria present in the

soil ecosystem and they interact with plant roots in terms of nutrients uptake (Zhang et

al., 2016). L. fusiformis 31MZR is already reported as P-solubilizer which promotes the

plant growth confirmed by various biochemical tests (Chauhan et al., 2016). It may

solubilize the P in biochar-amended soil and make it available for the plant roots and

mycorrhizal fungi for transportation into the root.

In our studies, biochar-amended soil increased the root colonization and improved

plants growth. Literature shows, the increase, and decrease of mycorrhizal fungi

abundance depending on biochar type and soil properties. Besides, increase in

mycorrhizal fungi abundance in biochar-amended soil is common as observed in our

studies with different types of soil and biochar (Lehmann et al., 2011). The commonly

known mechanism behind reduced utility of symbiosis in the presence of nutrients is

explained by (Lehmann et al., 2011). Our studies also showed the same response and

mycorrhizal fungi colonization was comparatively less in with-P soils. As the biochar is

considered soil conditioner, it improves soil physical and chemical properties. Thus,

mycorrhizal fungi inoculation has a linear trend with increasing dose of the biochar

(Elmer and Pignatello, 2011).

5.6. Conclusion

In the present study, PSB and mycorrhizal fungi were used as bioinoculants in two

different soils amended with two different biochars prepared form the plant

feedstock.The limited P condition was also induced to evaluate the performance of the

maize plants in terms of root colonization, plant biomass, root architecture

characterization, and macro and micronutrients concentration. Soil amended with SDB

reduced the root colonization in presence of phosphorus whereas in PhB amended soil it

was non-significant. Moreover, the shoot and root dry biomass were enhanced in the

SDB amended soil which also favored the nutrients uptake to the plant. Moreover, the

study concluded that:

Soil and biochar properties influence mycorrhizal fungi root colonization.

Combination of bacteria and mycorrhizal fungi enhances micronutrients

concentration in the plant.

Plant biomass enhanced in bacterial and mycorrhizal fungi combination.

Chapter 6

Mycorrhizal and Biochar Assisted

Phytostabilization of Cd

6.1. Introduction

Environmental pollution exacerbated a worldwide problem where heavy metal

toxicity attained primary concern (Fu and Wang, 2011). Increasing contamination of soil

and water by cadmium (Cd) appeared as a major threat to the ecosystem, food security

and environmental sustainability (Rizwan et al., 2017a). Cadmium is a most common

heavy metal coming from industrial activity, wastewater irrigation, overfertilization of

crops and improper waste disposal contaminate the farmland which risks human and

plants health (Yi et al., 2011). Moreover, it is among the most toxic environmental

pollutants for living things which enters in agricultural soils mainly through

anthropogenic activities such as the use of phosphate fertilizers, and application of

sewage sludge (Murtaza et al., 2015). It has a non-biodegradable property which allows

them to accumulate in soil and it enters to the food chain when plants cultivated on

contaminated soil uptake heavy metals (Liu et al., 2012). Several approaches have been

suggested to reduce the mobility of soluble heavy metal fraction in the soil. They include

the use of rhizospheric microbes and optimizing field management practices (Abhilash et

al., 2012; Rajkumar et al., 2012; Teng et al., 2015). Among rhizospheric microbes,

arbuscular mycorrhizal fungi (AMF) assist plant roots in enhancing access to water, and

nutrients. Moreover, AMF improves growth of the plant by reducing uptake of heavy

metal and translocation among parts of the plant, ultimately reducing metal toxicity

(Solís-Domínguez et al., 2011; Liu et al., 2018; Wazny et al., 2018). Therefore, AMF

plays a key role in phytostabilization where polyphosphate complexes are precipitated in

plant roots and fungal mycelium by retaining heavy metals. Moreover, AMF forms

specific structures known as radical mycelium, which improves plant adaptation to

environmental stress (Wu et al., 2016). Besides that, AMF also alters physicochemical

properties of rhizospheric soil and microbial community structuring in rhizosphere which

reduces metal phytoavailabilty (Ogar et al., 2015). Heavy metal remediation potential

depends on a number of factors such as plant tolerance to contaminants, AMF fungal

species, and bioavailability of heavy metal concentration (Yang et al., 2015).

In recent years, biochar has gained increasing attention as a soil amendment in

immobilizing the heavy metals such as Pb Cd, Cu, Cr, Zn, and Ni in soil (Zheng et al.,

2012). Moreover, biochar has also been studied for improving crop productivity and

enhancing nutrients in the soil (Xu et al., 2015) enhancing carbon sequestration (Molina

et al., 2009) and hovering nutrient retention in the soil (Sun et al., 2017). Studies reported

that physical properties of biochar such as large specific surface area and porous structure

might immobilize detrimental compounds (organic and inorganic) in soil (Al-Wabel et

al., 2015; Bian et al., 2016; Abbas et al., 2017). However, the presence of surface

functional groups (e.g., hydroxyl, carboxyl, phenol) on biochar contribute to the

reduction of heavy metal ions. Assuming that biochar has diversified surface structure

which makes it a good sorbent by significantly binding heavy metals to the functional

groups, complexation or exchange reaction and sorption of heavy metals on the biochar

surface takes place (Vithanage et al., 2015). There is a number of physical, chemical and

biological techniques to remove heavy metal pollutants from the environment. Among

them, use of immobilizing agents in stabilizing heavy metals on contaminated sites of the

soil are widely accepted due to economic and scientific feasibility. Biochar has been

emerged recently for stabilizing heavy metals in contaminated sites. It sorbs heavy metal

from the aqueous solution of soil and immobilizes for availability to the plant roots

(Ahmad et al., 2018). Application of biochar to the soil absorbs heavy metal pollutants

through various mechanisms such as ion exchange, precipitation, electrostatic

interactions, chemisorption and complexation (Beesley et al., 2011; Yuan and Xu, 2011).

Besides that, properties of high surface charge density, large surface area, porous

structure, and biochar pH absorb more heavy metals and immobilize them (Hossain et al.,

2010; Van Zwieten et al., 2010; Zeng et al., 2015). Biochar addition to soil also benefits

AMF, likely by modifying soil properties which assist in mycorrhizal spore germination,

hyphal branching and further growth (Hammer et al., 2014). Combination of biochar and

AMF in the polluted soil can alter nutrient cycling paradigm, and soil microbial

community structuring, therefore, influences heavy metal speciation which immobilizes

them in the soil (Hammer et al., 2014).

Maize (Zea mays L.), is a major staple food, is supposed as the noticeable source

of Cd intake by human beings (Anjum et al., 2016). Maize is a favorable AMF colonizer

(Cao et al., 2017) and it is frequently used in phytomanagement of polluted soils such as

Cd-contaminated. Maize plant tolerates Cd stress and produces high biomass (Rizwan et

al., 2017). Liu et al. (2014) studied the Cd effect in maize plant inoculated with G.

constrictum, G. intraradices, and G. mosseae separately. Mycorrhizal fungi inoculated

plants phytostabilized maize plant and reduced Cd uptake. Biochar application to the soil

also phytostabilized the Cd in maize plant depending on biochar application rate (Zhao et

al., 2016). Several studies showed that biochar addition to soils could significantly

enhance heavy metals adsorption and immobilization capacity (Rizwan et al., 2016).

However, biochar potential for Cd immobilization in alkaline soil has not been widely

explored to our knowledge (Abbas et al., 2017). Moreover, only one study (Liu et al.,

2018) has been reported to our knowledge where AMF inoculation in the presence of

biochar was evaluated for Cd uptake. It was hypothesized that biochar and AM fungi

alone or in combination might alleviate Cd toxicity in maize by improving plant

morphological, physiological parameters and altering Cd uptake by plants. Thus, the

present study was designed to explore the morpho-physiological growth of maize to

different Cd toxicity concentrations (0, 5 and 10 mg kg-1

) with biochar and microbial

inoculation alone and in combination.

6.2. Objective

Evaluation of gasous exchange in maize plant grown in biochar – mycorrhizal

fungi syetem under cadmium stress.

Macro and micronutrients quantification in plants to evaluate cadmium

influence on plant growth.

Quantification of cadmium in plant to evaluate biochar – mycorrhizal fungi

system efficiency under stress condition.


6.3.1. Biochar preparation and soil collection

The feedstock of common reed (Phragmites australis) was collected from the vicinity of

the research area of Cukurova University, Turkey. Before making the biochar, feedstock

was ground to small size and sieved. The obtained material was passed through a 50-

mesh sieve. Particle size was reduced to 0.7-0.8 nm, and it was dried at 110oC for 24 h.

The feedstock was charred at 550 °C for 2 h using a heating rate of 10oC min

-1 in a closed

container under oxygen-limited conditions in a muffle furnace (RD50, REF-SAN,

Turkey) (Sánchez et al., 2009). The residence time of preparing biochar was 1 hr.

Biochar was milled to pass through a 2 mm sieve and labeled for further analysis and

utilization.

The surface horizon (0-15 cm) of Kiziltapir (Lithic Rpodoxeralf Xeralf Alfisol)

soil series by USDA classification located at the research farms of Cukurova University

were collected, air-dried, passed through aperture sieve (2 mm mesh) and analyzed for its

physiochemical properties (Table 6.1). The soil was sterilized at 121 °C (20 min) before

using in the experiment.

6.3.2. Experimental design

An experiment was conducted in the greenhouse of the Cukurova University,

Turkey. Exactly 3 kg of soil placed in each pot (21 cm, D x 18 cm, H). Three

concentrations of Cd containing 0, 5, and 10 mg kg-1

Cd as CdSO4 were used in the

experiment. Cadmium was spiked in the soil by dissolving the salt in 50 ml distilled

water for each pot and then thoroughly mixed in the soil. After this, each set

concentration of Cd was divided into four sets containing control, biochar, AMF, and

biochar + AMF. For biochar treatment, 1% biochar was added to pots, and half of the

pots containing biochar were inoculated with Rhizophagus clarus. Sorghum (Sorghum

bicolor) was used for mycorrhizal fungi propagation as a host plant, and infected roots,

spores, hyphae, and substrates were collected. Each mycorrhizal fungi inoculated pot was

filled with 50 g (equivalent to ~ 700 spores) inoculum. Half of the inoculated pots were

without biochar. In total, there were 36 pots containing three replicates of each treatment.

Treatments were factorial combinations of three Cd concentrations, uninoculated and

non-treated control (Control), Phragmites biochar (Biochar) and the combination of

Phragmites biochar and R. clarus (Biochar + AMF) resulting in four treatments.

Each pot was provided with a basal dose of ammonium nitrate (34% N),

potassium dihydrogen phosphate (34% K2O and 52% P2O5) and muriate of potash (MOP,

60% K2O) at the recommended rates of 160 kg N ha-1

, 80 kg P2O5 ha−1

and 60 kg K2O

ha−1

equivalents (NARC, 2017). The study was conducted in a completely randomized

design with three replications and plants were harvested after 70 days of growth.

Environmental conditions of the greenhouse were 25 ± 3°C, 80 ± 3% relative humidity

and 16:8 h day:night cycle. Five seeds of maize (cv. LG 37.10, Anadolu) were sown in

each pot. After the thinning of germinated plants, only three of them were allowed to

grow further in each pot. The plants were irrigated with deionized water in maintaining

70% of field capacity moisture content in the soil. Another split dose (half of the

recommended dose) of N was given 5 weeks after germination in solution form.

After 10 weeks of plant growth, the aboveground and belowground maize plant

biomass was harvested. Plant roots and shoots were gently rinsed with deionized water

(Kachenko and Singh, 2006). Then, the root system of the plants was placed in a

transparent plastic tray filled with water to put on a scanner (Epson Perfection V700,

Photo Long Beach, CA, USA). Various parameters of root such as root length, root

surface area, and root volume were examined by WinRHIZO Pro 3.10 (Regent

Instruments Inc.) (Himmelbauer, 2004). Finally, the shoot and root biomass were oven-

dried at 60 °C in paper bags to achieve a constant weight. All dried plant tissues (above-

and belowground) were ground with a Tema mill, RM100 (Retsch Solutions in Miling

and Sieving, Haan, Germany) to pass through a 0.5 mm mesh sieve, samples were stored

in sealed containers for further analyses.

6.3.3. Gas Exchange Measurement

Two weeks after Cd-spiking in the soil, the gaseous exchanges were investigated.

Transpiration rate (E, mol m-2

s-1

), net assimilation rate of CO2 (A, μmol m-2

s-1

),

intercellular CO2 Ci, μmol mol-1

) and stomatal conductance to water vapor (gsw, mol m-

2s

-1) of maize plant leaves of all treatments were evaluated via a portable photosynthesis

system (LI-COR Model 6800, Lincoln, NE, USA) with an extra clamp-on leaf cuvette

that exposed 3 cm2 of leaf area. Temperature and light were 26 ± 0.2°C, and 1500 µmol

m−2

s−1

respectively. The LI-6800-01 CO2 injector (LI-COR Lincoln, NE, USA) with a

high-pressure liquefied CO2 cartridge source was used to keep a constant level of CO2 at

400 µmol m−2

s−1

. The light was executed using the LI-6800-02P light source (LI-COR).

Newly matured leaves were used to conduct these measurements (Dutt et al., 2018).

6.3.4. Tissue nutrient analyses and AMF root colonization





optical emission spectrometry (ICP-OES), Perkin Elmer, USA (Wheal et al., 2011). To

ensure the reliability of equipment, chemicals and digestion process, two blanks were

included in each digestion batch. In determining the AMF colonization range, maize plant

roots were cut into small pieces of 1 cm length

and further stained with Trypan Blue with

some modifications described by Phillips and Hayman (Koske and Gemma, 1989).

Further counting of colonized roots was done using the method described by Giovannetti

and Mosse (1980).

( )

Table 6.1: Soil properties before fertilization and biochar amendment

Kiziltapir Soil Biochar

Texture analysis (%)

Sand 6.0 --

Silt 67.0 --

Clay 27.0 --

Characteristics

SOM (%) 1.10 --

pHwater (1:1) 7.84 8.98±0.06

CaCO3 (%) 22.16 0.19±0.04

Total carbon -- 68.31±1.29

Bulk Density (g cm-3

) 1.0 --

CECest (meq [100g]-1

) 21.23 --

Total Cd (mg kg-1

) 0.04 --

Nutrient content (mg kg-1

)

NO3-N 8.0 --

Total N (%) -- 0.51±0.03

P2O5 (mg kg-1

) 1.45

Total P (%) -- 0.23±0.01

K2O (mg kg-1

) 82.05 --

Total K (%) -- 2.56±0.11

SOM soil organic matter, CECest estimated cation exchange capacity, (means and standard deviation; n =

3)

6.3.5. Cd extraction and determination in plant

An amount of 0.5 g of homogenized sample was weighed on the microanalytical

balance and processed with a mixture of 2 ml of hydrogen peroxide and 6 ml of nitric

acid in the microwave digestion system. The samples were digested by setting the

program as: step 1 (power: 250 w, time: 2 min), step 2 (power: 0 w, time: 2 min), step 3

(power: 250 w, time: 6 min), step 4 (power: 400 w, time: 5 min), and step 5 (power: 600

w, time: 5 min). The subsequent extracts were redissolved in 10 ml of ultrapure water for

succeeding analysis by ICP-OES. An amount of 0.5 g of certified reference material

(NIST 1573a tomato leaves) was digested in the microwave as mentioned above for

maize plant samples. The chemical solutions provided by the sample treatment and those

used to construct the calibration curves were made in water containing 0.5% (v/v) HNO3,

injected by the autosampler of the Optima 8000 ICP-OES (PerkinElmer Inc, USA).

6.3.6. Soil P analysis

Olsen extractable P content of soils was determined based on Olsen (1954).

Briefly, prepared 0.5M sodium bicarbonate (NaHCO3) solution adjusted to pH 8.5 with

NaOH. Shaken 5g soil with 100ml of NaHCO3 solution. After 30 min shaking, the

solution was filtered through Whatman No. 40. The molybdate reagent is modified by

adding an extra 50 ml. of concentrated HCI per liter to neutralize the NaHCO3 in a 5-ml.

Finally, took 5ml filterate to a 25ml volumetric flask and determined phosphorus.


Data were analyzed using Statistix software (Statistix, 2008). Statistical



estimate the relationships between different factors and the observed nutrients

concentration.

6.4. Results

6.4.1. Gaseous Exchange

In Cd 0 (mg kg-1

) concentration, the addition of biochar + AMF significantly

enhanced assimilation rate by 27%. In soil spiked with 5 mg Cd kg-1

, AMF, and biochar

+ AMF neutralized the Cd stress effect by enhancing assimilation to 22% and 24%

respectively (Figure 6.1a). Generally, the increase in Cd concentration decreases

assimilation. However, it increased by 10%, 17% and 15% in biochar + AMF, AMF and

biochar amended treatment in soil spiked with Cd (10 mg Cd kg-1

) respectively. Besides

that, transpiration rate was maximum for the biochar + AMF by 34% in Cd 0 (mg kg-1

)

(Figure 6.1b). Biochar + AMF significantly improved transpiration rate in soil spiked

with 5 mg Cd kg-1

. Intercellular CO2 in biochar + AMF (Cd 0 mg kg-1

) was enhanced by

27% and significantly improved in Cd 5 (mg kg-1

) than control and biochar treatments

(Figure 6.1c). Stomatal conductance was enhanced by increasing the Cd concentration

(Figure 6.1d).

Figure 6.1a: Assimilation rate of CO2 in maize plant leaves in Cd-spiked soil. Values are

mean of three replicates.

a-d

d

b-d

a

a-d cd

ab a

a-d

ab a a-c

0

5

10

15

20

25

30

Contr

ol

Bio

char

AM

F

Bio

char

+ A

MF

Contr

ol

Bio

char

AM

F

Bio

char

+ A

MF

Contr

ol

Bio

char

AM

F

Bio

char

+ A

MF

Cd (0 mg kg-1) Cd (5 mg kg-1) Cd (10 mg kg-1)

Ass

imil

atio

n r

ate

of

CO

2 (

µm

ol

m⁻²

s⁻¹

)

Figure 6.1b: Transpiration rate of the maize plant leaves in Cd-spiked soil. Values are


Figure 6.1c: Intercellular CO2 of the maize plant leaves in Cd-spiked soil. Values are


cd

d d

a

a-c b-d

a-c a a-c ab ab a-c

0.00000.00050.00100.00150.00200.00250.00300.00350.00400.00450.0050

Contr

ol

Bio

char

AM

F

Bio

char

+ A

MF

Contr

ol

Bio

char

AM

F

Bio

char

+ A

MF

Contr

ol

Bio

char

AM

F

Bio

char

+ A

MF


Tra

nsp

irat

ion r

ate

(mol

m⁻²

s⁻¹

)

a-d

d

b-d

a

a-d cd

ab a a-d ab

a

a-c

0.0

5.0

10.0

15.0

20.0

25.0

30.0

Contr

ol

Bio

char

AM

F

Bio

char

+ A

MF

Contr

ol

Bio

char

AM

F

Bio

char

+ A

MF

Contr

ol

Bio

char

AM

F

Bio

char

+ A

MF


Inte

rcel

lual

r C

O2 (

µm

ol

mol⁻

¹)

Figure 6.1d: Stomatal conductance of the maize plant leaves in Cd-spiked soil. Values

are mean of three replicates.

6.4.2. Shoot and root dry weight

The maximum increase in dry shoot weight was observed in biochar + AMF

treatment in Cd 0 (mg kg-1

) (32%) and Cd 5 (mg kg-1

) (39%) than respective controls

whereas, in the Cd 10 (mg kg-1

), 50% increase was observed (Figure 6.2). Besides that,

biochar-amended soil increased 52% shoot weight in Cd 10 (mg kg-1

). A similar trend

was observed for the dry root weight where a 29% increase in biochar + AMF followed

by AMF (27%) in Cd 0 (mg kg-1

). The indifferent trend was followed in Cd 5 (mg kg-

1)concentration, and biochar + AMF had a 54% increase in dry root weight than control

while biochar addition enhanced 22% dry root weight. In Cd 10 (mg kg-1

), root attributes

were changed tremendously. Biochar + AMF enhanced 40% dry root weight followed by

biochar (31%), and AMF (20%).

c-e

e de

a

a-d b-e a-c

ab a-d a-c a-c

a-d

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

Contr

ol

Bio

char

AM

F

Bio

char

+ A

MF

Contr

ol

Bio

char

AM

F

Bio

char

+ A

MF

Contr

ol

Bio

char

AM

F

Bio

char

+ A

MF


Sto

mat

al c

onduct

ance

to w

ater

vap

or

(mol

m⁻²

s⁻¹)

Figure 6.2: Dry weight of shoot and root of maize plant in Cd-spiked soil. Values are


6.4.3. Root colonization and characterization

The root colonization was high in Cd 0 (mg kg-1

). It was maximum of 58% for the

AMF treatment. Addition of biochar enhanced colonization by 62% (Figure 6.3).

Increasing Cd concentration decreased root colonization. In biochar + AMF, 95% root

colonization was reduced in Cd 10 (mg kg-1

). Besides that, control and biochar treatments

had <10% root colonization which could be due to the transportation of mycorrhizal

fungi spores through the wind.

a-c

bc

ab

a

c bc

a-c a-c

c

a

ab a

b-d d a-c ab

d cd a-d

a-c b-d ab

a-d a

0.0

2.0

4.0

6.0

8.0

10.0

12.0

Contr

ol

Bio

char

AM

F

Bio

char

+ A

MF

Contr

ol

Bio

char

AM

F

Bio

char

+ A

MF

Contr

ol

Bio

char

AM

F

Bio

char

+ A

MF

Cd (mg kg-1) Cd (mg kg-1) Cd (mg kg-1)

Wei

ght

(g 3

kg

-1 s

oil

)

DSW

DRW

Figure 6.3: Root colonization of AMF in maize. Values are mean of three replicates.

Plant root length was enhanced by increasing Cd concentration in comparison to

control. In biochar + AMF inoculated plants, 36% increase was noticed in Cd 0 (mg kg-1

)

while 38% enhancement was observed in Cd 5 (mg kg-1

). Only Cd 10 (mg kg-1

) reduced

root length by 32% in biochar + AMF inoculated plants (Table 6.1). Addition of biochar

enhanced 37% root length in Cd 10 (mg kg-1

) while it increased by 44% in AMF

inoculated plants of Cd 5 (mg kg-1

) concentration. A similar trend was followed in root

surface area where 35%, 37%, and 29% enhancement was observed by biochar + AMF in

Cd 0 (mg kg-1

), Cd 5 (mg kg-1

), and Cd 10 (mg kg-1

) respectively. The AMF inoculation

boosted root surface area by 15%, 42% and 19% in Cd 0 (mg kg-1

), Cd 5 (mg kg-1

)and Cd

10 (mg kg-1

) respectively. Root volume followed increased by 33%, 36%, and 25% in

biochar + AMF inoculated plants for Cd 0 (mg kg-1

), Cd 5 (mg kg-1

) and Cd 10 (mg kg-1

).

The AMF alone contributed by 19%, 39% and 20% enhancement in root volume for Cd 0

(mg kg-1

), Cd 5 (mg kg-1

)and Cd 10 (mg kg-1

) respectively.

.

c c

a a

c c

a a

c c

b

b

0

10

20

30

40

50

60

70

Contr

ol

Bio

char

AM

F

Bio

char

+ A

MF

Contr

ol

Bio

char

AM

F

Bio

char

+ A

MF

Contr

ol

Bio

char

AM

F

Bio

char

+ A

MF


Colo

niz

atio

n (

%)

Table 6.2: Root length, root surface area and root volume of maize plant in Cd-spiked soil treated with biochar and AMF. Values are

means of three replicates.

Concentration Treatment Root length (cm) Root surface area (cm2) Root volume (cm

3)

Cd (0 mg kg-1

) Control 9406.2 ± 2837.6 ab 736.7 ± 162.9 abc 4.6 ± 0.6 abcd

Biochar 6244.3 ± 1056.7 b 546.2 ± 81.7 bc 3.9 ± 1.0 bcd

AMF 10487.7 ± 1881.1 ab 869.4 ± 163.3 abc 5.7 ± 1.1 abcd

Biochar + AMF 14591.6 ± 1629.6 a 1125.4 ±134.4 a 6.9 ± 1.0 abc

Cd (5 mg kg-1

) Control 6655.6 ± 1609.2 b 548.1 ± 122.0 bc 3.6 ± 0.9 cd

Biochar 5949.9 ± 4099.5 b 484.9 ± 328.3 c 3.2 ± 2.1 d

AMF 11909.0 ± 2933.3 ab 938.7 ± 255.4 abc 5.9 ± 1.8 abcd

Biochar + AMF 10694.6 ± 4058.8 ab 871.3 ± 305.2 abc 5.7 ± 1.8 abcd

Cd (10 mg kg-1

) Control 9910.4 ± 5423.7 ab 834.2 ± 480.5 abc 5.6 ± 3.4 abcd

Biochar 15691.5 ± 3891.2 a 1217.2 ± 274.8 a 7.5 ± 1.5 a

AMF 12046.8 ± 1114.0 ab 1031.0 ± 92.0 ab 7.0 ± 0.6 ab

Biochar + AMF 14525.0 ± 2013.0 a 1167.7 ± 190.6 a 7.5 ± 1.4 a

All same values at probability (p ≤0.05)

6.4.4. Nutrients concentration in maize shoot and root

In the shoot, biochar enhanced P by 21% (Cd 0 mg kg-1

) in comparison to control.

Generally, P-uptake was reduced by increasing the Cd concentration. In biochar + AMF (Cd 10

mg kg-1

), P-uptake was reduced by 85% (Table 6.2). Similarly, biochar addition enhanced Ca

uptake. Maximum Ca uptake of 25% was noticed in biochar-amended soil (Cd 5 mg kg-1

).

Uptake of Mg was enhanced by 4-15% in all treatments, and Mg uptake was proportional to Cd

concentration. Similarly, Fe uptake was enhanced by 10-15% in all treatments (Cd 5 mg kg-1

)

while Cd 10 (mg kg-1

) inversely affected Fe uptake. Cu uptake was augmented by Cd

concentration, and Cd 10 (mg kg-1

) enhanced Cu uptake by 19% in biochar-amended soil.

Whereas, Cd 10 (mg kg-1

) promoted Mn uptake by 6% in AMF inoculated maize plant, while it

was reduced by 6% in AMF inoculated plant of Cd 5 (mg kg-1

).

In the root, biochar enhanced P by 17% (Cd 0 mg kg-1

) in comparison to control.

Generally, P-uptake was reduced by increasing the Cd concentration. In biochar + AMF (Cd 10

mg kg-1

), P-uptake was reduced by 60% (Table 6.3). Biochar + AMF inoculated plants

stimulated P uptake by 15% in Cd 5 (mg kg-1

). Biochar addition to the soil enhanced Ca uptake,

and biochar-amended soil had a 27% increase in Cd 10 (mg kg-1

). Combination of biochar +

AMF stimulated Ca uptake by 32%. Increasing Cd concentration reduced Mg uptake by 16% in

biochar-amended soil (Cd 0 mg kg-1

) followed by 3% (Cd 10 mg kg-1

). At Cd 5 (mg kg-1

), Fe

uptake was enhanced in all treatments by 16-27% while Cd 10 (mg kg-1

) inversely affected Fe

uptake. Uptake of Cu was augmented at Cd 5 (mg kg-1

) by a 31% increase in AMF inoculated

soil.

Table 6.3: Nutrients concentration in maize shoot in the Cd-spiked soil. Values are means of three replicates

Concentration Treatment P (%) Ca (%) Mg (%) Fe (ppm) Cu (ppm) Mn (ppm)

Cd (0 mg kg-1

) Control 0.8 ± 0.0 cd 0.8 ± 0.0 c 0.2 ± 0.0 c 96.5 ± 1.9 bcd 10.6 ± 0.3 bc 133.3 ± 2.1 bcd

Biochar 1.1 ± 0.0 a 1.0 ± 0.0 b 0.3 ± 0.0 a 110.6 ± 5.8 a 11.3 ± 1.8 b 138.7 ± 11.8 bcd

AMF 0.5 ± 0.0 h 0.5 ± 0.1 f 0.2 ± 0.0 e 80.4 ± 4.6 gh 9.2 ± 0.3 c 121.2 ± 3.8 d

Biochar + AMF 0.7 ± 0.0 fg 0.6 ± 0.0 ef 0.2 ± 0.0 cd 91.1 ± 4.1 cdef 9.4 ± 0.0 c 131.0 ± 1.2 bcd

Cd (5 mg kg-1

) Control 1.0 ± 0.0 ab 0.8 ± 0.0 c 0.2 ± 0.0 g 83.8 ± 3.0 efgh 10.5 ± 0.7 bc 128.7 ± 2.5 cd

Biochar 0.8 ± 0.1 de 1.1 ± 0.0 a 0.3 ± 0.0 f 92.8 ± 2.3 bcde 10.2 ± 0.6 bc 148.7 ± 16.8 bc

AMF 0.6 ± 0.1 gh 0.6 ± 0.1 e 0.2 ± 0.0 ab 94.0 ± 5.5 bcd 11.1 ± 0.3 b 138.6 ± 20.7 bcd

Biochar + AMF 0.7 ± 0.0 efg 0.6 ± 0.0 ef 0.3 ± 0.0 e 99.4 ± 0.9 bc 10.9 ± 0.5 b 151.4 ± 10.1 b

Cd (10 mg kg-1

) Control 0.9 ± 0.1 bc 0.7 ± 0.0 cd 0.2 ± 0.0 b 100.7 ± 1.2 b 11.0 ± 0.1 b 141.8 ± 2.4 bcd

Biochar 0.8 ± 0.0 cd 0.6 ± 0.0 ef 0.3 ± 0.0 e 83.0 ± 9.6 fgh 13.5 ± 1.0 a 176.7 ± 15.7 a

AMF 0.7 ± 0.0 def 0.7 ± 0.0 d 0.2 ± 0.0 d 89.0 ± 1.6 defg 11.1 ± 0.6 b 142.9 ± 11.2 bcd

Biochar + AMF 0.5 ± 0.1 h 0.5 ± 0.0 f 0.2 ± 0.0 e 78.3 ± 1.6 h 10.2 ± 0.4 bc 147.8 ± 9.8 bc


Table 6.4: Nutrients concentration in maize root Cd-spiked soil. Values are means of three replicates

Concentration Treatment P (%) Ca (%) Mg (%) Fe (ppm) Cu (ppm) Mn (ppm)

Cd (mg kg-1

) Control 0.3 ± 0.0 c 0.6 ± 0.0 fg 0.2 ± 0.0 d 3158.5 ± 44.5 ab 28.0 ± 0.3 c 128.6 ± 6.5 def

Biochar 0.4 ± 0.0 a 0.6 ± 0.0 efg 0.2 ± 0.0 a 2172.5 ± 45.3 f 31.0 ± 0.5 ab 124.7 ± 6.3 ef

AMF 0.2 ± 0.0 g 0.7 ± 0.0 cde 0.2 ± 0.0 cd 3028.0 ± 27.8 bc 24.6 ± 0.8 e 141.7 ± 2.1 bcd

Biochar + AMF 0.3 ± 0.0 d 0.5 ± 0.0 g 0.2 ± 0.0 d 2531.0 ± 10.6 e 27.2 ± 0.9 cd 120.0 ± 0.5 f

Cd 5 (mg kg-1

) Control 0.3 ± 0.0 e 0.6 ± 0.1 fg 0.2 ± 0.0 abc 2329.5 ± 51.0 f 21.3 ± 0.5 g 145.8 ± 6.8 bc

Biochar 0.3 ± 0.0 f 0.7 ± 0.1 cd 0.2 ± 0.0 ab 2760.5 ± 186.6 d 23.0 ± 0.6 f 153.9 ±18.5 ab

AMF 0.3 ± 0.0 f 0.7 ± 0.0 cde 0.2 ± 0.0 cd 3191.7 ± 23.2 a 30.7 ± 0.4 ab 136.0 ± 2.0 cde

Biochar + AMF 0.3 ± 0.0 c 0.8 ± 0.0 b 0.2 ± 0.0 a 2852.7 ± 97.4 d 24.4 ± 0.4 e 152.6 ± 4.4 ab

Cd 10 (mg kg-1

) Control 0.4 ± 0.0 b 0.7 ± 0.0 cde 0.2 ± 0.0 bcd 3075.7 ± 103.6 ab 29.6 ± 0.2 b 149.2 ± 6.6 abc

Biochar 0.4 ± 0.0 b 0.9 ± 0.1 a 0.2 ± 0.0 bcd 2254.0 ± 95.5 f 25.1 ± 0.4 e 148.7 ± 7.8 abc

AMF 0.3 ± 0.0 e 0.6 ± 0.0 def 0.2 ± 0.0 d 3059.7 ± 22.9 ab 26.9 ± 0.3 d 160.6 ± 3.7 a

Biochar + AMF 0.2 ± 0.0 fg 0.7 ± 0.1 bc 0.2 ± 0.0 bcd 2882.0 ± 84.1 cd 24.7 ± 0.5 e 136.2 ± 6.1 cde


6.4.5. Cd concentration in plant

In the whole plant, Cd uptake increased by enhancing the Cd concentration in

soil. The use of AMF strongly assisted Cd uptake by enhancing up to 43% in Cd 0 (mg

kg-1

). In biochar + AMF, it was enhanced by 15% only (Figure 6.4). Moreover, the AMF

enhanced uptake by 20% and 21% in Cd 5 (mg kg-1

) and Cd 10 (mg kg-1

) concentrations

respectively compared to the control. In assessment to AMF, biochar amendment

probably adsorbed the Cd and reduced the uptake in comparison to rest of the treatments.

Figure 6.4: Uptake of Cd in maize. Values are mean of three replicates.

6.4.6. Soil P concentration

During soil P estimation, reduction in soil P was noticed by increasing the Cd

concentration. The reduction of 36% soil P was noted in the biochar-amended soil at Cd 5

(mg kg-1

) concentration whereas 8% more soil P was residing in the biochar-amended

soil at Cd 10 (mg kg-1

) concentration than control (Figure 6.5). When AMF was

inoculated in the presence of biochar, soil P reduced by 26% in Cd 10 (mg kg-1

).

h h h h

f

g

e f

b

d

a

c

0.0

2.0

4.0

6.0

8.0

Contr

ol

Bio

char

AM

F

Bio

char

+ A

MF

Contr

ol

Bio

char

AM

F

Bio

char

+ A

MF

Contr

ol

Bio

char

AM

F

Bio

char

+ A

MF


Cd i

n m

g

kg

-1

Figure 6.5: Soil P concentration. Values are mean of three replicates.

a

ab

b-d c-e

a-c

e

b-e b-e

a-c ab

a-c

de

20

25

30

35

40

Contr

ol

Bio

char

AM

F

Bio

char

+ A

MF

Contr

ol

Bio

char

AM

F

Bio

char

+ A

MF

Contr

ol

Bio

char

AM

F

Bio

char

+ A

MF


Soil

P (

mg k

g-1

)

6.5. Discussion

In the study, a significant decline in photosynthetic attributes was observed in control

treatments of all Cd concentrations (Figure 6.1). Studies conducted on maize plant under

Cd stress concluded plant biomass reduction, assimilation rate alteration, the decline in

transpiration rate and modifications in intercellular CO2 Ńimonová et al., 2007; Akhtar et

al., 2017). The extent of decline in gaseous exchange parameters varied in accordance

with the Cd concentration. The plants inoculated with AMF and biochar + AMF had

more assimilation rate and stomatal conductance in comparison to the biochar-amended

soil. The reduction was aligned to increase in the concentration of Cd. The decline in

stomatal conductance of the plant is one of the critical approaches adopted by the plant to

bound assimilation rate with the aim of keeping cellular turgidity (Bertholdi et al., 2018).

Stomatal and non-stomatal activities that include stomatal closure and impairments of

metabolic processes are associated with decreasing assimilation rate on enhancing Cd

stress (Wu et al., 2004; Wu et al., 2006). It was corroborated that under Cd toxicity,

assimilation rate declines resulting to decrease in chlorophyll content and enzymatic

activity responsible for CO2 fixation Ńimonová et al., 2007). It is also attributed to a

decrease in intercellular CO2 subsequently reduced assimilation rate on exposure to Cd

stress (Cui and Wang, 2006). The results additionally proposed that a reduction in

gaseous exchange at higher Cd concentration could be linked with damages in

photosynthetic apparatus (Horváth et al., 1996; Akhtar et al., 2017).

Enhancement in Cd concentration decreased dry plant biomass with the exception of

AMF inoculated plants (Figure 6.2). The decrease in plant biomass could be due to drop

in photosynthetic activity and leaf photosystems severely affected by Cd (Rizwan et al.,

2016; Rizwan et al., 2017). Moreover, plants become stunted, and growth retardation

occurs due to denaturing of proteins, where Cd disrupts H-S (hydrogen-sulfur) bond (LIN

et al., 2007). Artiushenko et al. (2014) reported that during metal stress, plants develop

an effective defense system to alleviate its potential adversities. Such defensive system

may comprise chelate synthesis, production of an osmolyte, enhanced enzymatic and

non-enzymatic antioxidants, suberin lamella formation and cell wall lignification (Lux et

al., 2010).

Nutrient absorption by the root surface is strongly influenced by root surface area

(Zhou et al., 2018). Roots grown in the soil have root hairs which increase root surface

area. Generally, Cd has an inhibitory effect on root proliferation, but the present study

showed contrary results (Table 6.2). Increase in Cd concentration enhanced root surface

area linearly. The plants inoculated with AMF and biochar + AMF had more root

proliferation capability. Root hairs are primary sites for contact between plant root and

rhizosphere (Parker et al., 2000). Absorption of nutrients and heavy metals such as Cd,

Pb, Cr are absorbed on these sites (Nedelkoska and Doran, 2000). The rate of mineral

ions absorption by root cells is dependant on the distance between absorbing cells from

the root tip in the plant. Apical zone of the root is a most active zone for cations uptake

(Boominathan and Doran, 2003). It shows that proportion of apical surface area and

whole root surface area could be a key factor for enhancement of nutrients and heavy

metal absorption. Maize crop is easily colonized by AMF due to its property of high

mycorrhizal dependency (Cao et al., 2017). Several studies have been reported that AMF

can assist the host plants in alleviating Cd toxicity (Liu et al., 2014; Mau and Utami,

2014). Chemophytostabilization practice for heavy metals (e.g., Zn, Cu, Cd, and Pb)

enhance AMF colonization in plant roots (Gucwa-Przepióra et al., 2007). Studies showed

that soil with a high dose of P abate AM colonization to the plants. It further affects plant

growth, and heavy metal bioavailability (Nzanza et al., 2012; Qiao et al., 2015).

Our study showed that the concentration of P, Ca, Mg, and Fe in maize shoot was

decreased (Table 6.3) whereas, in maize root, the only P was decreased by increasing Cd

concentration (Table 6.4). Cadmium alters plasma membrane permeability, and related

membrane transporters which reduce micronutrients uptake changes nutrients

composition in plants (Sarwar et al., 2010). Consequently, plant observes nutrients

deficiency leading to nutrients imbalance (Gogorcena et al., 2011; Rizwan et al., 2012;

Liu et al., 2017). Additionally, the alteration in nutrient uptake in plants could be due to

inhibition in root growth and Cd-induced enzymatic activity (i.e., superoxide dismutase,

peroxidase, catalase, polyphenol oxidase) (Chen et al., 2003). In the present study, a

significant variation in macro and micronutrients concentration was observed in maize

root and shoot grown in Cd-spiked soil (Table 6.3 and Table 6.4).

Cadmium uptake varied significantly and increased linearly by enhancing the

concentration of applied Cd. Tanwir et al. (2015) further corroborated similar results for

Cd uptake against a number of maize cultivars. Plant cell wall accumulates Cd as the first

detoxification strategy to cope with Cd stress (Fernández et al., 2014). This metal

sequestration is further aggravated by the use of AMF inoculation, and once the metal

accumulated in the cell wall, their toxic effects are further mitigated by phytochelatins

(Fernández-Fuego et al., 2017). The negatively charged cell wall of maize plant has

significant potential in Cd+2

binding and retention for a longer time (Polle and

Schützendübel, 2003) Root being a primary contributor in rhizosphere played a key role

in transforming architecture, modifying nutrient mobility, solubility and their uptake

(Keller et al., 2015). This root structuring and their activities further influence Cd uptake

in plants (Stritsis et al., 2014). Besides that, the addition of biochar significantly reduced

the Cd uptake as shown in previous studies (Lu et al., 2014; Zheng et al., 2015).

Moreover, the porous structure of biochar, its high surface charge density, and large

surface area highlight its ability in sorbing inorganic pollutants (Xu et al., 2013). Large

surface area property of biochar and oxygen-containing functional groups on the surface

adsorb Cd (Peng et al., 2017). Biochar addition could have decreased Cd concentration in

maize plant through altering their availability in soil. Alkaline properties of biochar ash

influence Cd2+

hydrolysis by transforming Cd into Cd2+

as less mobile form (Novak et

al., 2009). Addition of biochar into the soil alters pH which reduces Cd-soluble form

(Bashir et al., 2018). Hydrolysis and dissolution of biochar in soil increases pH and

induce precipitation of Cd to Cd3 (PO4)2, resultantly increasing residual Cd in soil

(Mehmood et al., 2018).

6.6. Conclusion

Biochar and AM inoculation separately enhanced maize growth and decreased uptake

of Cd in maize tissues. By comparison, biochar application was potent in Cd stress

alleviation. Resultantly, increasing maize growth, and altering gaseous exchange. Biochar

as a soil amendment significantly persuades soil alkalinization which contributes to Cd

immobilization. Alkaline pH of the soil lowers available Cd concentration and root

colonization in biochar + AMF treatment which could assist as a tactic to be extensively

adopted in alleviating Cd toxicity. These results propose that biochar with AMF

inoculation could be considered as an operative technique for reducing Cd concentration

in maize plant as well as for phytostabilization of Cd-contaminated soil. Further findings

of the study are:

The AMF and biochar enhance maize growth and reduces Cd uptake in their

independent capacity.

Cd stress influence gaseous exchange in maize plant and biochar + AMF

assisted to mitigate Cd toxicity effect.

Root attributes were enhanced in biochar + AMF inoculated plant which makes them

more tolerant to Cd toxicity by enhancing nutrient uptake.

Concluding Remarks

Summary and Conclusion

Biochar is an emerging soil amendment benefiting the soil microbes, soil

physiochemical properties, promoting plant growth, remediating soil pollutants and

ultimately improving the environment by carbon sequestration. Considering paybacks of

biochar in the agricultural and environmental systems, different analytical and pot studies

were conducted on various combinations of biochar, phosphorus solubilizing bacteria

(PSB), arbuscular mycorrhizal (AM) fungi, phosphorus as an important nutrient and

cadmium as heavy metal. In every study, biochar endorsed its potential of promoting

plant growth of maize and onion. Nutrients (N. P and K) availability in plant root, shoot

and soil were enhanced when biochar was used with PSB strains. Results also endorsed

that, PSB may further mine the phosphorus bounded in biochar particles. Further

characterization of sewage sludge biochar, animal feedstock derived biochar and plant

biomass-derived biochar concluded interesting results about their future utilization in

agriculture and environment. Sewage sludge and animal feedstock derived biochar had

high nutrient contents which could be suitable in availability of nutrients to the plants.

Besides that, plant-derived biochar sequesters the carbon in the soil for a longer time

period. They also release the nutrients slowly for a longer time and plant roots can

approach to them for better uptake. If the combination of the animal and plant feedstock

derived biochar is used in future, it may fulfill both objectives of plant growth and carbon

sequestration, ultimately improving the soil quality in long run. Scanning electron

microscopy of the biochar additionally showed the porosity of biochar which is more in

plant-derived biochar. It may provide the habitat for bacterial and AM fungi which assist

in P-solubilization, its uptake along with other nutrients and plant growth promotion.

When two plant-derived biochar (sawdust and phragmites) were used for onion plant with

PSB and AM fungi, the physiological behavior of the plant was improved and

chlorophyll fluorescence was enhanced. It showed that addition of biochar in

combination with AM fungi assist the plant in C-assimilation. Similarly, maize plant was

tested with biochar, PSB and AM fungi combination and results showed that as biochar

improves soil quality, root colonization also increases. Moreover, nutrients concentration

in plant biomass was enhanced. Additionally, biochar was tested for maize plant in

cadmium stress in the presence of AM fungi. The Cd uptake was enhanced in presence of

AM fungi and biochar. Root attributes were also enhanced in supporting the Cd and

nutrients uptake. Biochar is important for sustainability in agriculture and the

environment. It‘s use in combination with PSB and AM fungi could be an ideal approach

in moving towards organic farming and enhancing plant and soil quality.

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Appendices

Onion nursery development

Onion growth in different soils

Onion root colonization

Maize plant growth in both soils

Chapter 4

Analysis of Variance Table for chlorophy

Source DF SS MS F P

Soil 1 0.00427 0.00427 2.52 0.1172

Biochar 1 0.05227 0.05227 30.90 0.0000

P 1 0.00602 0.00602 3.56 0.0638

Treat 3 0.01440 0.00480 2.84 0.0449

Soil*Biochar 1 0.00920 0.00920 5.44 0.0228

Soil*P 1 0.00350 0.00350 2.07 0.1550

Soil*Treat 3 0.03201 0.01067 6.31 0.0008

Biochar*P 1 0.00020 0.00020 0.12 0.7294

Biochar*Treat 3 0.00086 0.00029 0.17 0.9168

P*Treat 3 0.01802 0.00601 3.55 0.0192

Soil*Biochar*P 1 0.00167 0.00167 0.99 0.3247

Soil*Biochar*Treat 3 0.03382 0.01127 6.66 0.0005

Soil*P*Treat 3 0.01417 0.00472 2.79 0.0474

Biochar*P*Treat 3 0.01562 0.00521 3.08 0.0337

Soil*Biochar*P*Treat 3 0.00589 0.00196 1.16 0.3316

Error 64 0.10827 0.00169

Total 95 0.32020

Grand Mean 0.6598 CV 6.23

Analysis of Variance Table for N uptake

Source DF SS MS F P

Soil 1 113686 113686 187.60 0.0000

Biochar 1 1560 1560 2.58 0.1135

P 1 40775 40775 67.29 0.0000

Treat 3 6688 2229 3.68 0.0165

Soil*Biochar 1 1707 1707 2.82 0.0982

Soil*P 1 2958 2958 4.88 0.0307

Soil*Treat 3 9132 3044 5.02 0.0034

Biochar*P 1 48 48 0.08 0.7799

Biochar*Treat 3 3401 1134 1.87 0.1434

P*Treat 3 13488 4496 7.42 0.0002

Soil*Biochar*P 1 2823 2823 4.66 0.0347

Soil*Biochar*Treat 3 4054 1351 2.23 0.0932

Soil*P*Treat 3 4651 1550 2.56 0.0628

Biochar*P*Treat 3 3339 1113 1.84 0.1494

Soil*Biochar*P*Treat 3 3967 1322 2.18 0.0987

Error 64 38784 606

Total 95 251061


Analysis of Variance Table for P uptake

Source DF SS MS F P

Soil 1 774.35 774.35 37.34 0.0000

Biochar 1 127.63 127.63 6.15 0.0157

P 1 4817.09 4817.09 232.30 0.0000

Treat 3 71.31 23.77 1.15 0.3372

Soil*Biochar 1 3.69 3.69 0.18 0.6744

Soil*P 1 26.45 26.45 1.28 0.2630

Soil*Treat 3 201.09 67.03 3.23 0.0280

Biochar*P 1 65.13 65.13 3.14 0.0811

Biochar*Treat 3 73.16 24.39 1.18 0.3259

P*Treat 3 606.25 202.08 9.75 0.0000

Soil*Biochar*P 1 9.08 9.08 0.44 0.5104


Soil*P*Treat 3 112.52 37.51 1.81 0.1545

Biochar*P*Treat 3 107.24 35.75 1.72 0.1709


Error 64 1327.14 20.74

Total 95 8471.83


Analysis of Variance Table for RCu

Source DF SS MS F P

Soil 1 640.15 640.150 159.24 0.0000

Biochar 1 234.38 234.375 58.30 0.0000

P 1 445.91 445.913 110.92 0.0000

Treat 3 77.81 25.936 6.45 0.0007

Soil*Biochar 1 20.54 20.535 5.11 0.0272

Soil*P 1 362.32 362.315 90.13 0.0000

Soil*Treat 3 22.97 7.656 1.90 0.1378

Biochar*P 1 265.34 265.335 66.00 0.0000

Biochar*Treat 3 33.58 11.194 2.78 0.0479

P*Treat 3 95.17 31.725 7.89 0.0001

Soil*Biochar*P 1 66.00 66.002 16.42 0.0001


Soil*P*Treat 3 62.58 20.861 5.19 0.0029

Biochar*P*Treat 3 48.66 16.220 4.03 0.0108


Error 64 257.29 4.020

Total 95 2919.76


Analysis of Variance Table for RK

Source DF SS MS F P

Soil 1 16.236 16.2362 38.07 0.0000

Biochar 1 0.002 0.0024 0.01 0.9404

P 1 59.977 59.9768 140.61 0.0000

Treat 3 3.727 1.2422 2.91 0.0411

Soil*Biochar 1 2.089 2.0886 4.90 0.0305

Soil*P 1 37.101 37.1011 86.98 0.0000

Soil*Treat 3 16.264 5.4212 12.71 0.0000

Biochar*P 1 0.728 0.7280 1.71 0.1961

Biochar*Treat 3 3.071 1.0237 2.40 0.0760

P*Treat 3 10.686 3.5620 8.35 0.0001

Soil*Biochar*P 1 0.952 0.9520 2.23 0.1401


Soil*P*Treat 3 14.332 4.7775 11.20 0.0000

Biochar*P*Treat 3 0.173 0.0578 0.14 0.9385


Error 64 27.298 0.4265

Total 95 204.852


Analysis of Variance Table for root Length

Source DF SS MS F P

Soil 1 1.317E+08 1.317E+08 78.77 0.0000

Biochar 1 1461960 1461960 0.87 0.3534

P 1 4.981E+07 4.981E+07 29.78 0.0000

Treat 3 4.959E+07 1.653E+07 9.88 0.0000

Soil*Biochar 1 2914916 2914916 1.74 0.1915

Soil*P 1 8617070 8617070 5.15 0.0266

Soil*Treat 3 2.408E+07 8027976 4.80 0.0045

Biochar*P 1 4408493 4408493 2.64 0.1094

Biochar*Treat 3 873917 291306 0.17 0.9135

P*Treat 3 1.230E+07 4101056 2.45 0.0714

Soil*Biochar*P 1 321071 321071 0.19 0.6628


Soil*P*Treat 3 3475309 1158436 0.69 0.5600

Biochar*P*Treat 3 5899068 1966356 1.18 0.3261


Error 64 1.070E+08 1672788

Total 95 4.155E+08


Analysis of Variance Table for RMn

Source DF SS MS F P

Soil 1 613864 613864 3329.38 0.0000

Biochar 1 13199 13199 71.59 0.0000

P 1 162 162 0.88 0.3519

Treat 3 52680 17560 95.24 0.0000

Soil*Biochar 1 29335 29335 159.10 0.0000

Soil*P 1 2082 2082 11.29 0.0013

Soil*Treat 3 49323 16441 89.17 0.0000

Biochar*P 1 1691 1691 9.17 0.0035

Biochar*Treat 3 18812 6271 34.01 0.0000

P*Treat 3 4091 1364 7.40 0.0002

Soil*Biochar*P 1 1691 1691 9.17 0.0035


Soil*P*Treat 3 3866 1289 6.99 0.0004

Biochar*P*Treat 3 105891 35297 191.44 0.0000


Error 64 11800 184

Total 95 1031338


Analysis of Variance Table for RN

Source DF SS MS F P

Soil 1 10.2377 10.2377 578.47 0.0000

Biochar 1 0.3589 0.3589 20.28 0.0000

P 1 0.2763 0.2763 15.61 0.0002

Treat 3 0.4889 0.1630 9.21 0.0000

Soil*Biochar 1 0.0053 0.0053 0.30 0.5878

Soil*P 1 0.0231 0.0231 1.31 0.2572

Soil*Treat 3 0.6358 0.2119 11.98 0.0000

Biochar*P 1 0.0646 0.0646 3.65 0.0606

Biochar*Treat 3 0.2322 0.0774 4.37 0.0073

P*Treat 3 1.1036 0.3679 20.78 0.0000

Soil*Biochar*P 1 0.1313 0.1313 7.42 0.0083


Soil*P*Treat 3 0.2512 0.0837 4.73 0.0048

Biochar*P*Treat 3 0.0489 0.0163 0.92 0.4359


Error 64 1.1327 0.0177

Total 95 15.6786


Analysis of Variance Table for RP

Source DF SS MS F P

Soil 1 1.8454 1.84538 282.01 0.0000

Biochar 1 0.0013 0.00128 0.20 0.6603

P 1 9.6203 9.62033 1470.16 0.0000

Treat 3 0.2308 0.07695 11.76 0.0000

Soil*Biochar 1 0.0047 0.00468 0.71 0.4011

Soil*P 1 1.5991 1.59908 244.37 0.0000

Soil*Treat 3 0.2658 0.08859 13.54 0.0000

Biochar*P 1 0.0002 0.00018 0.03 0.8702

Biochar*Treat 3 0.2657 0.08856 13.53 0.0000

P*Treat 3 0.6869 0.22897 34.99 0.0000

Soil*Biochar*P 1 0.0001 0.00005 0.01 0.9299


Soil*P*Treat 3 0.2870 0.09568 14.62 0.0000

Biochar*P*Treat 3 0.2103 0.07009 10.71 0.0000


Error 64 0.4188 0.00654

Total 95 15.7653


Analysis of Variance Table for SurfArea

Source DF SS MS F P

Soil 1 3373068 3373068 92.35 0.0000

Biochar 1 40394 40394 1.11 0.2969

P 1 1157737 1157737 31.70 0.0000

Treat 3 1185168 395056 10.82 0.0000

Soil*Biochar 1 82496 82496 2.26 0.1378

Soil*P 1 160560 160560 4.40 0.0400

Soil*Treat 3 454007 151336 4.14 0.0095

Biochar*P 1 139455 139455 3.82 0.0551

Biochar*Treat 3 27497 9166 0.25 0.8604

P*Treat 3 246593 82198 2.25 0.0909

Soil*Biochar*P 1 17210 17210 0.47 0.4949


Soil*P*Treat 3 151919 50640 1.39 0.2550

Biochar*P*Treat 3 94988 31663 0.87 0.4630


Error 64 2337578 36525

Total 95 9776733


Analysis of Variance Table for RootVolum

Source DF SS MS F P

Soil 1 1.373E+11 1.373E+11 62.91 0.0000

Biochar 1 1.210E+09 1.210E+09 0.55 0.4593

P 1 4.799E+10 4.799E+10 21.98 0.0000

Treat 3 6.471E+10 2.157E+10 9.88 0.0000

Soil*Biochar 1 3.851E+09 3.851E+09 1.76 0.1888

Soil*P 1 1.562E+09 1.562E+09 0.72 0.4008

Soil*Treat 3 3.772E+10 1.257E+10 5.76 0.0015

Biochar*P 1 8.855E+09 8.855E+09 4.06 0.0482

Biochar*Treat 3 4.843E+09 1.614E+09 0.74 0.5323

P*Treat 3 1.697E+10 5.656E+09 2.59 0.0604

Soil*Biochar*P 1 1.889E+08 1.889E+08 0.09 0.7696

Soil*Biochar*Treat 3 5.567E+09 1.856E+09 0.85 0.4717

Soil*P*Treat 3 3.687E+09 1.229E+09 0.56 0.6414

Biochar*P*Treat 3 7.200E+09 2.400E+09 1.10 0.3559

Soil*Biochar*P*Treat 3 8.093E+09 2.698E+09 1.24 0.3041

Error 64 1.397E+11 2.183E+09

Total 95 4.895E+11

Grand Mean 80770 CV 57.85

Analysis of Variance Table for RZn

Source DF SS MS F P

Soil 1 58.1 58.1 0.08 0.7718

Biochar 1 1478.9 1478.9 2.16 0.1466

P 1 15225.8 15225.8 22.23 0.0000

Treat 3 1141.3 380.4 0.56 0.6464

Soil*Biochar 1 88.2 88.2 0.13 0.7209

Soil*P 1 1537.6 1537.6 2.24 0.1390

Soil*Treat 3 3128.3 1042.8 1.52 0.2172

Biochar*P 1 4877.8 4877.8 7.12 0.0096

Biochar*Treat 3 3338.3 1112.8 1.62 0.1924

P*Treat 3 5726.2 1908.7 2.79 0.0478

Soil*Biochar*P 1 1511.3 1511.3 2.21 0.1424


Soil*P*Treat 3 2717.8 905.9 1.32 0.2748

Biochar*P*Treat 3 1276.8 425.6 0.62 0.6038


Error 64 43838.6 685.0

Total 95 93793.3


Analysis of Variance Table for SK

Source DF SS MS F P

Soil 1 9.0038 9.00375 98.27 0.0000

Biochar 1 5.7135 5.71350 62.36 0.0000

P 1 0.1536 0.15360 1.68 0.2001

Treat 3 1.2834 0.42780 4.67 0.0052

Soil*Biochar 1 0.5370 0.53700 5.86 0.0183

Soil*P 1 1.0753 1.07527 11.74 0.0011

Soil*Treat 3 3.1181 1.03938 11.34 0.0000

Biochar*P 1 0.2147 0.21470 2.34 0.1308

Biochar*Treat 3 1.8318 0.61061 6.66 0.0005

P*Treat 3 3.6604 1.22014 13.32 0.0000

Soil*Biochar*P 1 0.8550 0.85504 9.33 0.0033


Soil*P*Treat 3 0.7410 0.24701 2.70 0.0532

Biochar*P*Treat 3 1.6631 0.55436 6.05 0.0011


Error 64 5.8641 0.09163

Total 95 39.1433


Analysis of Variance Table for SMn

Source DF SS MS F P

Soil 1 398694 398694 1899.71 0.0000

Biochar 1 5435 5435 25.90 0.0000

P 1 20284 20284 96.65 0.0000

Treat 3 34249 11416 54.40 0.0000

Soil*Biochar 1 7020 7020 33.45 0.0000

Soil*P 1 20290 20290 96.68 0.0000

Soil*Treat 3 34142 11381 54.23 0.0000

Biochar*P 1 2224 2224 10.60 0.0018

Biochar*Treat 3 11113 3704 17.65 0.0000

P*Treat 3 22239 7413 35.32 0.0000

Soil*Biochar*P 1 3731 3731 17.78 0.0001


Soil*P*Treat 3 19264 6421 30.60 0.0000

Biochar*P*Treat 3 36458 12153 57.90 0.0000


Error 64 13432 210

Total 95 675900


Analysis of Variance Table for SN

Source DF SS MS F P

Soil 1 10.3032 10.3032 480.19 0.0000

Biochar 1 0.1434 0.1434 6.68 0.0120

P 1 8.2427 8.2427 384.16 0.0000

Treat 3 3.7971 1.2657 58.99 0.0000

Soil*Biochar 1 0.4579 0.4579 21.34 0.0000

Soil*P 1 0.8012 0.8012 37.34 0.0000

Soil*Treat 3 1.1779 0.3926 18.30 0.0000

Biochar*P 1 0.0128 0.0128 0.60 0.4421

Biochar*Treat 3 0.4675 0.1558 7.26 0.0003

P*Treat 3 1.7043 0.5681 26.48 0.0000

Soil*Biochar*P 1 0.9540 0.9540 44.46 0.0000


Soil*P*Treat 3 1.7162 0.5721 26.66 0.0000

Biochar*P*Treat 3 0.9828 0.3276 15.27 0.0000


Error 64 1.3732 0.0215

Total 95 32.9696


Analysis of Variance Table for SP

Source DF SS MS F P

Soil 1 0.00010 0.00010 0.03 0.8605

Biochar 1 0.00184 0.00184 0.55 0.4614

P 1 1.81500 1.81500 542.30 0.0000

Treat 3 0.01847 0.00616 1.84 0.1489

Soil*Biochar 1 0.02600 0.02600 7.77 0.0070

Soil*P 1 0.10667 0.10667 31.87 0.0000

Soil*Treat 3 0.05502 0.01834 5.48 0.0020

Biochar*P 1 0.02282 0.02282 6.82 0.0112

Biochar*Treat 3 0.00150 0.00050 0.15 0.9294

P*Treat 3 0.27504 0.09168 27.39 0.0000

Soil*Biochar*P 1 0.07260 0.07260 21.69 0.0000


Soil*P*Treat 3 0.00673 0.00224 0.67 0.5737

Biochar*P*Treat 3 0.00466 0.00155 0.46 0.7084


Error 64 0.21420 0.00335

Total 95 2.69570


Analysis of Variance Table for SCu

Source DF SS MS F P

Soil 1 1.720 1.7200 15.41 0.0002

Biochar 1 12.434 12.4344 111.43 0.0000

P 1 26.935 26.9346 241.37 0.0000

Treat 3 6.309 2.1032 18.85 0.0000

Soil*Biochar 1 0.779 0.7794 6.98 0.0103

Soil*P 1 4.399 4.3990 39.42 0.0000

Soil*Treat 3 0.912 0.3040 2.72 0.0515

Biochar*P 1 13.091 13.0907 117.31 0.0000

Biochar*Treat 3 0.638 0.2125 1.90 0.1377

P*Treat 3 20.104 6.7013 60.05 0.0000

Soil*Biochar*P 1 4.314 4.3138 38.66 0.0000


Soil*P*Treat 3 0.223 0.0745 0.67 0.5752

Biochar*P*Treat 3 4.928 1.6428 14.72 0.0000


Error 64 7.142 0.1116

Total 95 114.226


Analysis of Variance Table for SZn

Source DF SS MS F P

Soil 1 177.67 177.67 27.62 0.0000

Biochar 1 1939.50 1939.50 301.47 0.0000

P 1 1846.26 1846.26 286.97 0.0000

Treat 3 504.72 168.24 26.15 0.0000

Soil*Biochar 1 455.45 455.45 70.79 0.0000

Soil*P 1 57.04 57.04 8.87 0.0041

Soil*Treat 3 104.61 34.87 5.42 0.0022

Biochar*P 1 517.55 517.55 80.44 0.0000

Biochar*Treat 3 195.31 65.10 10.12 0.0000

P*Treat 3 246.51 82.17 12.77 0.0000

Soil*Biochar*P 1 119.93 119.93 18.64 0.0001


Soil*P*Treat 3 96.35 32.12 4.99 0.0036

Biochar*P*Treat 3 27.36 9.12 1.42 0.2457


Error 64 411.75 6.43

Total 95 6923.73


Chapter 5

Analysis of Variance Table for Colonizat

Source DF SS MS F P

Soil 1 1 1.0 0.02 0.8919

Biochar 1 9 9.4 0.17 0.6838

P 1 704 704.2 12.58 0.0007

Treat 3 118784 39594.8 707.18 0.0000

Soil*Biochar 1 51 51.0 0.91 0.3433

Soil*P 1 204 204.2 3.65 0.0607

Soil*Treat 3 159 53.1 0.95 0.4224

Biochar*P 1 17 16.7 0.30 0.5872

Biochar*Treat 3 151 50.3 0.90 0.4466

P*Treat 3 819 272.9 4.87 0.0041

Soil*Biochar*P 1 17 16.7 0.30 0.5872

Soil*Biochar*Treat 3 201 67.0 1.20 0.3181

Soil*P*Treat 3 385 128.5 2.29 0.0862

Biochar*P*Treat 3 148 49.3 0.88 0.4559

Soil*Biochar*P*Treat 3 90 29.9 0.53 0.6611

Error 64 3583 56.0

Total 95 125324


Analysis of Variance Table for DRW

Source DF SS MS F P

Soil 1 45.584 45.5842 139.08 0.0000

Biochar 1 3.249 3.2487 9.91 0.0025

P 1 52.664 52.6644 160.68 0.0000

Treat 3 18.971 6.3237 19.29 0.0000

Soil*Biochar 1 0.626 0.6263 1.91 0.1717

Soil*P 1 8.849 8.8488 27.00 0.0000

Soil*Treat 3 2.595 0.8649 2.64 0.0570

Biochar*P 1 0.031 0.0312 0.10 0.7588

Biochar*Treat 3 0.318 0.1061 0.32 0.8081

P*Treat 3 0.376 0.1254 0.38 0.7660

Soil*Biochar*P 1 0.049 0.0491 0.15 0.6999


Soil*P*Treat 3 2.039 0.6797 2.07 0.1124

Biochar*P*Treat 3 0.295 0.0982 0.30 0.8255


Error 64 20.976 0.3277

Total 95 157.212


Analysis of Variance Table for DSW

Source DF SS MS F P

Soil 1 812.61 812.612 627.32 0.0000

Biochar 1 44.58 44.584 34.42 0.0000

P 1 493.55 493.553 381.01 0.0000

Treat 3 35.60 11.866 9.16 0.0000

Soil*Biochar 1 6.98 6.984 5.39 0.0234

Soil*P 1 1.40 1.403 1.08 0.3020

Soil*Treat 3 11.29 3.763 2.91 0.0414

Biochar*P 1 0.01 0.008 0.01 0.9361

Biochar*Treat 3 0.68 0.228 0.18 0.9123

P*Treat 3 4.05 1.349 1.04 0.3802

Soil*Biochar*P 1 0.86 0.863 0.67 0.4174


Soil*P*Treat 3 3.16 1.055 0.81 0.4908

Biochar*P*Treat 3 2.78 0.927 0.72 0.5464


Error 64 82.90 1.295

Total 95 1503.43


Analysis of Variance Table for Rca

Source DF SS MS F P

Soil 1 4.4419 4.44190 255.62 0.0000

Biochar 1 1.0438 1.04375 60.06 0.0000

P 1 0.0636 0.06355 3.66 0.0603

Treat 3 1.2982 0.43272 24.90 0.0000

Soil*Biochar 1 0.0546 0.05463 3.14 0.0810

Soil*P 1 1.1463 1.14625 65.96 0.0000

Soil*Treat 3 1.5632 0.52106 29.99 0.0000

Biochar*P 1 0.0009 0.00088 0.05 0.8231

Biochar*Treat 3 0.2867 0.09555 5.50 0.0020

P*Treat 3 1.4734 0.49113 28.26 0.0000

Soil*Biochar*P 1 0.5445 0.54451 31.33 0.0000


Soil*P*Treat 3 2.0587 0.68625 39.49 0.0000

Biochar*P*Treat 3 0.7177 0.23922 13.77 0.0000


Error 64 1.1121 0.01738

Total 95 19.1077


Analysis of Variance Table for Rcu

Source DF SS MS F P

Soil 1 312.843 312.843 104.42 0.0000

Biochar 1 0.413 0.413 0.14 0.7115

P 1 41.475 41.475 13.84 0.0004

Treat 3 17.379 5.793 1.93 0.1330

Soil*Biochar 1 0.128 0.128 0.04 0.8372

Soil*P 1 1.525 1.525 0.51 0.4781

Soil*Treat 3 1.262 0.421 0.14 0.9354

Biochar*P 1 39.398 39.398 13.15 0.0006

Biochar*Treat 3 5.716 1.905 0.64 0.5946

P*Treat 3 1.858 0.619 0.21 0.8914

Soil*Biochar*P 1 2.768 2.768 0.92 0.3401


Soil*P*Treat 3 37.533 12.511 4.18 0.0092

Biochar*P*Treat 3 5.148 1.716 0.57 0.6350


Error 64 191.747 2.996

Total 95 718.332


Analysis of Variance Table for RK

Source DF SS MS F P

Soil 1 0.2563 0.2563 10.87 0.0016

Biochar 1 0.1584 0.1584 6.72 0.0118

P 1 27.1575 27.1575 1151.86 0.0000

Treat 3 1.6717 0.5572 23.64 0.0000

Soil*Biochar 1 2.0768 2.0768 88.09 0.0000

Soil*P 1 4.9142 4.9142 208.43 0.0000

Soil*Treat 3 0.7448 0.2483 10.53 0.0000

Biochar*P 1 0.0135 0.0135 0.57 0.4514

Biochar*Treat 3 0.8159 0.2720 11.54 0.0000

P*Treat 3 2.0393 0.6798 28.83 0.0000

Soil*Biochar*P 1 1.1008 1.1008 46.69 0.0000


Soil*P*Treat 3 0.8635 0.2878 12.21 0.0000

Biochar*P*Treat 3 0.5634 0.1878 7.97 0.0001


Error 64 1.5089 0.0236

Total 95 44.1174


Analysis of Variance Table for RMg

Source DF SS MS F P

Soil 1 7.05250 7.05250 3273.89 0.0000

Biochar 1 0.16007 0.16007 74.31 0.0000

P 1 0.10667 0.10667 49.52 0.0000

Treat 3 0.05058 0.01686 7.83 0.0002

Soil*Biochar 1 0.05607 0.05607 26.03 0.0000

Soil*P 1 0.23207 0.23207 107.73 0.0000

Soil*Treat 3 0.11415 0.03805 17.66 0.0000

Biochar*P 1 0.04594 0.04594 21.32 0.0000

Biochar*Treat 3 0.08235 0.02745 12.74 0.0000

P*Treat 3 0.02202 0.00734 3.41 0.0228

Soil*Biochar*P 1 0.11070 0.11070 51.39 0.0000


Soil*P*Treat 3 0.04275 0.01425 6.62 0.0006

Biochar*P*Treat 3 0.09488 0.03163 14.68 0.0000


Error 64 0.13787 0.00215

Total 95 8.49420


Analysis of Variance Table for RMn

Source DF SS MS F P

Soil 1 304701 304701 786.39 0.0000

Biochar 1 39664 39664 102.37 0.0000

P 1 2243 2243 5.79 0.0190

Treat 3 2675 892 2.30 0.0856

Soil*Biochar 1 8182 8182 21.12 0.0000

Soil*P 1 5517 5517 14.24 0.0004

Soil*Treat 3 12834 4278 11.04 0.0000

Biochar*P 1 1258 1258 3.25 0.0763

Biochar*Treat 3 10934 3645 9.41 0.0000

P*Treat 3 2415 805 2.08 0.1119

Soil*Biochar*P 1 157 157 0.41 0.5261


Soil*P*Treat 3 7348 2449 6.32 0.0008

Biochar*P*Treat 3 6923 2308 5.96 0.0012


Error 64 24798 387

Total 95 435682


Analysis of Variance Table for RP

Source DF SS MS F P

Soil 1 0.88743 0.88743 3944.12 0.0000

Biochar 1 0.05753 0.05753 255.67 0.0000

P 1 2.48648 2.48648 11051.0 0.0000

Treat 3 0.01494 0.00498 22.13 0.0000

Soil*Biochar 1 9.375E-06 9.375E-06 0.04 0.8389

Soil*P 1 0.78663 0.78663 3496.12 0.0000

Soil*Treat 3 0.03126 0.01042 46.31 0.0000

Biochar*P 1 0.05273 0.05273 234.38 0.0000

Biochar*Treat 3 0.00364 0.00121 5.40 0.0022

P*Treat 3 0.04589 0.01530 67.98 0.0000

Soil*Biochar*P 1 1.041E-06 1.041E-06 0.00 0.9460


Soil*P*Treat 3 0.04418 0.01473 65.45 0.0000

Biochar*P*Treat 3 0.00159 5.288E-04 2.35 0.0807


Error 64 0.01440 2.250E-04

Total 95 4.46865


Analysis of Variance Table for RSA

Source DF SS MS F P

Soil 1 1.986E+07 1.986E+07 87.27 0.0000

Biochar 1 8944.73 8944.73 0.04 0.8435

P 1 1.774E+07 1.774E+07 77.96 0.0000

Treat 3 5449730 1816577 7.98 0.0001

Soil*Biochar 1 894993 894993 3.93 0.0517

Soil*P 1 6344525 6344525 27.88 0.0000

Soil*Treat 3 602329 200776 0.88 0.4551

Biochar*P 1 70312.6 70312.6 0.31 0.5803

Biochar*Treat 3 303902 101301 0.45 0.7216

P*Treat 3 859888 286629 1.26 0.2958

Soil*Biochar*P 1 186873 186873 0.82 0.3683


Soil*P*Treat 3 1628184 542728 2.38 0.0774

Biochar*P*Treat 3 453774 151258 0.66 0.5769


Error 64 1.456E+07 227586

Total 95 7.242E+07


Analysis of Variance Table for RZn

Source DF SS MS F P

Soil 1 32.20 32.202 2.25 0.1387

Biochar 1 1.00 1.000 0.07 0.7924

P 1 38.51 38.507 2.69 0.1060

Treat 3 249.97 83.325 5.82 0.0014

Soil*Biochar 1 3.41 3.413 0.24 0.6272

Soil*P 1 126.73 126.730 8.85 0.0041

Soil*Treat 3 61.61 20.537 1.43 0.2413

Biochar*P 1 288.08 288.080 20.11 0.0000

Biochar*Treat 3 105.34 35.115 2.45 0.0715

P*Treat 3 172.98 57.660 4.02 0.0110

Soil*Biochar*P 1 84.75 84.750 5.92 0.0178


Soil*P*Treat 3 92.29 30.764 2.15 0.1029

Biochar*P*Treat 3 28.15 9.385 0.66 0.5827


Error 64 916.92 14.327

Total 95 2479.05


Analysis of Variance Table for Sca

Source DF SS MS F P

Soil 1 0.0513 0.05134 6.53 0.0130

Biochar 1 2.7270 2.72700 347.07 0.0000

P 1 8.1201 8.12007 1033.44 0.0000

Treat 3 1.1637 0.38790 49.37 0.0000

Soil*Biochar 1 0.0030 0.00304 0.39 0.5363

Soil*P 1 0.5891 0.58907 74.97 0.0000

Soil*Treat 3 0.1442 0.04807 6.12 0.0010

Biochar*P 1 0.1634 0.16335 20.79 0.0000

Biochar*Treat 3 0.5180 0.17266 21.97 0.0000

P*Treat 3 1.0685 0.35615 45.33 0.0000

Soil*Biochar*P 1 0.0253 0.02535 3.23 0.0772


Soil*P*Treat 3 0.1675 0.05583 7.10 0.0003

Biochar*P*Treat 3 0.5736 0.19120 24.33 0.0000


Error 64 0.5029 0.00786

Total 95 16.7886


Analysis of Variance Table for Scu

Source DF SS MS F P

Soil 1 0.586 0.5859 1.09 0.3001

Biochar 1 12.255 12.2551 22.82 0.0000

P 1 0.788 0.7884 1.47 0.2301

Treat 3 10.212 3.4040 6.34 0.0008

Soil*Biochar 1 1.955 1.9551 3.64 0.0609

Soil*P 1 8.343 8.3426 15.54 0.0002

Soil*Treat 3 4.244 1.4145 2.63 0.0573

Biochar*P 1 4.996 4.9959 9.30 0.0033

Biochar*Treat 3 5.498 1.8326 3.41 0.0226

P*Treat 3 11.109 3.7032 6.90 0.0004

Soil*Biochar*P 1 0.100 0.1001 0.19 0.6674


Soil*P*Treat 3 5.910 1.9701 3.67 0.0167

Biochar*P*Treat 3 2.970 0.9901 1.84 0.1481


Error 64 34.367 0.5370

Total 95 111.617


Analysis of Variance Table for SK

Source DF SS MS F P

Soil 1 0.1114 0.1114 2.43 0.1242

Biochar 1 10.3294 10.3294 225.01 0.0000

P 1 10.9688 10.9688 238.94 0.0000

Treat 3 0.5372 0.1791 3.90 0.0127

Soil*Biochar 1 0.2552 0.2552 5.56 0.0214

Soil*P 1 2.6700 2.6700 58.16 0.0000

Soil*Treat 3 1.7865 0.5955 12.97 0.0000

Biochar*P 1 19.9382 19.9382 434.32 0.0000

Biochar*Treat 3 3.5407 1.1802 25.71 0.0000

P*Treat 3 1.8477 0.6159 13.42 0.0000

Soil*Biochar*P 1 0.1313 0.1313 2.86 0.0957


Soil*P*Treat 3 0.2875 0.0958 2.09 0.1106

Biochar*P*Treat 3 4.8151 1.6050 34.96 0.0000


Error 64 2.9380 0.0459

Total 95 68.8239


Analysis of Variance Table for SMg

Source DF SS MS F P

Soil 1 3.65820 3.65820 2052.53 0.0000

Biochar 1 0.10800 0.10800 60.60 0.0000

P 1 0.68344 0.68344 383.46 0.0000

Treat 3 0.14541 0.04847 27.20 0.0000

Soil*Biochar 1 0.00007 0.00007 0.04 0.8473

Soil*P 1 0.32202 0.32202 180.68 0.0000

Soil*Treat 3 0.12050 0.04017 22.54 0.0000

Biochar*P 1 0.03375 0.03375 18.94 0.0000

Biochar*Treat 3 0.03659 0.01220 6.84 0.0005

P*Treat 3 0.07635 0.02545 14.28 0.0000

Soil*Biochar*P 1 0.02470 0.02470 13.86 0.0004


Soil*P*Treat 3 0.04822 0.01608 9.02 0.0000

Biochar*P*Treat 3 0.03091 0.01030 5.78 0.0015


Error 64 0.11407 0.00178

Total 95 5.46358


Analysis of Variance Table for SMn

Source DF SS MS F P

Soil 1 88234 88233.6 525.02 0.0000

Biochar 1 16886 16885.8 100.48 0.0000

P 1 1860 1860.3 11.07 0.0015

Treat 3 14905 4968.4 29.56 0.0000

Soil*Biochar 1 2567 2566.8 15.27 0.0002

Soil*P 1 17 16.8 0.10 0.7527

Soil*Treat 3 9210 3069.9 18.27 0.0000

Biochar*P 1 282 282.2 1.68 0.1997

Biochar*Treat 3 1928 642.8 3.82 0.0139

P*Treat 3 970 323.5 1.92 0.1344

Soil*Biochar*P 1 70 69.7 0.41 0.5219

Soil*Biochar*Treat 3 3197 1065.7 6.34 0.0008

Soil*P*Treat 3 233 77.8 0.46 0.7091

Biochar*P*Treat 3 2981 993.8 5.91 0.0013

Soil*Biochar*P*Treat 3 3203 1067.7 6.35 0.0008

Error 64 10756 168.1

Total 95 157300


Analysis of Variance Table for SP

Source DF SS MS F P

Soil 1 3.4277 3.42770 3089.76 0.0000

Biochar 1 0.0043 0.00427 3.85 0.0542

P 1 9.3875 9.38750 8461.98 0.0000

Treat 3 0.1527 0.05090 45.88 0.0000

Soil*Biochar 1 0.2542 0.25420 229.14 0.0000

Soil*P 1 2.2204 2.22042 2001.50 0.0000

Soil*Treat 3 0.0137 0.00457 4.12 0.0098

Biochar*P 1 0.0002 0.00020 0.18 0.6694

Biochar*Treat 3 0.0368 0.01227 11.06 0.0000

P*Treat 3 0.4389 0.14628 131.86 0.0000

Soil*Biochar*P 1 0.2091 0.20907 188.45 0.0000


Soil*P*Treat 3 0.0258 0.00860 7.75 0.0002

Biochar*P*Treat 3 0.0436 0.01454 13.11 0.0000


Error 64 0.0710 0.00111

Total 95 16.3720


Analysis of Variance Table for SZn

Source DF SS MS F P

Soil 1 849.7 849.7 30.82 0.0000

Biochar 1 808.5 808.5 29.33 0.0000

P 1 13282.2 13282.2 481.75 0.0000

Treat 3 348.2 116.1 4.21 0.0088

Soil*Biochar 1 1138.5 1138.5 41.29 0.0000

Soil*P 1 2116.9 2116.9 76.78 0.0000

Soil*Treat 3 85.0 28.3 1.03 0.3863

Biochar*P 1 1208.4 1208.4 43.83 0.0000

Biochar*Treat 3 295.4 98.5 3.57 0.0187

P*Treat 3 501.1 167.0 6.06 0.0011

Soil*Biochar*P 1 1044.1 1044.1 37.87 0.0000


Soil*P*Treat 3 140.7 46.9 1.70 0.1756

Biochar*P*Treat 3 303.1 101.0 3.66 0.0168


Error 64 1764.5 27.6

Total 95 24940.7


Analysis of Variance Table for RV

Source DF SS MS F P

Soil 1 5.0849 5.08487 38.84 0.0000

Biochar 1 0.1668 0.16678 1.27 0.2632

P 1 8.4589 8.45892 64.62 0.0000

Treat 3 1.9905 0.66351 5.07 0.0033

Soil*Biochar 1 0.1302 0.13025 0.99 0.3223

Soil*P 1 2.2998 2.29977 17.57 0.0001

Soil*Treat 3 0.4915 0.16383 1.25 0.2986

Biochar*P 1 0.0262 0.02624 0.20 0.6559

Biochar*Treat 3 0.0790 0.02633 0.20 0.8952

P*Treat 3 0.2255 0.07517 0.57 0.6340

Soil*Biochar*P 1 0.1341 0.13411 1.02 0.3153


Soil*P*Treat 3 0.4993 0.16643 1.27 0.2917

Biochar*P*Treat 3 0.3410 0.11366 0.87 0.4623


Error 64 8.3781 0.13091

Total 95 30.7696


Analysis of Variance Table for RL

Source DF SS MS F P

Soil 1 3.817E+09 3.817E+09 99.48 0.0000

Biochar 1 9.326E+07 9.326E+07 2.43 0.1239

P 1 1.925E+09 1.925E+09 50.18 0.0000

Treat 3 7.443E+08 2.481E+08 6.47 0.0007

Soil*Biochar 1 2.711E+08 2.711E+08 7.07 0.0099

Soil*P 1 9.863E+08 9.863E+08 25.71 0.0000

Soil*Treat 3 4.306E+07 1.435E+07 0.37 0.7720

Biochar*P 1 7610849 7610849 0.20 0.6575

Biochar*Treat 3 1.380E+08 4.601E+07 1.20 0.3173

P*Treat 3 1.720E+08 5.732E+07 1.49 0.2246

Soil*Biochar*P 1 1.420E+07 1.420E+07 0.37 0.5451

Soil*Biochar*Treat 3 3.350E+08 1.116E+08 2.91 0.0411

Soil*P*Treat 3 3.089E+08 1.029E+08 2.68 0.0540

Biochar*P*Treat 3 5.385E+07 1.795E+07 0.47 0.7058


Error 64 2.456E+09 3.837E+07

Total 95 1.137E+10


Chapter 6

Analysis of Variance Table for X1

Source DF SS MS F P

Replicate 2 0.00549 0.00274

Factor 2 0.00107 0.00054 0.10 0.9054

Treatment 3 0.78281 0.26094 48.60 0.0000

Factor*Treatment 6 0.33342 0.05557 10.35 0.0000

Error 22 0.11811 0.00537

Total 35 1.24090



Source DF SS MS F P

Replicate 2 0.00015 0.00008

Factor 2 0.13382 0.06691 24.16 0.0000

Treatment 3 0.51836 0.17279 62.40 0.0000


Error 22 0.06092 0.00277

Total 35 1.09908



Source DF SS MS F P

Replicate 2 1.085 0.5425

Factor 2 50.314 25.1569 63.25 0.0000

Treatment 3 17.574 5.8580 14.73 0.0000


Error 22 8.750 0.3977

Total 35 319.382



Source DF SS MS F P

Replicate 2 0.00022 0.00011

Factor 2 0.00612 0.00306 5.35 0.0128

Treatment 3 0.00756 0.00252 4.41 0.0142


Error 22 0.01258 0.00057

Total 35 0.02807



Source DF SS MS F P

Replicate 2 0.3485 0.17424

Factor 2 10.7535 5.37674 6.72 0.0053

Treatment 3 11.6172 3.87241 4.84 0.0098


Error 22 17.6149 0.80068

Total 35 58.0439



Source DF SS MS F P

Replicate 2 41.29 20.643

Factor 2 301.17 150.584 5.41 0.0122

Treatment 3 339.52 113.172 4.07 0.0193


Error 22 611.89 27.813

Total 35 3585.80



Source DF SS MS F P

Replicate 2 506.2 253.09

Factor 2 2713.9 1356.97 7.78 0.0028

Treatment 3 2499.5 833.18 4.78 0.0103


Error 22 3835.6 174.35

Total 35 10938.9



Source DF SS MS F P

Replicate 2 0.00015 0.00008

Factor 2 0.01127 0.00563 41.54 0.0000

Treatment 3 0.04028 0.01343 99.01 0.0000


Error 22 0.00298 0.00014

Total 35 0.11630



Source DF SS MS F P

Replicate 2 0.00137 0.00069

Factor 2 0.13424 0.06712 27.40 0.0000

Treatment 3 0.08279 0.02760 11.26 0.0001


Error 22 0.05389 0.00245

Total 35 0.45163



Source DF SS MS F P

Replicate 2 0.00077 3.861E-04

Factor 2 0.00144 7.194E-04 5.60 0.0108

Treatment 3 0.00219 7.296E-04 5.68 0.0049

Factor*Treatment 6 0.00196 3.269E-04 2.54 0.0504

Error 22 0.00283 1.285E-04

Total 35 0.00919



Source DF SS MS F P

Replicate 2 45261 22630

Factor 2 55971 27985 3.17 0.0616

Treatment 3 2266274 755425 85.62 0.0000

Factor*Treatment 6 2076569 346095 39.23 0.0000

Error 22 194112 8823

Total 35 4638186



Source DF SS MS F P

Replicate 2 487.57 243.78

Factor 2 2942.92 1471.46 21.94 0.0000

Treatment 3 444.04 148.01 2.21 0.1159


Error 22 1475.55 67.07

Total 35 7183.23



Source DF SS MS F P

Replicate 2 2.308E+07 1.153E+07

Factor 2 1.123E+08 5.616E+07 4.04 0.0321

Treatment 3 1.202E+08 4.008E+07 2.88 0.0589

Factor*Treatment 6 1.246E+08 2.078E+07 1.49 0.2263

Error 22 3.060E+08 1.390E+07

Total 35 6.863E+08



Source DF SS MS F P

Replicate 2 186758 93379

Factor 2 778501 389251 4.41 0.0246

Treatment 3 730585 243528 2.76 0.0666

Factor*Treatment 6 532168 88695 1.00 0.4477

Error 22 1943605 88346

Total 35 4171617



Source DF SS MS F P

Replicate 2 9.834 4.9168

Factor 2 34.062 17.0309 4.45 0.0238

Treatment 3 27.988 9.3292 2.44 0.0914


Error 22 84.128 3.8240

Total 35 168.896



Source DF SS MS F P

Replicate 2 27.532 13.7658

Factor 2 58.290 29.1450 2.73 0.0872

Treatment 3 161.359 53.7864 5.04 0.0083


Error 22 234.707 10.6685

Total 35 699.108



Source DF SS MS F P

Replicate 2 151.4 75.69

Factor 2 1209.7 604.86 10.52 0.0006

Treatment 3 18700.0 6233.33 108.38 0.0000


Error 22 1265.3 57.51

Total 35 22405.6



Source DF SS MS F P

Replicate 2 6.329 3.1646

Factor 2 8.895 4.4474 2.09 0.1477

Treatment 3 34.255 11.4184 5.36 0.0063


Error 22 46.838 2.1290

Total 35 112.953



Source DF SS MS F P

Replicate 2 0.31732 0.15866

Factor 2 0.84552 0.42276 2.98 0.0718

Treatment 3 1.99787 0.66596 4.69 0.0112


Error 22 3.12522 0.14206

Total 35 7.08327



Source DF SS MS F P

Replicate 2 0.034 0.0172

Factor 2 160.280 80.1401 2838.82 0.0000

Treatment 3 7.726 2.5753 91.23 0.0000


Error 22 0.621 0.0282

Total 35 173.818



Source DF SS MS F P

Replicate 2 0.00000 0.00000

Factor 2 0.00000 0.00000 M M

Treatment 3 0.00000 0.00000 M M

Factor*Treatment 6 0.00000 0.00000 M M

Error 22 0.00000 0.00000

Total 35 0.00000

Grand Mean 0.0000

WARNING: The total sum of squares is too small to continue.

The dependent variable may be nearly constant.


Source DF SS MS F P

Replicate 2 21.127 10.5637

Factor 2 74.390 37.1952 2.77 0.0845

Treatment 3 167.596 55.8654 4.16 0.0177


Error 22 295.300 13.4227

Total 35 713.084



Source DF SS MS F P

Replicate 2 1589.7 794.87

Factor 2 2137.6 1068.79 1.96 0.1644

Treatment 3 7489.7 2496.56 4.58 0.0122


Error 22 11982.2 544.65

Total 35 30096.6



Source DF SS MS F P

Replicate 2 0.00161 0.00080

Factor 2 0.00267 0.00134 3.94 0.0345

Treatment 3 0.00354 0.00118 3.48 0.0331


Error 22 0.00746 0.00034

Total 35 0.02050


Exploration of Microbially Inoculated Biochar for...

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