366 Varo-Arguello 12-Revised-Black 120919

7/29/2019 366 Varo-Arguello 12-Revised-Black 120919

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TRIPHASIC SLURRY BIOREACTORS FOR THE BIOREMEDIATION OF

LINDANE-IMPACTED SOIL UNDER AEROBIC AND ANAEROBIC

CONDITIONS

Wendy E. Varo-Arguello1, Beni Camacho-Prez1, Elvira Ros-Leal2, Pedro A. Vazquez-

Landaverde3, Mara T. Ponce-Noyola4, Josefina Barrera-Corts5, Isabel Sastre-Conde6, Noem

F. Rindernknecht-Seijas7, Hctor M. Poggi-Varaldo1

1Environmental Biotechnology and Renewable Energies R&D Group, Department of Biotechnology

and Bioengineering, CINVESTAV del IPN, P.O. Box 14-740, 07000 Mxico D.F., Mxico;

2Central Analtica, Department of Biotechnology and Bioengineering, CINVESTAV del IPN

3CICATA-IPN, Qro., Mxico;

4Microbial Genetics Group, Department of Biotechnology and Bioengineering, CINVESTAV del

IPN;

5Control inteligente de Procesos, Department of Biotechnology and Bioengineering, CINVESTAV

del IPN

6IRFAP, Conselleria d`Agricultura i Pesca, Palma de Mallorca, Islas Baleares, Spain;

7ESIQIE del IPN, Mexico D.F., Mxico

Abstract

The objective of this study was two-fold: (i) to evaluate the effect of co-substrate supplementation

and possible synergistic effect of the indigenous population and a lindane-acclimated inoculum on

the removal of lindane in three-phase, aerobic slurry bioreactors (SB) , and (ii) to evaluate the

effect final electron acceptor (O2, CO2 and SO4-2, or A, M, and SR, respectively) and

supplementation with carbon source (sucrose, 1 and 0 g/L; C or NC, respectively) on the removal of

lindane in triphasic lab scale SB. In a first experiment lindane was significantly removed in the firstweek of operation (55-70%); its reduction further continued at a lower rate. Both factors had a

moderately significant effect; on the one hand, sucrose supplementation enhanced the removal of

In memoriam Dr. Y. M. Cabidoche who made outstanding contributions to the remediation of soils polluted with chlordecone and other

pesticides

Author to whom all correspondence should be addressed: e-mail: [email protected], Tel.: (5255) 5747 3800 ext 4324


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lindane (p < 0.08); on the other hand the indigenous microflora and lindane-acclimated inoculum

exhibited some kind of antagonism (p < 0.07), since removals in SB with sterile soil were higher

than those with live soil. In a second experiment, there was a significant effect of factor electron

acceptors on removal of lindane (p < 0.0001): lindane removal followed the order A > SR > M.

Supplementation with sucrose had a significant positive effect (p < 0.004). Main metabolites fromlindane degradation were chlorobenzene (CB), 1,2-dichlorobenzene (1,2-DCB) 1,3-

dichlorobenzene (1,3-DCB) and 1,2,4-trichlorobenzene (1,2,4-TCB) in aerobic and sulfate reducing

slurry bioreactors, only CB and 1,2-DCB were found in methanogenic units. Metabolites were

consistent with those reported in aerobic and anaerobic degradation pathways of lindane.

Keywords: bioremediation, clayish soil, lindane, slurry bioreactors, solvent, triphasic

1. Introduction

The application of lindane and technical HCH during the last six decades has resulted in

environmental contamination of global dimensions and concern (Camacho-Prez et al., 2012; Li et

al., 2003; Li, 1999; Li and MacDonald, 2005; Vijgen et al ., 2011) due to its widespread use in the

past, pronounced toxicity as well as persistence against biotic or abiotic degradation, and its trend to

bioconcentration. Production and agricultural use of lindane has been banned in the Stockholm

Convention on Persistent Organic Pollutants (POPs) held in 26th August 2009 (UNEP, 2009).

However, in some countries, lindane is still used in public health applications to control insect-borne

diseases, and there are some unofficial reports that point out to illegal agricultural use in several

developing countries. More importantly, past use of lindane has created an environmental burden

and contaminated sites that are still waiting for remediation initiatives (International HCH and

Pesticides Association, 2012; Ramos et al., 2011; Romero et al., 2004; Zhang et al., 2005).

Lindane is introduced into the environment mainly via diffuse sources (agricultural runoff),

but also from point sources like production sites and pesticide spills (Wauchope et al., 2002). In a

large set of countries (Austria, Brazil, China, Czech Republic, France, Germany, Hungary, India,

Italy, Japan, Macedonia, Mexico, Nigeria, Poland, Romania, Slovakia, South Africa, Spain,

Switzerland, Turkey, The Netherlands, UK, USA, and former USSR) between 4 and 7 million

tonnes of wastes of toxic, persistent and bioaccumulative lindane residues have been produced and

discarded during 60 years of lindane production. The amounts of lindane wastes and number of

countries with lindane problems increase if wastes and contaminated sites from lindane application


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are considered (Fuentes et al., 2010; Ramos et al., 2011; Vijgen et al., 2011; Zhang et al., 2005).

Human exposure to lindane and the negative effects on human health have been reviewed by

Nolan et al. (2011). The most important route of human exposure to lindane is ingestion of food

contaminated with this pesticide as well as dermal exposure. In Mxico, lindane has been detected

and its levels have been measured in human blood, human breast milk and human adipose tissue,demonstrating that humans still are being exposed to lindane (Lpez-Carrillo et al., 2002; Ramos et

al., 2011).

In soils and sediments, lindane is degraded primarily by biotransformation, although

volatilization may play a positive role. It ks known that lindane can be uptaken by natural microbial

populations owing to their extremely high heterogeneity and adaptability to unfavourable

environmental conditions (Fuentes et al., 2010; Phillips et al., 2004). Yet, the hydrophobic nature of

organic pesticides leads to a strong sorption with the organic matter and clay contents of the soil and

this, in turn, significantly slows down any remediation process (Cabidoche et al., 2006, 2009; Poggi-

Varaldo et al., 2002, Poggi-Varaldo and Rinderknecht-Seijas, 2003).

Bioremediation constitutes a feasible approach to clean up soils and water systems

contaminated by -HCH because of its advantages over other alternatives such as incineration,

storage, or soil washing (Lal et al., 2010; Phillips et al., 2005; Robles-Gonzalez et al., 2012). As

we mentioned above, one challenge faced by bioremediation is to overcome the strong, irreversible

sorption of hydrophobic contaminants onto solid matrices (Robles-Gonzlez et al., 2008 and 2006;

Zanaroli et al., 2010). In this regard, it is known that solvents added to a variety of biological

treatments allows increased mass transfer of oxygen and enhanced desorption of hydrophobic

pollutants sorbed onto soils . Former work of our Group has shown the advantages of using silicone

oil as a solvent in triphasic slurry bioreactors (Robles-Gonzalez et al., 2012)). Thus, the objective of

this study was two-fold: (i) to evaluate the effect of co-substrate supplementation and the synergistic

effect of the indigenouspopulation and a lindane- acclimated inoculum on the removal of lindane in

three-phase, aerobic slurry bioreactors, in lab scale units, and (ii) to evaluate the effect of final

electron acceptor (O2, CO2, and SO4-2) and biostimulation with a degradable carbon source (sucrose,

1 and 0 g/L; C and NC, respectively) on the removal of lindane in triphasic lab scale slurry

bioreactors.

2. Materials and methods

2.1. Soil and pesticide


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The soil used in the experiment originated from San Miguel Tequixtepec, Oaxaca (Table 1).

Its pH was 7.2, and had high contents of organic matter (8.1%). It contained 35, 33, and 42% of

sand, silt, and clay, respectively (Table 1). Lindane, or -1,2,3,4,5,6-hexachloro-cyclohexane

reagent grade from Aldrich Chemical Company, Inc., with 97% purity was used. Lindane is amoderately lipophilic, organo-chlorinated substance characterized by a high partition coefficient

octanol-water Kow 4*103 (Robles-Gonzlez et al., 2012), with a low solubility in water, approx. 7

mg/L at 20C, and slight polarity due to the strong electronegative effects of chlorine atoms bound

to the aliphatic ring.

Table 1. Main physico-chemical characteristics of mineral agricultural soil

used in this work

Parameter Value

Source San Miguel Tequixtepec, Oaxaca

Soil type Cambisol

pH 7.2 0.1

Organic matter (%) 8.1 0.1

COD a (mg COD/ kg dry soil) 5100 436

BOD b (mg BOD5/kg dry soil) 3725 350

Clay (%) 42.3 0.8

Sand (%) 36.5 2.7

Silt (%) 21.2 3.3

Texture ClayishHydraulic conductivity LowNotes: aCOD: Chemical Oxygen Demand, bBOD: Biochemical Oxygen Demand

2.2. Preparation and contamination of soil

The soil was sieved through a 20 mesh screen. Afterwards, it was sterilized by tyndalization

(three times at 121C for 60 min, with a 24 h period of incubation between treatments) except for the

soil used with live indigenous microbial population (LS). All soil samples were contaminated with adose of 100 mg lindane/kg dry soil: the adequate amount of lindane was dissolved in 500 mL of

acetoneand thoroughly mixed. The polluted soil was vacuum-dried at 40C in order to evaporate the

acetone.

2.3. Seed bioreactors


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Lindane-acclimated inocula for the slurry bioreactors were drawn from continuous suspended

growth reactors (aerobic, methanogenic, and sulfate-reducing units). The aerobic seed bioreactor

consisted of an aerated Eckenfelder cell of 14 L capacity operated at ambient temperature. It was fed

an influent containing (per liter of tap water): lactose (1g), KH2PO4 (0.17 g), K2HPO4 (0.57g)Na2HPO4 (0.668g), NaHCO3 (0.02g), NH4NO3 (0.1855g), MgSO4 (0.022g); CaCl2 (0.0275g) and

lindane (7 mg). The methanogenic seed reactor was a 4 L capacity glass bottle and working volume

3 L that operated at 35C. It received an influent containing (per liter of tap water): sucrose (5g),

NaHCO3 (1.5g), K2HPO4 (0.6g), Na2CO3 (3g), NH4Cl (0.6g), CH3COOHglacial (1.5mL) and lindane

(7mg). The sulfate-reducing seed reactor was a 4 L capacity glass bottle and working volume 3 L

that operated at 35C. Its influent contained (per liter of water) sucrose (5 g), NaHCO3 (1.5 g),

K2HPO4 (0.6 g), Na2CO3(3 g), NH4Cl (0.6 g), Na2SO4 (13g), sulfuric acid (0.75 mL) and lindane (7

mg). Influents to seed bioreactors were prepared in batches of 2 L that were kept under refrigeration

until use. 14 mg of lindane in powder (Aldrich Chemical Co., Inc., 97% purity) was weighed in an

electronic balance Sartorius brand, model Micro, capacity 1500 mg, with precision 0.001 mg. The

lindane was added to each batch and solubilized by energic magnetic mixing.

All the seed bioreactors were operated for 1 year or more with lindane in their feeds.

2.4. Experimental design

2.4.1. First experiment

A 22 factorial experiment with two replicates was carried out. The effect of (i) co-substrate

sucrose concentration at 0 to 1 g/L (abbreviations NC, C, respectively), and (ii) indigenous soil

microbial population (live soil, LS; and sterile or dead soil, DS) were evaluated. Also an abiotic

control was made (sterile both soil and inoculum) was implemented.

The experimental setup for evaluating the kinetics of lindane removal was carried-out in lab

scale slurry bioreactors, which were prepared as follows: 20 g of soil contaminated with lindane

(100 mg/kg), 30 mg of volatile solids suspended (VSS) of inoculum acclimatized to lindane, 60 mL

of mineral medium and 20 mL of silicone oil, either with or without sucrose. Bioreactors were

incubated under aerobic conditions and stirred at 120 rpm at 25 C for 4 weeks; samples were

taken every week by destructive sampling (0,1,2,3,4 weeks).

The medium for aerobic slurry bioreactors contained the following (per liter): 33 mL of

NaH2PO4 (0.2 M), 67 mL Na2HPO4 (0.2M), 180 mg (NH4)3PO4, 31.5 mg CaSO4, 23 mg FeSO4 and


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22.5 mg MgSO4 (Pesce and Wunderlin, 2004).The pH of the medium (before mixing with soil) was7.2.

2.4.2. Second experiment

The effect of 2 factors at 3 levels was evaluated: co-substrate sucrose (1 and 0 g/L; C and NC,respectively) and final electron acceptor (O2, CH4 and SO

-4; or aerobic A, methanogenic M, and

sulfate-reducing SR, respectively). Abiotic controls were run (sterilized soil and inoculum).

Bioreactors were implemented and operated as described elsewhere (Robles-Gonzalez et al., 2012).

Methanogenic mineral medium was prepared as follows (per liter of deionized water) (modified

from Bachmann et al., 1988): 60 mg of K2HPO4, 100 mg of (NH4)3PO4. Sulfate reducing mineral

medium was prepared as follows (per liter of desionized water): 100ml phosphate buffer; 100mg

(NH4)3PO4; 10ml of vitamin stock, with a ratio COD/SO4= 2.5.

Both anaerobic media were formulated with a ratio 16C:1N depending of co-substrate

supplementation and 10mL of resazurin at (1g/L) as redox indicator. Lab scale slurry bioreactors

were seeded with corresponding inocula sampled from either aerobic, methanogenic or sulfate

reducing seed bioreactors. Liquors from seed bioreactors were sedimented (under anoxic conditions

when required) and decanted, and the biomass of concentrated liquors was determined as

concentration of VSS in mg/L. Taking into account the target seed biomass per slurry bioreactor (30

mg VSS per bottle), and the concentration of biomass in the concentrated liquors, the volumes of

concentrated liquor to be transferred to slurry bioreactors could be easily found with the mass

balance

VSL = 30 mg/CSL (1)

where

VSL stands for the volume of sedimented liquor, in L; and

CSL is the concentration of biomass in the sedimented liquor, in mgVSS/L

The slurry bioreactors were also loaded with 20 g of soil polluted with 100 mg lindane/kg dry soil,

60 mL of medium, and 20 mL of silicone oil.

2.5. Analytical methods

The concentration of lindane was measured by Headspace-Solid Phase Microextraction-Gas


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Chromatography- Electron Capture Detector in a Perkin-Elmer chromatograph equipped with

electron capture detector, and a Supelco capillary column (30m x 0.25mm x 0.5m). Temperatures

of column, injector, and detector were 210, 250C and 350C respectively. Nitrogen at 8 mL/min

was the carrier gas. A sample of 10 mL slurry was placed in a vial and stirred and heated at 80C for

10 min. After that, a solid phase microextraction fiber was immersed into it for 10 min at 80C. Thefiber was then placed into the injector of a gas chromatograph for thermal desorption of adsorbed

compounds for 5 min. Limits of detection for quantification was of 0.001 mg lindane/L level

(Prosen et al., 2002); lindane metabolites were extracted and analized as described by Quintero et al.

(2006).

Microbial counts of aerobic lindane-clastic microorganisms were carried out by plating. Petri

dishes were prepared by triplicate for each dilution (from 1X101 to 1X105); they were incubated at

30C for 10 days in a static incubator. After incubation, colonies were counted and results were

expressed on the basis of colony-forming unit (CFU) per mL of sample.

The medium R2A agar described in APHA (1987) Method 9215 A section 6, p. 9-55 was

used (per L of distilled water): K2HPO4, 0.3g; MgSO47H2O, 0.05g; agar, 15g; lindane, 7mg.

The liquid mixture pH was adjusted to 7.2 with either 2N HCl or NaOH before adding agar.

Afterwards, the medium was heated to dissolve agar and further sterilized at 121C for 15min.

While warm, lindane was added and quickly dissolved by mixing with a sterile magnetic mixer in a

laminar flow hood surrounded by four burners to keep sterile and warm conditions. While warm,

the medium was poured into Petri dishes.

The soil pH was determined in a slurry soil/deionized water 1:2 (w/w) (Robles-Gonzlez et

al., 2012), soil texture was measured by the hydrometer method, soluble biochemical oxygen

demand (BOD) and soluble chemical oxygen demand were s estimated according to the Standard

Methods (American Public Health Association, 1981) in a warm water soil extract 1:5. Total soil

organic matter content was estimated by the method of oxidation with K2Cr2O7 (Robles-Gonzlez et

al., 2006). In the seed bioreactors were determined pH, sulphate (for the sulphate-reducing unit),

organic matter content as COD and biomass according to the Standard Methods (methods 423,

426C, 508 and 209E respectively). Lindane and metabolites sorbed onto biomass of seed bioreactors

were determined intermittently. The alkalinity and ratio were determined according to Poggi-

Varaldo and Oleszkiewicz (1992).

2.6. Statistical analysis


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Results of the experiment were subjected to analysis of variance using the software

Design-Expert (Stat-Ease, Inc., Minneapolis, MN, USA). Duncan test of means was performed

according to procedures described in Montgomery (1997).

3. Results and discussion

3.1. Seed bioreactors

Table 2 shows a summary of the performance of the seed bioreactors. Sulfate-reducing

SBs displayed the highest lindane removal. This is consistent with reports of Elango et al. (2011)

who mentioned that there are many sulfate reducing bacteria such asDesulfovibrio gigas, D.

africanus and Desulfococcus multivorans, able to dechlorinate organic compounds. Concentration

of lindane and its metabolites sorbed onto biomass in the seed bioreactors was negligible (data not

shown). Thus, lindane removal in the seed bioreactors was essentially biologically-mediated. The

specific removal rates in seed bioreactors were 336, 155, and 120 mg lindaneremoved/(kgbiomassd) for

the A, M, and SR seed bioreactors, respectively. These values were estimated on the basis of

average mass of biomass, other average data from Table 2 for the seed bioreactors, and an input

concentration of lindane of 7 mg/L, using the corresponding mass balances.

3.2. First experiment

3.2.1. Lindane removal

The general trend of lindane concentration in all treatments was a drastic removal of lindane in

the first week (55-70%); lindane reduction further continued at a lower rate (Fig. 1). In the

discussion below, treatments have received convenient abbreviations as follows: NC-DS stands for

without sucrose and sterile soil, C-DS means supplemented with sucrose and sterile soil; NC-LS

stands for without sucrose and live soil, and C-LS represents supplemented with sucrose and live

soil. At the end of the experiment, the highest lindane removal was observed in treatment C-DS

(90%). NC-DS, C-LS, and NC-LS treatments showed removals of 87, 87, and 83%, respectively

(Fig. 1). Both factors had a moderate significant effect; sucrose supplementation slightly enhanced


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Table 2. Average performance of suspended growth biomass seed reactors used for

inoculation of slurry bioreactors

Parameter Average Standard deviation

Aerobic Methanogenic Sulfate reducing

Lindane (%)a 76.3 3.4 70.1 4.5 80.7 6.2

pH (-) 7.95 0.25 7.69 0.31 8.03 0.27

COD (%)b 69.8 9.2 67.3 10.1 59.9 9.5

VSS (mg/L) 1318 251 1463 274 1893 295

Coefficient NAd 0.20 0.09 0.15 0.07

sulfate (%)c NA NA 76.5 7.9

CH4 (%) NA 44.7 13.1 NA

Notes: aLindane removal efficiency, bChemical oxygen demand removal efficiency, csulfate removal efficiency, VSS:

Volatile suspended solids, NA: Not applicable. Standard deviations calculated with respect to time.

the removal of lindane (p < 0.08). Indigenous microflora and lindane-aclimated inoculum exhibited

some kind of antagonism (p < 0.07), since removals of DS treatments were higher than those of LS

units. In contrast, Di Toro et al. (2008) reported an improved degradation of initial 10 g diesel/kg

soil in polluted soils using slurry reactors bioaugmented and biostimulated with a commercial

preparation Enzyveba, after 4.5 months of treatment. They suggested the occurrence of some kind of

synergism between indigenous and exogenous microflora.

Our abiotic control showed a low-to-moderate removal of lindane (11%). This indicates that

biotic removal contributed up to 79% of the overall lindane reduction.

3.2.2. Lindaneclastic bacteria counts

Lindaneclastic bacteria counts in our slurry bioreactors in treatments with sterilized soil and

live acclimated inoculum were in the order of 5 log CFU (Fig. 2). In contrast, the counts in the units

with live soil (live indigenous microflora) and live acclimated inocula were in the order of 3.5 log

CFU, independently of sucrose or no sucrose supplementation. This points out to some kind of

antagonism that was more evident after the second week of operation of the slurry bioreactors.

3.2.3. Intermediate metabolites

Fig. 3 shows a typical chromatogram of the aerobic, triphasic slurry bioreactor supplemented


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0

0.2

0.4

0.6

0.8

1

1.2

0 1 2 3 4

nane

t (wk)

a) b)

Fig. 1. (a) Time course of remaining lindane concentration in polluted soils treated in aerobic,

triphasic slurry bioreactors; (b) average lindane removals after 30 days batch operation treated in

aerobic, triphasic slurry bioreactors (Keys to cadre (a): ( ) Abiotic control (AC); () supplemented

with cosubstrate, live indigenous soil microflora plus acclimated inoculum (C-LS); () non

supplemented, live indigenous soil microflora plus acclimated inoculum (NC-LS); () non

supplemented, sterilized soil plus live acclimated inoculum (NC-DS); () supplemented with co-

substrate, sterilized soil plus live acclimated inoculum (C-DS);

Keys to cadre (b): the error bar represents the standard error of the experiment SEE = (MSE/r)1/2,

where MSE is the mean square error and r is the number of replicates. AC: abiotic control; C-LS:supplemented with cosubstrate, live soil and live acclimated inoculum; NC-LS, non supplemented,

live soil and live acclimated inoculum; NC-DS, non-supplemented, sterile soil and live acclimated

inoculum; C-DS: supplemented with cosubstrate, sterile soil and live acclimated inoculum)

with sucrose and bioaugmented with lindane-acclimated inoculum. The soil was sterilized.

Chlorobenzene (CB), 1,2-dichlorobenzene (1,2-CB) and a couple of trichlorobenzenes were found.

These metabolites are consistent with those found in the last stages of lindane aerobic degradation

(Camacho-Prez et al., 2012).

Interestingly, the highly chlorine substituted metabolites characteristic of the early stages of

lindane transformation pentachlorocyclohexane (PCCH) and tetrachlorocyclohexane TCCH (Lal et

al., 2010) were not detected in our SBs (Table 3).


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0

2

4

6

0 1 2 3 4 5

LogCFU

Time (week)

Fig. 2. Time course of colony-forming units (CFU) of lindaneclastic microorganisms in aerobic

slurry bioreactors (Keys: () supplemented with co-substrate sucrose, sterilized soil plus live

acclimated inoculum (C-DS); () non supplemented, sterilized soil plus live acclimated inoculum

(NC-DS); () supplemented with cosubstrate, live indigenous soil microflora plus live acclimated

inoculum (C-LS); (), not supplemented, live indigenous soil microflora plus live acclimated

inoculum (NC-LS)

3.3. Second experiment

3.3.1. Lindane removal

Lindane removal followed the order A > SR > M (Fig. 4). The ANOVA indicated that there

was a significant effect of factor electron acceptors on removal of lindane (p < 0.0001, Fig. 5).

The order of lindane removal in our work was slightly different to that reported by Robles-Gonzlez

et al. (2012) who found lindane removals in the order SR > A >> M in experiments with lab scale

slurry bioreactors without silicone oil. The difference was possibly due to the positive effect of

silicone oil on oxygen transfer (Daugulis, 2001) in A-SB of our current work. Sucrose

supplementation had a significant effect on the removal of lindane (p < 0.004, Fig 5), particularly in

the anaerobic treatments. Interestingly the increase of lindane removal was lower than expected

probably due to the important amount of indigenous soluble and degradable organic matter in the

soil (3.6 g soluble BOD/kg, Table 1). Interestingly, soluble organic removal efficiencies in sucrose-

supplemented SBs were consistently higher than those of the corresponding non-supplemented units

(methanogenic SB with sucrose, 74%; methanogenic SB without sucrose, 60%; sulphate-reducing


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Fig. 3. GC-MS detection of intermediate metabolites of lindane degradation in aerobic triphasic SB

at 30 day of batch operation (Supplementation with 1 g/L sucrose; sterile soil, bioaugmented withlindane-acclimated inoculum from seed bioreactors)

SB with sucrose, 76%; sulphate-reducing SB without sucrose, 66%; aerobic SB, with sucrose, 78%;

and the aerobic SB without sucrose, 68%.) This was somewhat expected because sucrose is more

easily degradable than natural soluble organic matter of the soil.

The average specific removal rates of lindane rL/S in our best slurry bioreactors were approx.

rL/S = 90 mg removed lindane/((kgsoil)(30 d)) = 3 mg removed lindane/(kgsoil.d) (2)

This value is in the middle to high side of the range of specific removal rates reported in the

literature dealing with bioremediation of soils polluted with lindane (Table 3).

Quintero et al. (2006) observed nearly 100% removal of lindane in anaerobic slurry

bioreactors; their high results could be ascribed to the use of a massive inoculum (8 g VSS/L), the


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Table 3. Treatment of contaminated soil with lindane in slurry bioreactors

Microorganism

External

sources of

carbon and

energy/

Electron

donors/

Electronacceptors

Initial

concentration

of -HCH

MatrixExperimental

conditions

Intermediate

metabolites

Removal

(%)and

removal rate

Ref.

Anaerobic

Granular sludge(8 g VSS/ L)

Starch (2gCOD/L)

100 mg/kgsoil

Soil slurry

(Sandyslime soil)

pH 7

Temp. 30C

350 RPM

V: 4000mL

PCCH

TCCH

1,2,3-TCB

1,3-DCB

CB

100 % in 3 days

33.33mg/kg*d

1

Anaerobic

granular sludge

--------- 4.1 mg/kg soil Soil slurry

(sandyclay

loam)and 2.3%

organicmatter

Temp. 30C

V:30mL

ND 95% in 2 weeks

0.24 mg/kg*d

2

Lindaneacclimated

inocula

(500 mg

VSS/L)

Sucrose/sulphate 100mg/kg Soil slurry(clayish

soil with8%organicmatter)

pH 7

Vt: 100 mL

120 RPM

PCCH

1,2,4-TCB;

1,2,3-TCB;

CB,

B

88% in 30 days

2.93 mg/kg*d

3

Lindane

acclimatedinocula

(500 mgVSS/L)

Sequential

M-SR

100 mg/kg Soil slurry

(clayishsoil with

8%organic

matter)

pH 7

Vt: 100 mL

120 RPM

PCCH,

1,2,4-TCB

98% in 30 days

3.26 mg/kg*d

4

Lindane

acclimatedinocula

(500 mgVSS/L)

Sucrose/CH4 100 mg/kg Soil slurry

(clayishsoil with

8%organic

matter)

pH 7

Vt: 100 mL

120 RPM

NR 47% in 30days

1.57 mg/kg*d

5

Mixed native

microbialpopulation

aclimated

glucose plus

acetate (1:1) atof 630 mg/liter;

SO4 ; HNO3

-HCH400mg/kg

Soil

Slurry

dark at 30C

in rotaryshaker (8 rpm;

amplitude, 20cm)

(30% water;

CB 3,5-DCP,

TCP

6% in 100 days;

0.24 mg/kg*d;nitrifying

insignificanttransformation

6


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Microorganism

External

sources of

carbon and

energy/

Electron

donors/

Electron

acceptors

Initial

concentration

of -HCH

MatrixExperimental

conditions

Intermediate

metabolites

Removal

(%)and

removal rate

Ref.

wt/wt),

acclimatedinocula

Sucrose / SO24 -HCH

100mg/kg

Soil slurry Triphasicreactor: 20%

v/v silicone oil

1,2,4-TCB

1,2-DCB

1,3-DCB

CB

84% in 30 days;2.8mg/kg*d

Thiswork

acclimatedinocula

NC / SO24 -HCH

100mg/kg

Soil slurry

(clayish


Triphasicreactor: 20%

v/v silicone oil

1,2,4-TCB

1,2-DCB

1,3-DCB

CB

78% in 30 days;

2.6 mg/kg*d

Thiswork

Mixed nativemicrobial

populationaclimated

glucose plusacetate (1:1) at

of 630 mg/liter

CH4

-HCH400mg/kg

SoilSlurry

dark at 30Cin rotary

shaker (8 rpm;amplitude, 20cm)

(30% water;wt/wt),

CB 3,5-DCP,TCP

85% in 100 days;

3.4 mg/kg*d.

6

Lindaneacclimatedinocula

Sucrose/

CH4

-HCH

100mg/kg

Soil slurry

(clayishsoil with

8%organicmatter)

Triphasicreactor: 20%v/v silicone oil

1,2-DCB

CB

35 % in 30days;

1.16 mg/kg*d

Thiswork


NC/CH4 -HCH

100mg/kg

Soil slurry(clayishsoil with

8%organicmatter)


1,2-DCB

CB

22% in 30 days;

0.73 mg/kg*d

Thiswork

Aerobic


15/26

Microorganism

External

sources of

carbon and

energy/

Electron

donors/

Electron

acceptors

Initial

concentration

of -HCH

MatrixExperimental

conditions

Intermediate

metabolites

Removal

(%)and

removal rate

Ref.

Pseudomonasaeruginosa

ITRC-5

Without

additionalsource of carbon Isomers of

HCH

2000mg/kg

soil

slurry

rotary shaker

at 28C at200rpm

-PCCH

1,2,4-TCB

CHQ

98% in 15 days

130.67 mg/kg*d 7

Microbacteriumsp. ITCR 1

Isomers ofHCH and the

intermediatesas its solesource ofcarbon and

energy

Soil slurry 2,5 DCP 96% in 28 days

6.85 mg/kg*d

8

Lindane

acclimatedinocula

O2 -HCH

100 mg/L

Soil slurry

(clayishsoil with8%organic

matter)

NR 86% in 30 days

2.86 mg/L*d

5

Lindaneacclimated

inocula

Sucrose/ O2 -HCH

100mg/kg

Soil slurry(clayish

soil with

8%organicmatter))

Sequential M-A with

silicone oil

1,4 DCB

CB

82% in 30 days

2.73 mg/kg*d

9

Lindaneacclimated

inocula

O2 -HCH

100mg/kg

Soil slurry

(clayish


Partially-aerated slurry

bioreactor.


NR 39% in 30 days

1.3 mg/kg*d

5

Mixed nativemicrobial

population

O2 -HCH400mg/kg

Soil slurry dark at 30Cin rotary

shaker (8 rpm;

amplitude, 20cm)

(30% water;

wt/wt),

-PCCH 100% in 100days

4 mg/kg*d

6

Pandoraea

species

O2 -HCH, -HCH

100 mg/ L

Soil slurry

(sandyloam soil)

orbital

incubator (160rpm) at 25, 30,

and 35 C) at7-9pH

NR -HCH 60% in30 days;

2 mg/L*d

10


16/26

Microorganism

External

sources of

carbon and

energy/

Electron

donors/

Electron

acceptors

Initial

concentration

of -HCH

MatrixExperimental

conditions

Intermediate

metabolites

Removal

(%)and

removal rate

Ref.

of each

isomer

Pandoraea

species

O2 150 mg/L Soil slurry

(15%)

(30C,

160rpm)

NR 59.6 and 53.3%

-HCH and -HCH in 9 weeks

11


Sucrose/ O2 -HCH

100mg/kg

Soil slurry

(clayishsoil with8%organic

matter)


1,2,4-TCB

1,2-DCB

1,3-DCB

CB

90% in 30 days

3 mg/kg*d

Thiswork

Lindane

acclimatedinocula

O2 -HCH

100mg/kg

Soil slurry

(clayishsoil with

8%organic

matter)

Triphasic

reactor: 20%v/v silicone oil

1,2,4-TCB

1,2-DCB

1,3-DCB

CB

88% in 30 days

2.9 mg/kg*d

This

work

Notes: -HCH: hexachlorocyclohexane; 1,2,3.TCB:1,2,3-trichorobenzene,1,2,4-TCB:1,2,4-trichlorobenzene; 1,2-

DCB:1,2-dichlorobenzene; 1,3-DCB:1,3-dichlorobenzene; CB: Clorobenzene; COD: chemical oxygen demand; M-SR:methanogenic-sulfate reducing; ND: Not detected; NR: Not reported; PCCH: Pentachlorocyclohexene; UASB: upflow

anaerobic sludge blanket; VSS: Volatile Suspended Solids; TCCH: Tetrachlocyclohexene; THCH: technical grade

hexachlorocyclohexane; References: 1Quintero et al.(2006); 2Baczynski et al. (2010);; 3Robles-Gonzalez et al. (2008);4Camacho-Prez et al. (2010); 5Robles-Gonzalez et al. (2008); 6Bachman et al. (1988) ; 7Manickam et al. (2008) ;8Manickam et al. (2006a) ; 9Camacho-Prez et al. (2010) ; 10Siddique et al., (2002); 11Okeke et al. (2002)

coarse texture of their soil (sandy) where lindane is more easily desorbed and available for further

degradation, as well as the semicontinuous supplementation with organic co-substrate (Table 3).

Bachmann et al. (1988) also reported high removals of -HCH (100%) although after a long lag

time (about 25 days) in methanogenic microcosms loaded with polluted soil (Table 3). In contrast,

Robles-Gonzalez et al. (2012) found that methanogenic slurry bioreactors exhibited a poor

performance with a low 20 to 30% of lindane removal. Our results and those of Robles-Gonzalez et

al. (2012) with M-SB disagree with several works where it was observed that -HCH could be

more easily degraded by anaerobic cultures than aerobic ones (Bachmann et al., 1988; Buser and

Muller, 1995). Okeke et al. (2002) carried out experiments with SB inoculated with the bacterium


17/26

Pandorea sp., with a presumably anaerobic operation of 9 weeks duration. Initial lindane

concentration was 100 mg/kg; they found removals of 59.6 and 53.3% for - and -HCH,

respectively (Table 3). Lindane removals in our A- and SR-SB (90 and 88%, respectively) were

significantly higher and 2.4 times faster than results reported by Okeke et al. (2002).

Quintero et al. (2005) treated a sandy soil polluted with a mixture of isomers , , y -HCH(100 mg/kg each isomers) in anaerobic SBs (Table 3). Starch was supplemented at 2 g/L every 3

days. They observed high removals of nearly 100% for the isomers and of HCH after 60 day

treatment.

a) b)

Fig. 4. Time course of lindane concentration in slurry bioreactors (a) supplemented with sucrose; (b)

without sucrose (Keys: ( ): abiotic control; ( ): methanogenic bioreactor; (): sulfate reducing

bioreactor; (): aerobic units)


18/26

Fig.5. Overall lindane removal efficiency in slurry bioreactors (Keys: black bars supplemented with

sucrose and dotted bars without sucrose. A: aerobic SB; M: methanogenic SB; SR: sulfate-reducing

SB. *Error bars represent the standard error of the experiment SEE= (MSE/r) 1/2)

3.3.2. Intermediate metabolitesMetabolites from lindane degradation found in A- and SR-SBs were chlorobenzene, 1,2

dichlorobenzene, 1,3 dichlorobenzene 1,2,4-trichlorobenzene (Fig. 6 and 7). In M slurry

bioreactors we found chlorobenzene and 1,2- dichlorobenzene (Fig. 8). There was no apparent

accumulation of these metabolites; they are present at trace levels so it is likely that the lindane

molecule was significantly dechlorinated, although lindane removal did not reach 100%. Most

metabolites identified in the final stage of operation of the A-SB are consistent with lindane aerobic

degradation pathways reported by Camacho-Prez et al. (2012) (Table 3).

Several microorganisms such as Pseudomonas sp (Tu, 1975; Sahu et al. 1995) can transform

-HCH to -pentachlorocyclohexene (-PCCH) in aerobic culture.Furthermore, Nagata et al. (1999)

found that the -HCH-dehydrochlorinase (LinA) of Sphingomonas paucimobilis catalyzes the

bioconversion of -HCH to 1,2,4-trichlorobenzene (1,2,4- TCB) via -1,3,4,5,6-pentachloro-

cyclohexene. On the other hand, Quintero et al. (2006) in a study on the degradation of HCH

isomers in anaerobic slurry reactors, found traces of diverse intermediate metabolite, such as

pentachlorocyclohexane isomers, tetrachlorocyclohexene, 1,2,3-trichlorobenzene, 1,3-

dichlorobenzene and clorobenzene. They concluded that low concentrations of the metabolites

indicated that intermediate compounds were not accumulated and they proceed to their further

degradation to CB, the end product in the degradation mechanism.

Interestingly, we could not find PCCH and TCCH in our SBs; it is known that these highly-

substituted metabolites are characteristic of the first stages in aerobic and anoxic pathways of

lindane degradation (Camacho-Prez et al., 2012). This would be consistent with our hypothesis

mentioned above regarding that the lindane molecule was significantly dechlorinated in our SBs,

although lindane removal did not reach 100%. Camacho-Prez (2010; reported also inCamacho-Prez et al., 2012) studied the effect of adding silicone oil (as a desorption-aid) on the

performance of slurry sequential bioreactors treating a heavy soil polluted with lindane (Table 3).

They found that the sequential methanogenic-sulfate-reducing slurry bioreactors without silicone oil

showed the highest lindane removal efficiency 98%. Unexpectedly, in their work the units added

with silicone oil showed a close removal (up to 93%), although slightly lower than that of SB


19/26

without silicone oil. The sulfate-reducing stage of operation contributed the most to lindane

reduction: 41% of lindane was removed in the 15-d methanogenic stage whereas the sulfate-

reducing stage was responsible for 57% of lindane elimination. After 15 d operation PCCH was

detected in the slurries, whereas traces of 1,2,4-trichlorobenzene was present after 30 d incubation.

In another work Robles-Gonzalez (2008) assessed the bioremediation of a heavy soil pollutedwith 100 mg lindane/kg in full (dominant) sulfate-reducing slurry bioreactors with no silicone oil.

Lindane removal was 88% whereas the detected metabolites after 30 d operation were PCCH; 1,2,4-

TCB; 1,2,3-TCB; CB, and benzene.

Fig. 6. GC-MS detection of intermediate metabolites of lindane degradation in aerobic -SB (a) at 15

days and (b) at 30 days of batch operation (Peaks after 55 min retention time are tetradecamethyl-

hexasiloxane, piperidine, 1-(5-trifluoromethyl-2-pyridyl)-4-(1H-pyrrol-1-yl), hexadecamethyl-

Heptasiloxane, 1,1,3,3,5,5,7,7,9,9,11,11,13,13,15,15-hexadecamethyl-octasiloxane, probably from

the chromatographic column phase or silicone oil)


20/26

Fig. 7. GC-MS detection of intermediate metabolites of lindane degradation in sulfate-reducing SB

(a) 15 days and (b) 30 days of batch operation


21/26

600000 -

500000 -

400000 -

300000

200000 -

100000

Abundance

y

x

z

Fig. 8. GC-MS detection of intermediate metabolites of lindane degradation in methanogenic SB at

30 day of batch operation

4. Conclusions

From the first experiment, we can conclude that:

Sucrose supplementation had a slight positive effect on lindane removal from soil with high

contents of clay and organic matter in aerobic, triphasic slurry bioreactors.

Indigenous soil microflora and inocula acclimated to lindane seem to be somewhat

antagonistic regarding lindane removal.

Overall, aerobic triphasic slurry bioreactors bioaugmented with a lindane-acclimated inoculum

and supplemented with sucrose could achieve a fast bioremediation of an agricultural soil with

high contents of clay and organic matter polluted with lindane.

In the second experiment, we also found that

There was a significant effect of factor electron acceptors on removal of lindane (p< 0.0001):

lindane removal followed the order A > SR > M (90%, 84% and 35% with sucrose

supplementation, and 88%, 78 and 21% without sucrose, respectively).

Supplementation with sucrose also had a significant positive effect, particularly in the

anaerobic slurry bioreactors (M- and SR-SB). Interestingly the increase of lindane removal was

lower than expected probably due to the important amounts of soluble and degradable organic

matter in the soil (3.6 g soluble BOD/kg ).


22/26

Main metabolites from lindane degradation were chlorobenzene (CB), 1,2-dichlorobenzene

(1,2-DCB) 1,3-dichlorobenzene (1,3-DCB) and 1,2,4-trichlorobenzene (1,2,4-TCB) in aerobic

and sulfate slurry bioreactors; only CB and 1,2-DCB were found in methanogenic units.

Highly-Cl-substituted metabolites such as PCCH and TCCH were not detected in our SBRs. It is

likely that a high extent of dechlorination occurred for the removed lindane in our work.

Acknowledgements

The authors express their sincere recognition to the Editor and the anonymous Reviewers of the

Journal; their insightful comments and suggestions helped to considerably improve the article. The

authors also wish to thank financial support from CINVESTAV-IPN. The excellent technical help of

Mr. Rafael Hernndez-Vera, MSBiol (GBAER-EBRE, Dept. of Biotechnology and Bioengineering,

CINVESTAV del IPN) Mr. Cirino Rojas and Mr. Gustavo Medina-Mendoza, both BSChemEng

(Central Analtica, ibidem) is sincerely appreciated. Two of the authors (WEV-A and BC-P) wish to

acknowledge graduate scholarships from CONACYT, Mexico. Design-Ease Inc. kindly provided a

free license of its software Design-Expert v8 to HMP-V, which is warmly appreciated.

Nomenclature

1,4 DCB 1,4-dichlorobenzeneA Aerobic

AC Abiotic control

ANOVA Analysis of varianceBOD Biochemical oxygen demandC Supplemented with co-susbtrate

CB Chlorobenzene

C-DS Supplemented with co-substrate, sterile soil and live acclimated inoculumCFU Colony-forming unit

C-LS Supplemented with co-susbtrate, live soil

COD Chemical oxygen demandCSL Concentration of biomass in the sedimented liquor

DS Sterile soil

GC-ECD Gas Chromatography- Electron Capture Detector

HCH HexachlorocyclonexaneHS-SPME Head-space with solid-phase microextraction

LS Live soil

M MethanogenicMSE Mean of the sum of squares of the error

NC Non-supplemented with cosusbtrate

NC-DS Non-supplemented sterile soil and live acclimated inoculumNC-LS Non supplemented, live soil and live acclimated inoculum

PCCH Pentachloro cyclohexene


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POPs Persistent organic pollutants

r Number of replicates

rL/S Average specific removal rate of lindane in slurry bioreactors

SR Sulfate reducingTCCH Tetrachloro cyclohexene

VSL Volume of sedimented liquor

VSS Volatile suspended solids

Greek characters

Alpha coefficient, ratio of alkalinities

lindane Lindane removal efficiency

sulfate Sulfate removal efficiency

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