Chapter 3 Materials and Methods -...

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Chapter 3

Materials and Methods

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3.1. Study area:

Mysore District is the second largest city in Karnataka after Bangalore

and is situated in the southern part of the state. The administrative area of

the Mysore City Corporation (MCC) is around 128 square kilometers. Mysore

is also well known for Dasara festival, a hallmark of the old Kingdom of

Mysore, which occurs during the month of September or October every year.

The average minimum and maximum temperatures vary from 21.4 to 340C

in April, 16.4 to 28.50C in January. Relative humidity ranges from 21 to 84

%. The highest temperature recorded in Mysore was 38.50C on 4 May 2006,

and the lowest was 7.70C on 16 January 2012. The summer season is from

April to May, followed by the rainy season from June to September and the

winter season from December to January (Raman, Afried 1994). The

relevant data about Mysore is given in Table No.3.1.

Table 3.1. Mysore city description

Source: Mysore city action plan 2001

3.2. Municipal Solid Waste Sources and Composition:

According to MCC, the estimated waste generation is around 300 tons

per day. The Maximum amount of MSW generates from the residential area

(212.5 tons per day). Residential area has more amount of MSW because,

Mysore city has a strong geological setting, no natural calamities, cool

Sl.No. City description Position

1 Latitude 120 30' N

2 Longitude 760 65' E

3 Population (2011 Census) 9,20,550

4 Area 128 km2

5 Number of Wards 65

6 Average rainfall per annum 804.2 mm (31.7 inches).

7 Annual Potential Evapotranspiration 1533.5 mm

8 Elevation 763 m (2,503 ft)

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weather and abundant water resources (river Cauvery, Kabini and tube well

water), so that, the area is more suitable for residential purpose. The

minimum waste generated industrial sources is 1.4 tons per day, which may

be due to the non-hazardous waste and hazardous waste can be treated by

the industries themselves (Table 3.2 and Fig. 3.1).

Table 3.2. Municipal Solid Waste generated according to sources

SL.No. Waste source Estimated waste in TPD

Estimated waste in %

1 Residential (Slum & non slum) 212.5 70.62

2 Shops & establishments, schools, etc.

20.5 6.80

3 Hotels, function halls, etc. 26.7 8.87

4 Vegetable Markets, meat shops, etc. 28.8 9.57

5 Hospitals, clinics, etc. 5.9 1.96

6 Industries 1.4 0.46

7 Others 5.1 1.69

Total 300.9 100

212.5

20.526.7

28.85.9

1.4

5.1

300.9

Source wise estimated waste (%) in Mysore city

Residential

Shops & establishments, Schools, etc.

Hotels & Function halls, etc.,

Vegetable markets & meat shops, etc.,

Hospitals & clinics, etc.,

Industries

Others

Total

Fig. 3.1. Source wise generation of Municipal Solid Waste in Mysore city

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The quantity and characteristics of solid waste vary from place

to place. Factors that influence the quantity and composition depends

on the average income level, the sources, population, social behavior,

climate, industrial production and the market for waste materials. The

waste generation and economic levels of the society has been related

to the quantity of domestic solid waste generated from three socio-

economic groups such as, Low Income Group (LIG), Middle Income

Group (MIG) and High Income Group (HIG). A positive correlation

between higher income and waste generation has been noted. The HIG

people throw away more plastic, metallic, glass and hazardous wastes.

The present composition of MSW being generated from the

Mysore city is 22% of paper and wood product wastes, 22% plastic

waste, 5% of food waste, 6 % of textile waste, 10% of garden waste, 8%

of glass waste, 3% of metal waste and other waste is 24%. This result

shows that, the total organic waste composition on an average is 43%

and recyclable material is 33% and other waste is 24% (Table 3.3).

Table 3.3. Physical characteristics of Municipal Solid Waste

Sl. No Physical characteristics Units (%)

1. Wood, Wood products, Pulp, Paper,

Cardboard

22

2. Food, Food Waste, Beverages & Tobacco 5

3. Textile 6

4. Garden yard & Park waste 10

5. Plastic 22

6. Glass 8

7. Metal 3

8. Other waste 24

9. Total 100

Source: Mysore Municipal Corporation Office 2010

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3.3. Municipal Solid Waste Management Practices in

Mysore city

The Mysore city administration has been decentralized in 8

zones. There are 65 wards in the city. Mysore City Corporation (MCC)

takes the initiation for management of solid waste. The MCC and

private sector jointly work for collection, transportation and disposal

of MSW. Around 30 wards are managed by contract to private

contractors, authorized by MCC.

3.3.1. Collection:

The MSW is collected from different sources by various

methods. Two types of dust bins are used. These are cylindrical

bottomless cement concretes of 0.45-0.8 m3

capacity and rectangular

bins with bottoms made of masonry of 2-10 m3

capacity. There are

2879 dustbins placed in Mysore city. Fig.3.2 has shown that,

household waste collection by Pourakarmikas using door to door

collection system and then disposal in masonry bins. Commercial

establishments, Hotel and restaurants waste collection was done by

masonry bins. However, Street Sweeping collection method by

Pourakarmikas sweeps masonry the road and drains and transfers

the waste into small heaps on the road or in to the bins.

The collection of waste from these dustbins is planned in

accordance with the frequency for container becoming full. The

present location of dustbins and the waste collection points have been

classified into daily collection (A type), weekly twice collection (B type)

and weekly once a collection (C type) as part of the Nirmal Nagara

Program. In addition, there are 20 dumper place containers used as

primary collection containers in commercial areas and bulk

generators. The contract involves sweeping of the wards, transfer of

waste to the bins and other collection points.

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3.3.2. Transportation:

MCC and partly by private contractors is managing

transportation of MSW. Out of the 65 wards, private contractors are

responsible for transportation of about 40 to 45% of generating solid

waste, while MCC is responsible for the 50 to 55% of the waste in the

city. Various types of vehicles (tipper, tractor, modern hydraulic

vehicles) are used for transportation of waste. However, the MSW is

stored temporarily in the dustbins and then transported to the

disposal site. This stored garbage lifting is done manually or modern

hydraulic lifting method attached to the vehicle.

3.3.3. Disposal and Composting processes:

A composting plant was set up under the ADB assisted

Karnataka Urban Infrastructure Development Project at

Vidyaranyapuram in the year 2001 to generate compost from the city

refuse, based on the quantity and composition of Municipal solid

waste generated in Mysore city. The plant is located about 6 Km from

the Mysore city and has the area of 12.5 acres. The plant was set up

by IL & FS Environmental Infrastructure and Service Limited

Company, New Delhi. The plant can able to handle 200 to 1000 tons

of waste per month (Table 3.4). The remaining waste is being dumped

beside the Excel plant. The city doesn’t have a sanitary landfill facility

for disposal of MSW at present. Management of biomedical waste

(BMW) is taken care by a private agency that has set up a biomedical

waste processing facility on T.Narasipura road. There is also a small

vermicompost unit in Mysore Zoo and now, another compost

treatment plant is under process in Kumbara Koppal village of Mysore

city. Usually the contraction debris is placed in low-lying areas.

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HouseholdsCommercial

Establishments &

Small Markets

Hotels and

Restaurants

Street

Sweeping

Construction

Debris

Door to door

Collection / Disposal in

RCC & Masonry BinsRCC &

Masonry Bins

RCC &

Masonry BinsPushcarts

Compost FacilityLow Laying Areas

Rejects

Open Dumping

(Adjacent to Compost

Facility)

Transportation TransportationTranspo

rtation

Transportation

Fig.3.2. Schematic representation of MSW Management practices in Mysore city

Table 3.4: Monthly-wise data for MSW received and compost production in Windrow composting treatment plant at

Vidyaranyapuram.

MSW received in tons Compost production in tons

2010-11

2011-12

2012-13

2013-14

2010-11

2011-12

2012-13

2013-14

April 6514 4005 5657 3775 348 467 345 395

May 7113 4497 5961 4132 259 22 181 307

June 6547 4970 5614 4307 186 181 401 208

July 6588 6899 3898 4648 169 479 275 506

August 6560 6542 4281 4194 201 758 452 492

September 5949 5663 5164 3817 249 702 413 512

October 5389 5945 5643 4207 239 543 261 408

November 6362 5943 4654 3915 92 514 316 446.

December 4900 5975 3944 4001 475 803 617 526

January 4174 5704 3528 3960 531 791 677 691

February 4207 5279 3599 3813 525 863 774 808

March 5015 5645 3922 4063 920 810 917 771

Total in tons 69318 67067 55865 48832 4194 6933 5629 6074

Source: Mysore City Corporation Office (2014)

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Solid waste treatment/ processing plant:

Fresh Municipal Solid Waste received 200 MT/day

Formation of windrows

4-6 meter width and 3 meter height

Spraying Cowdung’s slurry and culture

Windrows mixing once in a week

(5 times mixing up to 40 days)

After 50 days decomposed MSW Screening Process

More than 65 to 35 mm thickness materials was screening

through rotary screen drum

20 mm thickness material was screened

4 mm thickness material was screened

Fine grade compost enrichment with additives

rejects sent to landfill site

Packing, Loading and dispatch through Lorries/trucks

Source: Mysore City Corporation Office (2014)

Fig. 3.3. Flow Chart: Solid Waste Processing

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In the first step, waste was processed in open windrows of about

4-6 m width and 2-3 m height of the compost pad. The compost pad is

a concrete platform on which waste is kept and allowed to undergo

decomposition and spraying water for up to 7 days. It helps to avoid

seepage of leachate into the groundwater and also to prevent mixing of

earth/inerts with the waste. Later culture with cow dung’s slurry will

be added to the bacterial and fungus formation which starts the

decomposition, the culture was added based on the quantity of the

solid waste (Fig.3.4). The composting of municipal solid waste

complete cycle takes 60 days. However, materials were left for 50 days

for complete decomposing and these windrows are periodically mixed

and shifted once a week using JCBs for aeration and temperature

control to enable aerobic decomposition of waste. At the same time,

fresh stack culture with cow dung’s slurry is sprayed. On the 60th day,

these materials are dried and put into the hydraulic system through

conveyor belt with the help of a JCB and a device known as Bobcat. In

the Bobcat and convey belt 3 steps of the segregation process are

carried out. In the first process, the material with more than 65 to 35

mm thickness (like plastics, bottle and glass) are separated through

rotary screen drum, later it passes through second sieve drum which

separates the secondary process. The 20 mm materials are moved

through the 1.7 Bucket Elevator. By the Destonar machinery, the

small stones are separated which contain the air blower. Screening

through 4 mm sieves fine grade, stored for some days and finally

packed and sold (Fig. 3.3).

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One liter of Mother Culture + One Kg Jaggery + 48 Liter Water

Filter mix and closed

One week

pH checked (3-4) range ready to use

Diluted

50 liters culture + 450 liters water added

Can be used for 10 to 15 tons waste

Fig. 3.4: Flowchart of culture preparation

3.4. Sampling sites:

The research has been carried out by collecting the samples

from solid waste treatment plant that is from aerobic Windrow

composting plant set up by Vidyaranyapuram, Mysore (Fig.3.5).

Fig. 3.5: Open Windrow compost sampling site (Vidyaranyapuram, Mysore).

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Fig 3.6. Windrow composting (initial and final product)

3.4.1. Open Windrow compost plant and sample collection

method:

MSW compost samples (10, 20, 30, 40, 50 and 60 days old

compost samples) of different degradations stages were collected from

open windrow composting treatment plant, during summer, rainy and

winter seasons (2010, 2011, 2012). These samples were analysed for

physico-chemical, biological characteristics and heavy metals

concentrations through seasonal variations studies.

For compost stability and maturity study, samples were

collected during 2012-13. The comparative studies of nutritional

status of MSW and agricultural compost samples study purpose, the

agricultural compost were collected during 2013 (Hosur, K.R. Nagar

Taluk, Mysore District).

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At each heap, five compost samples were taken randomly within

a 0.5 meter quadrat and mixed into a composite sample representative

of that particular heap. The physico-chemical, biological

characteristics and heavy metals concentrations analysis were carried

out as per standard methodology (Jackson 1973; Mani, et.al., 2007

and Saha Arun Kumar 2008).

3.5. Methods of analysis:

3.5.1. Test of physico-chemical characteristics and heavy metals

concentrations:

The physico-chemical characteristics and heavy metals

concentrations at different degradation stage compost samples were

transported in sealed aluminium foil to the laboratory

(http://compost.tamu.edu/docs/pubs/compost_sample_guideline.

pdf).

The segregation of stones, plastic, etc., were carried out

manually. Further, the samples were dried using laboratory oven at

700C or sunlight and powdered and passed through 2 mm mesh

sieves to obtain a uniform powder. All experiments were carried out in

triplicates and the average value was presented.

Table 3.5. Method for physico-chemical characteristics and heavy

metals concentrations

Sl.No Characteristics Methodology

1. Thermometer Thermometer (Manivaskam, 1996)

2. Moisture content

Gravimetric method (CPCB-Manual for analysis of MSW 2002)

3. Particle density Pycnometric method (Saha Arun Kumar 2008)

4. Bulk density Pycnometric method (Saha Arun Kumar 2008)

5. pH pH meter (CPCB-Manual for analysis of MSW 2002)

6. Electrical Conductance

Conductivity meter (CPCB-Manual for analysis of MSW 2002)

7. Total Water Soluble Solids

Gravimetric method (CPCB-Manual for analysis of MSW 2002)

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8. Calcium Ammonium acetate extracts with EDTA titrimetric method (Heald, 1973).

9. Magnesium Ammonium acetate extracts with EDTA titrimetric method (Heald, 1973)

10. Chlorides Argentometric method (APHA 1976)

11. Calcium carbonate

Titration method (IS: 2720 Part XXIII, 1976) Reaffirmed 2001.

12. Total organic carbon

Walkely and Black 1934, method (Jackson, M.L. 1967)

13. Organic matter The organic matter content was calculated by determining Total Organic Carbon (TOC) using conversion factor 1.724 (Sahlemedhin and Taye, 2000)

OM=TOC X 1.724

14. Total nitrogen Kjeldhal method (Mani, et.al., 2007).

15. Nitrate nitrogen Brucine method (Trivedy and Goel, 1986)

16. Ammonia nitrogen

Nessler’s reagent method (Trivedy and Goel, 1986)

17. Soluble sulphates

BaCl2 method (IS: 2720 (Part XX VII 1977), (Reaffirmed 1995) and (CPCB-Manual for analysis of MSW 2002)

18. Total Phosphorus

Vanadomolybdate yellow color method

(Mani, et.al., 2007).

19. Sodium Flame photometric method (APHA 1976)

20. Potassium Flame photometric method (APHA 1976)

21. C/N ratio The carbon-nitrogen ratio of the compost was determined from the ratio of total organic carbon to total nitrogen (Martin,1991)

22. Copper Triacid digestion method (CPCB-Manual for analysis of MSW 2002)

23. Cadmium Triacid digestion method (CPCB-Manual for analysis of MSW 2002)

24. Lead Triacid digestion method (CPCB-Manual for analysis of MSW 2002)

25. Mercury Triacid digestion method (CPCB-Manual for analysis of MSW 2002)

26. Arsenic Triacid digestion method (CPCB-Manual for analysis of MSW 2002)

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3.5.2. Test of bacteriological characteristics:

The samples were collected in a separate sterilized polyethene

bag for biological characteristics and the media used for the

bacteriological study of compost includes Nutrient Agar (NA), Plate

Count Agar (PCA) and Eosin Methylene Blue agar (EMB) etc. 1g of

each compost sample was suspended in 10 ml of sterile saline

solution (0.9 g NaCl in 100 ml of distilled water) and serially diluted

further up to 10-10 dilution aseptically. 0.1 ml of each diluted compost

suspension was poured into Nutrient and Plate count agar plate using

the spread plate technique. Plates were incubated for 24 hr at 370C.

The evaluation of bacterial diversity in a compost sample was

determined by plate count by serial dilutions method, with the

equation given below.

CFU/g = Colonies Numbers X dilution /100

The number of mesophilic bacteria determined by dilution plate

count technique (Hernesmaa, et.al., 2008).

After the bacterial diversity calculation, selected pure bacterial

colonies were subjected to phenotypic characterizations and

biochemical tests. These phenotypic characterizations were compared

to phenotypic data of known organisms described in the Bergey’s

Manual of Systematic Bacteriology (Buchanan and Gibbons 1986).

Biochemical tests:

A number of biochemical tests and gram staining were

performed for the identification of bacterial isolates with the help of

Bergey’s Manual (Buchanan and Gibbons 1986). The principal tests

used for this purpose were Catalase Test (CAT), Oxidase Test (OXT),

Indole Test (INT), Methyl Red Test (MRT), Voges-Proskauer Test (VPT),

Citrate Utilization Test (CUT), Urease Test (UT), Nitrate Reduction Test

(NRT), Hydrogen Sulphide Production (H2S), Starch Hydrolysis Test

(SHT) and Gelatine Hydrolysis Test (GHT).

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Catalase test: This test was performed by adding a small amount of

bacterial isolate into freshly prepared 3% hydrogen peroxide and if the

bubbles of oxygen appeared, the isolate was considered as positive for

catalase test.

Oxidase test: Oxidase test was used to assess the bacteria which

produce the enzyme Cytochrome Oxidase. Trypticase soy agar was

inoculated and incubated the plates in an inverted position for 24 to

48 hours at 370C. After incubation, a few drops of 1% tetramethyl-p-

phenylenediamine dihydrochloride were added. A positive result was

the development of purple color. No color change indicated a negative

result.

Indole test: The Indole test was performed by culturing the

microorganisms in peptone water medium containing tryptophan in a

screw capped tube, incubated for 24 h at 370C and then Kovac’s

reagent (0.5 ml) was added, where, the positive results was indicated

by the formation of pink red layer on the broth within seconds of

adding Kovac’s reagent.

Methyl red test: Methyl red test was performed by inoculation of the

glucose phosphate peptone water in a screw capped tube, incubation,

for 24 to 48 h and then addition of 5 drops of methyl red, where the

change in color of the medium to cherry red was considered as

positive.

Voges-Proskauer test: This test was performed by inoculating glucose

phosphate peptone water with the microbial isolates in a screw

capped tube, incubating for 24-48 h, then adding 0.6 ml of alpha-

naphthol solution and 0.2 ml of Potassium hydroxide solution

(Barritti’s reagent). The tubes were then allowed to stay for 5-10 min

after shaking well. The red color formation was taken as the positive

result.

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Citrate utilization test: For the Citrate utilization test, the Simons

citrate agar was used and incubated at 370C for 24-48 h. The positive

slants were noted to change color from green to blue.

Urease test: This test, urea broth was inoculated and incubated at

370C for 24 to 48 h. The change of color of the broth from yellow-

orange to bright pink was considered as positive.

Nitrate reduction test: The samples were inoculated in nitrate broth

incubated at 370C for 24-48 h. After incubation, 5 drops of Sulfanilic

acid and 5 drops of N, N -dimethyl-1-naphthylamine were added. The

change of color of the broth to deep red within 5 min, show that, the

bacteria had produced nitrate reductase. If color did not change, the

result was indecisive. Small amount of Zinc was added to the broth. If

the solution remains colorless, then both nitrate reductase and nitrite

reductase are present. If the solution turns red, nitrate reductase was

not present.

Hydrogen sulphide production test: The Hydrogen sulphide

production test was used to differentiate species of the family

Enterobacteriaceae. Sulfide-Indole-Motility (SIM) media was used for

the H2S production test. SIM media was inoculated with bacterial

cultures by stabbing SIM media with inoculating needle. The tubes

were then incubated at 350C for 24 h. After incubation, a positive

result was indicated by a black precipitate formation because of the

reaction of H2S with the iron or ferrous sulfate; while the negative

result was indicated by without black precipitate.

Starch hydrolysis test: The starch hydrolysis test was used to

differentiate bacteria based on their ability to hydrolyze starch with

the enzyme alpha-amylase or Oligo-l, 6-glucosidase. Starch agar was

used to inoculate and incubated the plates in an inverted position for

48 hours at 350C and then iodine 0.5 ml was used to detect the

presence or absence of starch in the vicinity around the bacterial

growth. A positive result was the development of a blue or dark brown

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color; therefore, any microbial starch hydrolysis will be revealed as a

clear zone surrounding the growth.

Gelatine hydrolysis test: The gelatine hydrolysis test was used to

determine the ability of microbes to produce gelatinases. The presence

of gelatinases can be detected using nutrient gelatine and the tubes

were then incubated at 250C for one week. After incubation, a positive

result indicated solid form of gelatine to liquid form because of the

gelatinases-positive organism. secreted by gelatinase. The negative

results have that, the solid form of gelatine media remains as solid

only. Tables 3.6 & 3.7 shows the morphological and biochemical

characteristics.

Table 3.6. Morphological characteristics of isolates

Isolate Morphological Characteristics Organism

W1

Gram negative, circular, low convex, with

entire margin, mucoid, opaque, small, non

endospores forming rod shaped, pinkish

glittering with a metallic shine colony on EMB

Agar; grown at pH 7, 370C to 450C.

Escherichia

coli

W2 Gram negative rods on Nutrient Agar. Klebsiella sp.

W3 Spore forming, gram positive rods, a creamy

white colony on Nutrient Agar with entire

margin.

Bacillus sp.,

W4 Dark centered, gram negative, non endo-spores

forming colony on Salmonella-Shigella Agar.

Salmonella sp.,

W5

Non spore forming and non-motile gram negative rod colony on Nutrient Agar that

appeared translucent and feather-like margins.

Proteus sp.,

W6 Gram negative rods that appeared yellowish

with entire margin on Nutrient Agar.

Flavobacterium

sp.,

W7 Gram negative, non endospores forming rods,

light-yellow colony with feather like margin.

Enterobacter

aerogenes

W8 Nonspore forming, Gram negative short rods,

colorless colony on Nutrient Agar, grown at 40C

and 420C.

Pseudomonas

aerugionosa

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Table 3.7. Biochemical characteristics of isolates

Biochemical Test W1 W2 W3 W4 W5 W6 W7 W8

Catalase Test (CAT) + + + + + + - -

Oxidase Test (OXT) - - - + - - - -

Indole Test (INT) + - - + + - - -

Methyl Red Test (MRT) + - - - + + (+) -

Voges-Proskauer Test (VPT) - + + + - + + +

Citrate Utilization Test (CUT) - + - + - - + +

Urease Test (UT) - + - - + - + -

Hydrogen Sulphide Production (H2S)

ND - + + - - - +

Starch Hydrolysis Test (SHT) - + - - - - - -

Gelatine Hydrolysis Test (GHT) - (+) + + + + (+) +

Nitrate Reduction Test (NRT) + + + + + (+) + -

W1 = Escherichia coli W2 = Klebsiella sp.,

W3 = Bacillus sp,. W4 = Salmonella sp., W5 = Proteus sp,. W6 = Flavobacterium sp.,

W7 = Enterobacter aerogenes W8 = Pseudomonas aerugionosa,

ND = Non- detected (+): Postive (-): Negitive

3.5.3. Test of fungal characteristics:

The samples were collected in a separate sterilized polythene

bag for fungal characteristics and the media used includes Potato

Dextrose Agar (PDA) and Czapek Dox Agar (CDA). 1g of each compost

sample was suspended in 10 ml of sterile saline solution and serially

diluted further up to 10-10 dilution aseptically. 0.1 ml of each diluted

compost suspension was poured into Czapek Dox Agar and Potato

Dextrose Agar plate using the spread plate technique. Plates were

incubated for 7 days at 370C and fungal colonies were isolated and

further pure cultures were subjected to various morphological studies

using a Sterezoom microscope (Buchanan, et.al., 1974 and Dubey

et.al., 2002). The evaluation of cellular concentrations (fungal

diversity) in a compost sample was determined by plate count by

serial dilutions method, with the equation given below:

CFU/g = Colonies Numbers X dilution/100 (Hernesmaa, et.al., 2008).

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Fig 3.7. Biochemical Tests

71

Fig 3.8. Biochemical Tests for bacteriological analysis

72

Identification of fungal morphology by staining:

Each fungal colony was picked up on the 7th day of culture

incubation, placed onto a glass slide, stained with a few drops of

cotton blue dye (6 µg/ml) by spreading the sporulated fungi with the

help of a sterile needle. The stained material was covered with a glass

cover slip and visualized under the compound microscope by the

LYNX Trinocular Stereozoom microscope model LM-52-3622 and

studied the morphological characteristics of the fungi.

Fig 3.9. Fungal colonies on Czapek Dox Agar (CDA)

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3.5.4. Test of compost stability:

Jar test (Odour development): 500 gm of compost sample was added

into enough water to make the compost sample moist. The sample

was placed in a sealed plastic bag and kept for a week at room

temperature (Trautmann, Richard and Krasny, 1996).

Self-heating test (Heat production): A gallon-size container was

filled with compost having 40 to 50% moisture content and the

container was sealed and insulated with insulating material

(Trautmann, Richard and Krasny, 1996). Record the temperature of

the compost sample.

3.5.5. Test of compost maturity test:

Germinations bioassay test:

During the test, about 10 g of powered and dried (room temperature

condition around 250C for 24 hours) compost sample was taken in a

beaker. 50 ml distilled water was added and stirred the contents for

one hour (1:5 ratio, 1 part sample and 5 parts distilled water) and

allowed to settle approximately for 20 minutes. The contents were

centrifuged to obtain aqueous extracts, further they were filtered

through a double layered muslin cloth and the extracts were called as

100% full strength. At the same time 10times dilution samples were

prepared using 1 ml of 100% full strength sample was diluted with 10

ml of distilled water. Both these samples were used as germination

media. 8 Fenugreek (Trigonella foenum graecum) seeds were placed on

a Whatman filter paper (Number 42) which is placed inside a 9 cm

diameter sterilized, disposable Petri dish, wetted with 1 ml of each

germination solution. Distilled water was used as a control and five

replicates were kept for each treatment. The Petri dishes were placed

in sealed plastic bag to minimize water loss while allowing air

penetration and were then kept in the dark for 24 hours at a steady

warm temperature of 270C. After the incubation period, number of

germinated seeds and the primary radical length were measured and

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expressed as a percentage of the control (germination index). Data

were analyzed statistically against compost age and concentration of

organic carbon in the growing media. The germination index (GI) was

calculated using the following equation (Zucconi et.al., 1981).

%G × %L GI= 10,000

Where, %G = percent germination, %L = percent radical length

The same procedures were used for compost stability and

maturity study during the windrow composting process and

comparative study on nutritional status for different compost samples.

Plant bioassay test:

A) Compost stability and maturity study during windrow composting process:

The experiments can be performed indoors using pots. The

experiment designed to include the various combinations of municipal

solid waste compost and soil, as shown in Table 3.8 which was carried

out for 21 days. Initially, the Fenugreek seeds were planted in the

specific location of the pot and watered regularly (10 Fenugreek seeds

were used in each of the steps). The pots were properly maintained to

minimize the variations in temperature, light and other environmental

factors. Data was recorded daily for the number of seeds germinated;

the growth pattern of the seeds and the health indicators like color,

diseases, etc. The indicators of healthy plants is manily due to the

growth of the plants shoot and root systems.

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Table 3.8: The various treatment combinations of municipal solid waste compost and soil for plant bioassay test

Sampling days

Composition Sample

code

Sampling

days Composition

Sample

code

Soil 100% Soil S1-100%

10

100% MSW sample

D1-100%

40

100% MSW sample

D4-100%

75% MSW sample + 25% Soil

D1-75% 75% MSW sample

+ 25% Soil D

4-75%



+ 50% Soil D

4-50%



+ 75% Soil D

4-25%

20

100% MSW sample

D2-100%

50

100% MSW sample

D5-100%



+ 25% Soil D

5-75%



+ 50% Soil D

5-50%



+ 75% Soil D

5-25%

30

100% MSW sample

D3-100%

60

100% MSW sample

D6-100%



+ 25% Soil D

6-75%



+ 50% Soil D

6-50%



+ 75% Soil D

6-25%

B) Comparative study on nutritional status for different compost

samples:

In this study, final stage MSW and agricultural compost samples

were collected during 2013.

For plant bioassay, comparative study on nutritional status of

MSW and agricultural compost samples analysis using plot method

and the treatments as given below (Table. 3.9).

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Table 3.9: The different compost samples with soil combination of plant bioassay tests

Table 3.10. Instruments and Equipments:

Instruments Model Name

Thermometer 0.0 to 1000C HERMES Brand Mercury thermometer was used to record the temperature of water at sampling sites expressed in0C

pH meter Elico LI 613 pH meter

Conductivity meter Systronics Conductivity meter 304 model

Spectrophotometer Systronics Flame Photometer 118 model

Flame Photometer Systronics Flame Photometer 128 model

Atomic Absorption

Spectrophotometer

GBC Avanta Ver 1.31

Miscellaneous

Equipment’s

Whatman filter paper 42, musline cloth, Pre-calibrated accurate pipette, burettes and measuring cylinder was employed for volumetric measurements.

Name of the sampling

Composition Remarks

Control 100% Soil

(20 Fenugreek seeds were used)

After 7th week in each treatment,

the 10 plants were picked and

analysed for the test like root,

shoot, total length, No. of leaves,

no. of flowers and no. of pods of the

plant. At, the same time, fresh and

dry weight of the plant root, shoot

and leaves, pods weight were

calculated (mean values).

MSW compost sample

(Final stage)

25% of MSW

compost sample +75% Soil (20 Fenugreek seeds were used)

Agricultural compost sample

(Final stage)

25% of Agricultural compost sample + 75% soil (20 Fenugreek seeds were used).

Chapter 3 Materials and Methods -...

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