Chapter - I1

MATERIALS AND METHODS

COLLECTION OF STUDY MATERIALS

Coirpith

Coir pith is separated as waste in a colr fibre manufacturing industry Coir pith

as an agro-industrial organic waste gets generated in bulk quantcties and gets accumu-

lated in industrial yards causing environmental pollutioti besides insanitary conditions

Coir pith is collected from coir fibre industry located In Anyankuppan Pondicheny,

South India

Coffee husk

Coffee husk IS the main byproduct of the coffee pulp industry Usually coffee

husk is dumped In large open areas or near water bodies, causlng soil and water

contamlnatlon Coffee husk IS collected from TATA Coffee lnd~a (P) Ltd , Coorg,

Karnataka. South India

Collection of earthworms

Different specles of earthworms are collected from d~fferent sampl~ng sites

(Figure 7) of Pondicherry reglon The collect~on was done In the followng hab~tats-

paddy field. coconut field. municipal solid waste dumped area, groundnut field, poultry

waste dumped area. vermiculturing area, near fresh water bodies, sewage water canals,

and tndustr~al area Collection was done by hand sorting method (Nelson and

Satchell. 1962) From among the ten species of earthworms ~dentcfied from Pondicheq

(Sathyanaray- and Anisa, in press) four spectes are employed (Plate.1, a,b,c &d)

for the recycling ofboth coir pith and coffee husk They are I~rnprro maurrtrr (Platc1,aX

Eudr~lus eugeniae (phtcl ,b). pertonyx excavatus (Pla te. 1 ,c), atd (krc~haetom serrata

(Plate. 1 ,d).

Figure 'l: Map showing eoUeetion sita of earthworm specka in Pondkherry region.

Lu-Long 12"N - 7PE, on the Eui Collt of I d a '' Ucntionof Ism rbovethemakud

Msm Daily Tomparturr 25% to 32% Awn@ mnul ramfill l27an

'0 M4paottosuk

Collection sltes u w m

Collection and culturing of microbes

Microorganisms like Pseudomonasfluorescens, Bacrilus subtrlres, Trrchoderma

vrrrde, Actmomycetes-G2, Rhrzopus stolonrfer and Rhrzocotonra solanr were

collected from the department ofBotany, Kanchi Mamuniver Centre for Porn Graduate

studies, Pondicherry Anaerobic bactena (Sugar mill anaerobic waste water treatment

plant sludge) is obtaned from ELD- Pany, Nellikuppam, Cuddalore, South India

Culturing of microbes

Five mlcrob~al lnoculants - Pseudomonas, Bacrllus sp , lirchoderma.

Aclrnomycetes and Anaerobic bactena are used for compost expenments The compo-

sctlon of the microbial media used for the culturing of the selected m~crobes IS pre-

sented below

Pseudomonas /Bacillus sp (Plate.2, a) culture:- log of peptone ,lSg of sod~um

chlonde, and Sg of yeast extract dlssolved In 800ml of distilled water. 1s adjusted to pH-

7 to 7 2 at 25" C and made upto 1000 ml wlth distilled water Th~s media was then

sterilized, cooled and inoculated with the Pseudomonas Bacillus Culturing was

carried out for 3 days in a shaker Duplicates of culture media IS prepared

Trichoderm viridiae (Plate 2, b): To prepare the culture medium 1 g of Potasslum

dl-hydrogen phosphate. 2g of Sod~um n~trate. 0 Sg of Magnesium sulphate. 0 Sg of

Potasslum chlonde, 0 Olg of Ferrous sulphate and 30g of Sucrose . were dlssolved In

one litre of distilled water

Actinomycetccl -G2 (Plate 2, C) log of Starch, 2g of Potassium nltrate. 2g of Sod~um

chloride, 2g of Di- potassium hydrogen phosphate. 0 0Sg of Magnesium sulphate.

0 02g of Calcium carbonate, 0 Olg of Ferrous sulphate, and 0 3g of Casein are

dlssolved in one litre of distilled water for the preparation of the culture media

(8

) Lam

pilo

man

riti

i (b

) En

dri

lus

enge

niae

(c)

Orr

och

aer~

~n

a .ser

ra~

a (d

) Pe

rir~

n.~

.~

ercu

~~

acu

s

For Anaerobic bacteria (Plate.2, d): The sludge was obtained from the anaerobic

bio-gas plant of EID-Parry. Cuddalore, South India

DESIGNING OF VERMICOMPOST AND COMPOST REACTORS

Designing and setting up of verrnlreactor is shown in Figure 8(a) and

Plate 3(A) Recycling of coir pith / coffee husk IS done with 4 species of earthworms

providing different feed ratios separately in these reactors For wir p~th the feed ratios

Introduced into the reactors (in duplicates) are 100% cowdung (CD), 3 1 cowdung

colr pith ( I 3 CD CP). 1 1 cowdung co~rpi th ( 1 1 CD CP), 1 3 cowdung

coir pith (1 3 CD CP) and 100% colr pith (CP) This is presented in Plate 3(A)

For coffee husk the feed ratlos introduced are 100% wwdung (100% CD),

3 1 cowdung coffee husk (3 1 CD CH). I 1 cowdung coffee husk ( I 1 CD CH),

1 3 cowdung coffee husk ( 1 3 CD C H ) and 100% coffee husk (100% CH)

Plate 3(A) presents the reactors (in duplicate) each with these different feed

combinations

Figure $(a): Des~gn of Vermicompost reactor

3 cm Garden soil + 20 numbers of Earthworm + I OOg feed

Nylon mesh

2 cm Sand

/ I \

COMPOST REACTOR

Cornposting materials i.e, coffee husk I coir pith were filled in nylon bags

marked A-I. Duplicates run were marked A,-I,. The materials for composting

(coir pithlcoffee husk) and the inoculants are arranged in alternate layers as shown

in Figure 8(b).

Fipre B(b): Design of Compost reactor

, I cm sand

I Layer 6 I The microbial inoculants into each of the nylon bags (compost reactors, A-I and A,-I,)

are mentioned below:

A&AI -Pseudomo~sJuorescem.

B&B 1 -Bacillus subnlls

CCC1-Trichoderma viride,

DCD1- Actinomycetes G2

E&E I -Anaerobic bacteria,

F&F 1 -Control

GCGI -Pseudomonar sp.,+Bacillus sp.,

HBHI-7kichodPrma sp.,+Actinornycetes G2

I& 11-Pseudomonus sp.,+BaciNus sp+Trichoderm sp.,+ Actinomycetes G2

BLACK GRAM SEED MATERIAL

The seeds of Y i g ~ mungo (L.) Hepper cv. T-9 w e n obtained from Krishi

b y a n Kendra (KVK), Pondicheq, South India 35

PETRIDISH EXPERLMENT

Black gram seeds were kept in 1 percent HgCl solution for two minutes

and washed thoroughly under running water Nine types of vermicasts i .e ,

1 0 0 % C D V C , 3 I C D C P V C . I I C D C P V C , I 3 C D CPVC, lOO%CPVC,

100%CHVC, 3 ICD CHVC,I ICD CHVC and 1 3CD CHVC were each (10ym)

dissolved In IOOml of water and kept in petridishes, into which 10 seedslper

each petridish were Introduced A control was also maintained with farmyard

manure (as also used in pot study) All these experlments were run In duplicate

The expermental set up is as shown in Table 3. The germlnatlon of black gram

seeds in each petridish was observed for elght days lnlt~atlon of germination was

noted after 48 hours On the fourth and elghth day, growth parameters like number

of lateral roots, length of root and shoot were measured and fresh and dry weight of

the root and shoot were taken Chlorophyll-a & b, total chlorophyll and carotenoid

content of prlrnary leaves were also est~rnated on the 4'h and 8 I h day o f the

experiment The experlments were repeated thr~ce and the results were subjected

to s ta t~st~cal analysis as well

POT STUDY EXPEFUMENT

Pot study expenrnent was camed out from December 2001 to March 2002

Clay loamy soil is used for pot study experlments For each type of vermtcasts,

two pots were used Verm~cast of each type was mlxed w~th the garden so11 thoroughly

separately before filling into respective pots For the control. pot farmyard manure

(FYM) was added to the soil and rntxed thoroughly The seeds were sown at a depth of

2crn and at a distance of 5crn to each Totally 10 seeds were sown In each pot

Each pot was imgated with one litre water extract of the same type of vermicast

(100% CD/CP/CH, 3 1 CD CPICH, 1 I CD CPICH, 1 3 CD CPICH) The pots

were irrigated at an interval of every three days

DESIGN OF THE PETRIDISHPOT STUDY EXPERIMENT OF

THE BLACKGRAM Rgna mungo (L ) Hepper cv T-9

Petridish Experiment

Expenment penod Bdays

Petridish size 9 cm diameter

Number of petrldishes IO(5x2)

Number of seedsipetndish Ten seeds

Crop under study C b a mlmngo (L ) Hepper cv T-9

Treatment Petnd~shes and pots were labelled as glven in table 3 and the

treatment provided IS llsted below (Tab1 3)

Table 3: Set up of Petndish I Pot study expenment wlth Blackgram Irrgna mungo (L ) Hepper cv T-9

VC-Vermicast. CD-Cowdung. CP-Coir pith. CH-Coffee husk

Treatments Control

100% CDVC 3 I CD CPVC 1 1 CD CPVC 1 3 CD CPVC 100% CPVC 3 1 CD CHVC 1 1 CD CHVC 1 3 CD CHVC 100% CHVC

SI No I 2 3 4 5 6 7 8 9 10

Petr~d~shPot study VC-00 VC-OI VC-02 VC-03 VC-04 VC-05 VC-06 VC-07 VC-08 VC-09

Pot study experiment

Experiment period 3 months,

Pot size 5 Lit ,

Number of Pots 10 pots (5x2)

Number of plantslpot 10 plants

Crop under study Kgna mungo (L ) Hepper cv T-9

Treatment As per Petridish experiment

PHYSICO-CHEMICAL ANALYSIS OF

VERMICASTICOMPOSTICO1R PITWCOFFEE HUSK SAMPLES

The physic0 chemlcal parameters such as pH, Electrical conductivity,

total organic carbon, total organlc mtrogen, C N ratro, ava~lable nitrogen, phosphorus

and potasslum were determined by the following standard methods outllned

pH

pH IS a notation used for expressing the ac~d~ty or alkaliluty of a solution of

so11 sample The pH meter electrode when In contact wlth H+ ions of the solution

under test acquires an electrode potential which depends on the concentration

of H+ Ions pH of the sample IS expressed In pH units (Gupta,ZOM))

Reagents

1 Buffer solution pH - 4 0

2 Buffer solution pH - 7 0

3 Buffer solution pH - 9 2

Procedure:

Each buffer tablet is dissolved in lOOml of distilled water to obta~n three

buffer solutions This buffer solution is used to standardize the pH electrode

The samples of compost and vermicompost were dissolved separately in distilled

water in the ratio of 1 5 (WIV) and the pH of the samples was recorded w ~ t h an

electrical pH meter

ELECTRICAL CONDUCTIVIm

Electrical conductlvity was studied uslng electr~cal conduc t~v~ ty meter

model- TDS Scan-3 wlth ATC Eutech Cyber nets Electrical conductivity IS measured

In terms of the resistance offered to the flow of current uslng a conductivity

bridge (Gupta, 2000) An aqueous solut~on conta~ning Ions will conduct an

electric current

Reagents

Standard solut~on 0 01N KC1 solutlon

Procedure

The conduct~vlty meter IS checked with standard solution of 1 413m mohdcm

of KCL solution before proceeding w ~ t h the sample testlng Dr~ed vermlcast and

compost samples weighing 10g each were dissolved in 50ml of distilled water by

constant stirring This solution IS used to study conductlvity with the help of an

electrical conductivity meter

ESTIMATlON OF TOTAL ORGANlC CARBON (TOC)

Principle

The carbon in the organic matter gets oxidized in a mixture of YCr,O, + %SO,

and is converted to CO, The unused potassium dichromate of the oxidation reaction

was estimated by back titration against standard ferrous ammonium sulphate solution

(Gupta, 2000)

Preparation of reagents

I . Potassium dichromate solution: 12 26g of the K2Cr2 0 7 is dried at

105" C for 2 hours dissolved in 250ml of dlstilled water and then made

upto one litre wlth distilled water

2. Ferrous ammonium sulphate (FAS) solution 196 Ig of FAS is

dissolved In 800ml of distilled water and then 20ml of concentrated

sulfunc acid 1s added and made upto one litre with distilled water

3. D~phenyl amine Indicator 0 5g of diphenyl amne is dissolved in 20ml

of dlstilled water and IOOml of concentrated sulfurlc ac~d

4. 96% concentrated sulfunc acid

5. 85% Onho phosphoricac~d.

Procedure

1g of compost I vermicompost sample is taken separately In a 500ml

conical flask lOml of I N K,Cr:O, 1s added. shaken well and keeping the flask on

an asbestos sheet, 20mi of conc &SO, IS added and mlxed well After 30 minutes,

200ml of distilled water, lOml of 85% orthophosphor~c acld, and one ml of

dlphenylarnine indicator are added It is then tltrated against ferrous ammonium

sulphate till the blue colour turns green A blank 1s run s~multaneouslv

Calculation

Percentage of organlc carbon = [IO(B-T) / B] X [0 003 X 100 1 wt (g)]

B - Vol of ferrous ammomum sulphate for blank

T - Vol of ferrous ammonium sulphate for sample

ESTIMATION OF TOTAL ORGANIC NITROGEN (TON)

Preparation of reagents

1. Borate Buffer Solut~on. To 500ml of 0 025M Sod~um tetra borate (Na, B, 0,) 88ml of 0 IN NaOH is added and made upto one l~t re wlth

distilled water

2 Sodium hydrox~de Solutlon (6N NaOH) 240g of NaOH IS dlssolved

In one l~tre of dlst~lled water

3 Dechlonnatlng agent 3 5g of sodlum thlo-sulphate IS dissolved In one

litre of dlstllled water

4 Neutralmt~on agent Ammonia free water, IN NaOH. IN \SO4

5 lnd~catlng Bonc acld 200mg of methyl red IS dissolved In IOOml of

95% of ethyl alcohol Then lOOmg of methelene blue ind~cator is

dlssolved III 50ml of 95% ethyl alcohol Both the solutions are m~xed to

get a mlxed lndlcator 20g of bonc acid (H,BOJ IS d~ssolved In lOml of

mixed indicator solut~on and made upto one l~tre with distilled water

6 Mercuric sulphate 8g of red mercuric oxide dissolved In IOOml of

concentrated sulfunc acld

7 Digestion reagent 134g of potasslum sulphate is dlssolved In 650ml of

distilled water 200ml of conc H,S04 and 25ml of mercunc sulphate

solut~on are added to ~t and made upto one litre \nth d~st~lled water and

stored at 20°C

8 Sodlum hydroxide and sod~um th~o- sulphate reagent SOOg of NaOH

pellets and 25g of Na,S,O, 5H,O are dlssolved in one lltre of d~stilled

water

Procedure

The pH of the sample was adjusted to 9 5 uslng 6N NaOH and is then

digested with lOml of NaOH solut~on for approximately one hour 20ml of Sodium

thio-sulphate reagent is added after d~gest~on The digested sample is then dlstillated

with boric acid indicator1 nesseler's reagent Then it is titrated with standard

0 02N %SO, unt~l the ind~cator turns a pale lavender (APHA, 1998) in colour

Calculation

A - B x 280 Mg NH, - Nitrogen 1 kg=

(g) of dry wt of sample

A - Vol of H,SO, titrated w~th Sample B - Vol of H2S0, t~trated with Blank

ESTIMATION OF AVALLABLE NITROGEN CONTENT BY

ALKALINE KMnO, METHOD

Principle

The available nitrogen IS acted upon by alkal~ne KMn04 solution releasing

ammonla This ammonia IS est~mated volumetr~cally by tltratlng agalnst standard

H,SO, a c ~ d (Tandon. 1993)

Reagents

1 0 32% KMn04 solut~on

2 2 5% NaOH solution

3 Double (Mixed) Indicator Methyl red (0 Ig) and Methyl blue (0 5g) are

dissolved in IOOml of ethyl alcohol

4 Standard H,S0, (0 02N) (N/50)

Procedure

20g of vermicompost 1 compost samples were taken separately in distillation

flasks to which 20ml of distilled water and lml of liquid paraffin are added along

with a few glass beads Then lOOml of 0 32% KMnO, solution and IOOml of

2 5% NaOH solution are added These contents are distilled adding 20ml of Bonc acid

indicator solution In an Ice bath, till IOOml of distillate is collected in the beaker

Th~s is then titrated against standard H,SO, (0 02N) solut~on

Calculation

A-Bx280 Mg NH, - N~trogen 1 kg=

(g) of dry wt of sample

A - Vol of H,SO, titrated wth Sample

B - Vol of H,SO, t~trated w~th Blank

ESTIMATION OF AVAILABLE PHOSPHORUS

Principle

Phosphate ions in the soil sample can be extracted with 0 002N H,SO, in

the ratlo of 1 100(soil su lh r~c acld) In the presence of phosphates, the extract

reacts with ammonium molybdate and stannous chloride reagent giving a blue color

The reading is then taken at 690nm in a spectrophotometer (Tandon. 1993)

Prepamtion of magents

1 To 0 002N sulfuric acid solut~on, 3g of ammonlum sulphate or

potassium sulphate 1s added and made upto I l~tre wlth distilled water

Final pH is adjusted to 3 0

2. Ammonium molybdate solution:

Solution-A: 25g of ammonium molybdate are added to 175ml of

distilled water

Solution-B To 280ml of concentrated sulhric acid, 400ml of distilled

water is added Then solutions A and B are mixed and made upto one litre

3. Stannous chlonde solut~on: 2 5g of stannous chionde is rmxed with

100ml of glycerol and heated In a water bath

4. Standard phosphate solut~on: 4 388g of anhydrous potassium

hydrogen phosphate is dtssolved in distilled water to make one litre of

stock solut~on 100ml ofthis stock solution is mixed wtth distilled water

to make one lttre solution, so that one ml of this solution contains

0 0 l mg of phosphate

Procedure

To 50ml of sample (compostivermicasts extract), a spoonful of phosphate

free charcoal powder IS added, shaken well and filtered through whattman No-40

filter paper 2ml of ammontum molybdate solution and 5 drops of stannous

chlor~de, are added to thls solutton In the presence of phosphates when blue colour

appears, the r ead~ng I S taken at 690nm In a spectrophotometer Then the

concentratton of phosphate IS found out ustng a standard curve A blank IS run

to compare w ~ t h the standard curve

DETERMINATION O F POTASSIUM CONTENT IN COMPOST 1

VERMlCAST SAMPLES

Potassium was determtned using flame photometer (Systronic-FLAME

PHOTOMETER model-128 with FPM-COMPRESSOR-126) following the

Procedure of Tandon. (I 993)

Principle

Flame photometer is most commonly used to estimate potassium content

based on the principle that atoms of a specific element take energy from flame and

get excited to the higher orbit Such atoms release energy of a specific wavelength

which 1s proportional to the concentration of atoms of that particular element

Preparation of reagents

I. Ammonlum acetate solution 77g of ammonium acetate salt 1s d~ssolved

In 900ml of dlstllled water pH is adjusted to 7 using liqu~d Ammoma or

Acetlc acld and finally made upto one litre w th distilled water

2. Potasslum stock solutron 1 907gms of Potasslum chlonde was dned

at 1 I O°C and dissolved first In 250ml of de~onised dlstllled water and

finally made up IOOOml w t h deion~sed dlstllled water to get lOOOppm

3, lnterrned~ate stock solut~on lOml of stock solution 1s diluted wlth IOOml

w~th de~ontsed water to get IOOppm

4 Standard potassium solution lOml of Intermediate stock solution 1s

diluted with lOOml deionised distilled water to get lOppm

Procedure

Flame photometer was first standardized using specific standard solutions

5g of compost/vermicompost sample was taken separately and IOOml of distilled

water was added and stlrred for Sm~nutes It is then filtered through whattman-No 1

filter paper Then the potasslum content was estlmated In the flame photometer,

uslng the speclfic filters Potasslum concentration was expressed in ppm whlch

was later converted into percentage

MORPHOLOGICAL, BIOCHEMICAL AND YIELD PARAMETERS

OF BLACK GRAM Vigna mungo (L ) Hepper cv T-9

(SEED GERMINATION STUDY AND POT STUDY)

The following are the morphological, biochemical and yreld parameters

analysed Percentage of germcnation, number of lateral roots, shoot and root length,

fresh and dry weight of the roots, shoot, leaves, seed and fru~ts, total leaf area,

p~gment content of the primary leaves, chlorophyll-a, chlorophyll-b, carotenoids

and total chlorophyll, estrmatcon of carbohydrates, estimation of reducing and

non-reduc~ng sugars, , estimat~on of total sugar, estimation of starch content,

estlmatron of proteins, estimatcon of total free amino acids, and estimatcon of total

phenols

GERMINATION PERCENTAGE

At the rnrtiatron of germination of seed alter 48hours of setting up the experiment, the

seeds were examlned to determine the germinatron percentage Germlnatlon was

assumed to be complete when radical p~erced through the seed coat (Mayer and Mayber,

1982) The germrnated seeds were counted and germination percentage was calculated

uslng the following formula

Number of seeds germcnated

Germrnat~on percentage = X 100

Total number of seeds sown

LATERAL ROOTS

The number of lateral roots were counted for each of the seedlings grown in

different samples of the vermicasts

SEEDLING LENGTH

On the fourth and eighth day after setting up the experiment, the seedlings from

different samples of vermlcasts were removed carefully and washed in water

The length of root and shoot was measured w~th the help of wetted twine (for flexibility)

and a scale

FRESH AND DRY WEIGHT

Seedl~ngs were removed from petnd~shes and washed thoroughly They were

separated into root and shoot, and weighed For dry we~ght, the parts of seedlings were

wrapped with aluminum foils and kept in the oven at 80°C for 24 hours A top pan

electrical balance was used for weigh~ng the fresh and dry weight of the seedl~ngs of

black gram

ESTIMATION OF PIGMENT CONTENT OF PRIMARY LEAVES

The plgments were extracted following the procedure of HISCOX and Israelstam

(1979) An amount of IOOmg of leaf tlssue was kept In a vial containing 7 rnl of

dlmethyl sulphoxtde (DMSO) and kept In an incubator In the dark at 65°C until the

leaf materials h e colourless The extracted llqu~d was transferred to a graduated

tube and the volume was made upto lOml wth DMSO and stored at 0-4°C until

requ~red for analysis The absorbance of the l~qurd IS measured at 480, 645 and

663 nm with spectronic-20 spectrophotometer

The pigment content (mglml) was estimated as per the formula given by Harborne

(1 984)

Total Chlorophyll (mglml) = (0 0202 x OD 645) + (0 0082 x OD 663)

Chlorophyll- a (mglml) = (0 0427 x OD 663) - (0 00269 x OD 645)

Chlorophyll- b (mdml) = (0 0229 x OD 645) - (0 00468 x OD 663)

Carotenoid = (OD 480 - (0 01 14 x OD 663) - (0 0636 x OD 645)

Where OD = Opt~cal Density,

The pigment contents were expressed as mgig fresh weight of the leaf

LENGTH OF ROOT AND SHOOT

The length of roots and stem was measured once in 15 days with the help of a

scale and wetted twine

NUMBER OF ROOT NODULES

The number of root nodules was counted once In 15 days with the help of a

hand lens

NUMBER OF LEAVES

The number of leaves was calculated once in every 15 days The total number of

leaves per plant represents the average number of leaves from 10 different plants of

each samples of vermicast added pots

TOTAL LEAF AREA

The l u f area was calculated followng the formula of Kemp(1960) Every time

the secondq leaf from the tip of the plant was taken for measurement The total leaf

area was obtained by multiply~ng with the total number of leaves per plant

Total leaf area = L x B x K x N

Where L = Length of leaf

B = Breadth of leaf

K = Kemps Constant (0 66 for dicots)

N = Number of leaves per plant

FRESH AND DRY WEIGHT

Root, stem and leaves of the plants were separated For dry weight, the plant

parts were kept in the oven at 80°C for 24 hours The fresh and dry we~ghts of the plant

parts were found out with the help of electric top pan balance

YIELD PARAMETERS

On the 75' day, the fru~ts were removed from each plant that were grown w ~ t h

d~fferent verrnlcast samples The number of h ~ t s per plant, number of seeds per plant,

we~ght of fruits and seeds per plant, 100 seeds welght and y~eld per hectare were calcu-

lated

CARBOHYDRATES

Reducing and non-reducing sugars, total sugars and starch were extracted from

oven dried and powdered plant matenals grown for the exper~ment

Preparation of Ethanol extract

Sugars were extracted and estimated following the methods of Loomis and Shull

(1937) and Nelson (1944) IOOmg of plant leaf sample was boiled in 5ml of 80%

ethanol for 10 minutes and then centrifiged The supernatant was evaporated to I ml In

a boillnB water bath MIer cooltng it to room temperature. Sml of saturated lead acetate

was added to this alcoholic extract to precipitate the protelns 10ml of saturated

49

aqueous di-sodium phosphate was added to precipitate excess of lead Then 0 2 gram of

activated carbon was added and shaken at intervals of 30 minutes and then filtered

The filtrate was made up to 20ml with distilled water and then used for the estimation

of sugars

ESTIMATION OF REDUCING SUGARS

Reducing sugars were estimated from the alcoholic extract follow~ng the method

of Snell and Snell (1957)

Preparation of reagent

1 Reagent A 25g of anhydrous Na2C03, 25g of sodium potassium

tartrate (Rochelles salts), 20g ofNa HCO,, and 200g of anhydrousNa,SO,

were dissolved in 800 ml of dlst~lled water and the final volume was

made upto one litre w~th dlstllled water It IS then filtered and stored in

glass-stoppered brown bottles

2 Reagent B 15g of Cu SO, was drssolved on one litre of dlst~lled water

and one or two drops of conc , H?S04 were added

3 Nelson ' s reagent 25 parts of reagent-A and one part of reagent-B were

nuxed thoroughly to prepare Nelson's reagent

Procedure

To I d of ethanol extract, Iml of fresh Nelson's reagent was added The mix-

ture was heated for 20 minutes m a boiling water bath, and then after coollng. Iml of

arseno molybdate reagent was added The solution was diluted to 251111 w~th distilled

water The intens~ty of the resulting blue colour was read at 620nm In a

Spectrophotometer. A blank reagent containing lml of ethanol was also run

simultaneously The reducing sugar content of the samples under study was calculated

from the glucose standards

ESTIMATION OF NON-REDUCING SUGARS

Non-reduc~ng sugars present in the ethanol extract were hydrolyzed with sulk-

rlc acid (Malhotra and Sarkar, 1979) to reducing sugar The reducing sugar in the

hydrolysate was estimated following the method of Nelson (1944) The difference

between the total sugars and the reduc~ng sugars est~mated w~thout hydrolysis

corresponds to the non-reduclng sugars

ESTIMATION OF TOTAL SUGARS

To Iml of ethanol extract. 4ml of anthrone reagent was added and the test tube

was shaken well and kept In boding water bath for 10 mlnutes The test tubes were then

transferred to ice bath to terminate the reactlon The colour lntenslty was read at 630nm

In the Spectrophotometer

ESTIMATION OF STARCH CONTENT

The residue left beh~nd after alcoholic extractlon of the original sample material

was taken for the extraction of starch and it was estimated by the method of Mc Cready

et al .(1950) The residue was dissolved in 6 5ml of 52 % perchloric ac~d for one hour,

centrifuged, and made upto 100 ml In a volumetric flask wth distilled water One ml of

lhls solution was further diluted wth 5ml of distilled water and to this, lOml of Freshly

prepared anthrone reagent was added The test tube along w~th ~ t s contents was heated

for 7 5 minutes at 100°C in a boiling water bath The tube was then cooled

rapidly, shaken well and the colour whlch appeared was read at 630nm in the

Spectrophotometer A standard curve was prepared with known quantities of

glucose and the quantity of starch was calculated by multiplying the

glucose equrvalents present in the sample with 0.9

ESTLMATION OF TOTAL PROTEINS

The estimat~on of total proteins was by Lowry method (Lowry et al,

1951)

Prepartion of protein extract

100mg of fresh leaves were homogenized In lOml of 20% tri-chloro acetic acid

(TCA) and then centrifuged at 6000 rpm for 1 5mlnutes The supernatant was discarded

and 5ml of 0 IN NaOH was added After stlrrlng well ~t is again centrifuged

The supernatant was stored and used for proteln estlmatron

Preparation of reagents

1. Reagent-A 2g of Na2C03 In 1 OOml of 0 IN Na OH solution

2 Reagent-B 0 5% of Cu SO4 solut~on IS mixed with 1% of sod~um

potasslum tartrate solutron In the ratlo of 1 1

3 Reagent-C M~xture of 5Oml of reagent-A and one ml of Reagent-B

4. Fohlin-ciocalteu reagent- The reagent 1s d~luted at 1 1 ratlo with

distilled water

Procedure

To 0 Sml of protein extract Sml of reagent-C was added It was allowed to react

for 10 minutes at room temperature Then 0 5ml of Fohlin-ciocakteu reagent was added

with vigorous shaking and kept In dark for 20 minutes The colour intensity was read at

660nm in the spectrophotometer

ESTIMATlON OF TOTAL FREE AMINOACIDS

Total free aminoac~ds were extracted and estimated following Moore and

Steln(1948) method IOOmg of fresh plant material was ground in pestle and mortar

wlth 5 0 ml of 80% ethanol It was centr~fuged and the supernatant was used for estima-

tion of total free amino acids

Reagents

1 Ninhydrrn reagent was prepared by mlxlng freshly prepared equal

quantltles of solutron -A and solution-B

2 Solut~on-A 800ml of stannous chloride is dissolved in 500ml of 0 2M citrate buffer at pH-5 0

3 Citrate buffer 21g of cltrlc acld IS added to 200 ml of IN sodium

hydroxide and final volume was made upto 500ml with dist~lled water

4 Solution-B 20g of ninhydnn 1s d~ssolved in 500ml of methyl cellulosolve

Diluant solution was preparted by mixing equal quantltles of (vlv) water

and n-propanol

Procedure

To one ml of supernatant one ml of freshly prepared ninhydrin reagent was

added, and heated at 60' C in water bath for 20minutes. 5 Oml of diluant solution

was added to the test tube when it was still in the water bath. Then it was cooled and

as the colour developed it was read at 570nm in the spectrophotometer against

water blank. A standard graph was prepared using the aminoacid-leucine.

ESTIMATION OF TOTAL PHENOLS

One ml of alcoholic extract was taken and mixed with one ml of Folin-phe-

no1 reagent. Then two ml of 20% sodium carbonate solution was added mixed well

and boiled in water bath for one minute. Then the solution was made upto 251-111 with

distilled water. Reading was taken at 650nm in the spectrophotometer.

DETERMINATION OF PERCENTAGE OF INHIBITION OF

PLANT PATHOGENS

The two plant pathogens used in this experiment are Rh~zopus stolonifer /

Rhlzoctonia solani

Culturing of Rhizopvs stolonijcr /Rhizoctonio soloni:

250g of potato extract, 20g of dextrose and 20g of agar were dissolved in

800ml of distilled water, pH was adjusted to between 6.8 to 7.0, and then made upto

one litre with distilled water. Vermicasts of coir pith and coffee husk obtained on

recycling by Eudrilus eugeniae were used. This media is mixed with 20% of water

extract of different vermicasts in separate petridishes each of 9 cms in diameter.

The experiment was run in duplicate. It is then inoculated with these two

pathogen in separate petridishes Percentage of inhibition of growth of these

pathogens is calculated as per the following the method of Vincent, (1927)

Percentage of inhibition (%) I=C-TICX100

where,

I= Percentage of inhibition C= growth of mycelia in control (in diameter)

T= growth of mycelia in treatment (in diameter)

S1 GEL - COLUMN CHROMATOGRAPHY

The water extract of the vermicasts of E eugenlae were tested against plant

pathogens like Rhrzoclonra solanr and Rhrzopus stolonifer. Vermicast samples

exhlb~ting h~gher lnh~b~tion of growth of the plant pathogens were selected The

compound was separated by sil~ca gel column chromatography Acetone, petroleum

ether, chloroform, methanol, and n-hexane are the organlc solvents used for further

separation of the compounds present in the vermlcast samples, by column

chromatography

THIN LAYER CHROMATOGRAPHY (TLC)

The water extract of the vermicasts o fE eugenroe which ind~cated the high-

est Inhibition of the pathogen was selected for TLC and Sll~ca gel chromatography

experiments The TLC plates (0 2mm thick glass plates precoated w~th sillca gel)

were used for the Identification of different fractions The compound was spotted at

0 2cm from the edge of the silica gel plate Then the TLC plate was developed In

a mixture of n - hexane and ethyl acetate to a distance of 4cm and dr~ed at room

temperature (28 5 2°C) The TLC plate was vlsuallzed In iodlne chamber

INFRA RED SPECTRUM

The water extract of the compost and vermicast samples were filtered sepa-

rately through whattman No l filter paper The filtrate was dried at room tempera-

ture and the crystal sample is mixed with KBr. Then the crystal was subjected to

analysis by infrared spectroscopy The IR spectra were recorded on a BOMEM

MB 104 FT-IR Instrument

STATISTICAL ANALYSIS

The data obtalned in the present study was analyzed using standard

statistical methods-employing Two-way ANOVA (without replication) and the

results are presented In the specific chapters The test of significance refers to

the probability of obtaln~ng slgnlficant results, P=O 01 (1%) (Ostle and Menslng

1975) Null hypothesis was applied to the data wherever applicable and the F- value

and P-value are presented along wlth data In the respective chapters