Chapter-3 Materials and Methods - INFLIBNETshodhganga.inflibnet.ac.in/bitstream/10603/3269/13/13_chapter 3.pdf · Chapter-3 Materials and Methods 32 ... organism to ferment various

Chapter-3 Materials and Methods


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3.1 Microbial Cultures Microbial isolates used in this study were collected in our laboratory from diverse

environmental habitats. These isolates were selected under different physiological

parameters such as pH from 2-12 with each incremental unit, salt concentration

(NaCl, % - nil, 0.5, 2 and 5), sensitivity to metals i.e. (Nickel and cobalt: 1 mM) and

their susceptibility to 12 different antibiotics (Ampicillin (A), Carbenicillin (Cb),

Chloramphenicol (C), Gentamycin (G), Kanamycine (K), Nalidixic acid (Na),

Pecicillin (P), PolymyxinB (Pb), Rifampicin (R), Streptomycin (S), Tetracycline (T)

and Vancomycin (Va): Contaminated food sample (Pickle waste), Marine coastal

Water (Goa), MRL sludge sample (Madras Refinery Limited), IIT ETP sludge sample

(Pesticide ETP, Mumbai), Pesticide ETP sludge sample (Gardha ETP, Chennai)

(Porwal et al., 2008; Rani et al., 2008). These were grown and maintained on nutrient

agar at pH 7 and 37 ºC. 20% glycerol stock of the cultures was maintained in -80 ºC.

3.2 Feed materials 3.2.1 Sugars solution

The following sugars: Glucose, fructose, lactose, maltose, sucrose, starch and CM-

cellulose at concentrations of 0.5, 1, 2, 3 and 5% were used as feed material for H2

production. The total volume of the feed was 250 mL in 300 mL BOD reactor bottle,

unless otherwise mentioned.

3.2.2 Biowaste material

Vegetable wastes - green PS were collected from the local municipal market in Delhi,

India.

3.2.3 Chemicals

All the chemicals used in the study were of analytical grade and were purchased from

Himedia, MERCK, BDH, SIGMA (USA), SRL and SD fine chemicals.

3.2.4 Gases

All the gases [H2, N2, Argon (Ar) and Zero air (O2)] of high purity were procured

from M/s Laser Gases, New Delhi, India.


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3.3 Biochemical characterization of microbes 3.3.1 Metabolic characterization

Bacterial strains selected as H2 producers were tested against 47 different substrates

(35 different carbohydrates as carbon source, 5 five proteins as nitrogen source and 7

enzyme activities carbohydrates) to check their metabolizing abilities by KB009 Hi

CarbohydrateTM Kit. The tests are based on the principle of pH change and substrate

utilization. On incubation organisms undergo metabolic changes, which are indicated

by a spontaneous colour change in media. A single colony is picked up and inoculated

in nutrient broth media. The inoculum were incubated at 37 oC for 24 h and then used

for further testing. Each well of kit was inoculated with 50 μL (OD660 = 1) of

inoculum by surface inoculum method. Alternatively, the kit can also be inoculated by

stabbing each individual well with a loopful of inoculum. Incubate kits at 37 oC for 24

h. Observations were recorded and analyzed.

Carbohydrate fermentation tests: This test is used to determine the ability of an

organism to ferment various simple carbohydrates (sugars) as substrate. Fermentation

is a metabolic process in which the final electron acceptor is an organic molecule. The

indicator used is phenol red, which turns yellow below pH 6.8 and a darker pinkish

red above pH 7.4. If the organism does not ferment the carbohydrate, the pH may

remain neutral. If metabolizes the carbohydrate subsequent acid product ion will

result in lowered pH and hence positive test (yellow from red).

Esculin test: Esculin is a glycoside composed of glucose and dihydroxycoumarin

compound. On esculin hydrolysis, the original cream colour of the medium changes to

black.

Trehalose test: Trehalose is a disaccharide composed of two glucose molecules

bound by an α-1, 1-linkage. It has no reducing power. It acts as a source of energy in

most organisms e.g. - bacteria, fungi, insects, plants and invertebrates. It protects

organism against various stresses such as dryness, freezing and osmopressure.

Anhydrobiotic organisms are able to tolerate the lack of water due to trehalose

synthesis in large amounts and trehalose plays a key role in stabilizing membranes

and macromolecular assemblies under extreme environmental conditions.


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Citrate utilization test: This test is used to determine the ability of an organism using

enzyme citrase, to use citrate as it’s sole source of carbon. Test is done on Simmon’s

Citrate Agar containing sodium citrate as C-source and ammonium ion as the sole N-

source. The pH indicator bromothymol blue will turn from green at neutral pH (6.9) to

blue when a pH higher than 7.6 is reached (basic or alkaline). If the citrate is utilized,

the resulting growth will produce alkaline products (pH > 7.6), changing the colour of

the medium from green to blue.

Decarboxylase test: This test is used to detect the ability of an organism to

decarboxylate an amino acid. Decarboxylation is a reaction, which removes the

carboxyl group (-COOH) of an amino acid, producing an amine and CO2. The

amino acid is added to the test medium, along with the pH indicator bromocresol

purple. The decarboxylation of the amino acid by the decarboxylase enzyme then

results in alkaline end products. These in turn will cause the pH indicator to turn

purple (positive). Lysine and ornithine are commonly used amino acids for test.

Arginine and other amino acids are tested in different chemical reaction

dehydroxylation.

Methyl Red test: It is used to identify bacteria that produce stable acid end

products by means of mixed acid fermentation of glucose. Bacteria those are able

to perform mixed-acid fermentation of glucose and produce large amounts of

stable acids. The pH indicator used methyl red. If the pH is less than 4.4, the

indicator will turn red. A red colour is read as positive, a yellow colour (pH> 6) is

negative, and an orange colour indicating a pH between the two will usually

require further incubation.

Voges-Proskauer (VP) test: This test is used to identify organism able to produce

acetoin from the degradation of glucose during 2,3 butanediol fermentation. In some

fermentative organism, the chief end products of glucose metabolize are acetoin and

2,3 butanediol. After incubation Barritt’s reagent A (α-naphthol) and Barritt’s reagent

B (potassium hydroxide) are added to the sample. Formation of red colour will

indicate a positive reaction. No color change or a copper colour indicates negative

results.


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Nitrate reduction test: This test detects the ability of an organism to reduce NO3-

to

NO2- or some other nitrogenous compounds, such as molecular N2 using the enzyme

nitrite reductase. NO3- may be reduced to several different compounds, either by

anaerobic respiration or by denitrification. This test is used to detect whether or not

the reduction has taken place. The NO3- medium contains KNO3 as the substrate. If

the organism reduces the NO3- to NO2

-, the NO2- will react with added reagents

sulphanillic acid and α-naphthalamine to produce a red colour. If no colour is

produced, this can indicate either of two reactions: (i) the NO3- was not reduced and

(ii) the NO3- was reduced even further to compounds other than NO2

-.

Phenyalnine deamination: This test is used to identify bacteria possessing the

enzyme phenylanine deaminase. This medium contains the amino acid phenylalanine.

The enzyme will remove the amine group and release it as free ammonia (NH3). This

leaves phenyl pyruvic acid, which can be detected by adding an oxidizing reagent

such as ferric chloride to the incubated tube. If the acid is present, a green colour can

be detected.

ONPG test: This test is used to identify bacteria possessing the enzyme β-

galtosidase. It catalyzes the breakdown of the substrate lactose (the major sugar

present in milk) to galactose and glucose, which were feed into the glycolytic

pathway. Ortho-nitrophynyl-β-galtoside (ONPG) is used as artificial substrate for the

enzyme and in the presence of β-galtosidase, is converted to galactose and ortho-

nitrophenyl (ONP). It is colourless and also at neutral or acidic pH, but in alkaline

solution it is bright yellow.

Urease test: It is used to detect the presence of enzyme urease. It breaks the C-N

bond of amides to form CO2, NH3 and H2O. When urea is broken down, ammonia is

released and pH of the medium increases (becomes more basic). This pH change is

detected by a pH indicator that turns pink in a basic environment. A pink medium

indicates a positive test for urease (Results pertaining to these experiments have been

presented in Table 4.1).


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3.3.2 Growth curve of microbial cultures

Mixed microbial strains were grown in NB and M-9 (0.05x) media and initial OD660

for the growth curve was 0.05. Curve was plotted on the basis of incubation period (h)

versus OD660 (spectrophotometer: Lambda 35 Perkin-Elmer) taken against blank (NB

and M-9 without inoculum) with spectrophotometer.

3.3.3 Antibiotic susceptibility assay

The most common method for antibiotic susceptibility test is Kirby-Bauer method or

Disc diffusion method. In this test bacterial isolate is inoculated uniformly onto the

surface of an agar plate. A filter disc impregnated with a standard amount of an

antibiotic is applied to the surface of the plate and the antibiotic is allowed to diffuse

into the adjacent medium. The result is a gradient of antibiotic surrounding the disc.

Following incubation, a bacterial lawn appears on the plate. Zones of inhibition of

bacterial growth may be present around the antibiotic disc. The size of the zone of

inhibition is dependent on the diffusion rate of the antibiotic, the degree of sensitivity

of the microorganism, and the growth rate of bacterium. Discs with very small zones

or no zones of inhibition mean that the bacterium is not susceptible to the antibiotic.

Large zones indicate three levels of susceptibility (Figures 3.1 and 3.2): Susceptible,

Intermediate, or Resistant. Each tested organism is rated using these criteria.

Sometimes the production of the antibiotic involves genetically changing or

modifying the potential antibiotic. From there the extensively research is going on.

3.3.3.1 Characterization

Morphology of colony, shape, size, margins, appearance, colour, etc. were used as

initial criteria to distinguish strains by growing them on nutrient media. Microbial

growth rate was measured on nutrient broth at 37 °C, 200 rpm over a period of 24 h.

3.3.3.2 Requirements

Overnight grown culture, Nutrient agar plates, Spectrophotometer and Antibiotic

discs - A: 25 mcg; Cb: 100 mcg; C: 10 mcg; G: 10 mcg; K: 30 mcg; Na: 30 mcg; P:

10 units; Pb: 100 units: R: 15 mcg; S: 10 mcg; T: 10 mcg and Va: 10 mcg.

3.3.3.3 Procedure

Isolates were grown overnight in nutrient broth media (NB) at pH 7.

100 μL bacterial culture (OD660 = 1, i.e.105 CFU) was spread on NA plates.


37

Antibiotic discs of known concentrations were equidistantly placed on three NA

plates at the rate of 4 discs/plate.

Plates were incubated overnight at 37 °C.

Zone of inhibition of bacterial growth was measured (diameter in mm).

On the basis of zone of inhibition isolates were segregated.

3.3.4 Hydrolytic enzyme activities

The biochemical tests were done basically to identify the secretion of three

exoenzymes, viz. protease, amylase and lipase. Agar plates were prepared containing

protein, starch and lipid for testing protease, amylase and lipase activity respectively.

If the inoculated bacterium secretes the respective exoenzymes, a clear zone of

hydrolysis is observed around the inoculum (Results pertaining to these experiments

have been presented in Table 4.2).

3.3.4.1 Materials required

Lipase, amylase and protease plate of pH 7, nichrome straight wire and bacterial

culture.

Media compositions

A) For protease activity

Solution A: Composition g/100 mL: Beef extract, 0.15; Yeast extract, 0.15; Peptone,

0.5 and NaCl, 0.5.


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Solution B: 30 mL (1 g of milk powder (casein) was dissolved in DW to make final

volume of 30 mL).

Note: Solution A and solution B were autoclaved separately and mixed at the time of

pouring.

B) For amylase activity

Medium composition g/100 mL: Beef extract, 0.15; Yeast extract, 0.15; Peptone, 0.5;

NaCl, 0.5 and soluble starch, 0.2.

C) For lipase activity

Solution A: Composition g/200 mL: Beef extract, 0.3; Yeast extract, 0.3; Peptone, 1

and NaCl, 1 (150 mL).

Solution B: 50 mL (Gum acacia (1%)- 2 g, Calcium chloride- 20 μL, DW -50 mL).

Note:

1. Stock solution of calcium chloride was 200 mg/mL.

2. Solution A and solution B were autoclaved separately and mixed.

3. Later, 1% tributyrin (2 mL) was added and mixed vigorously before pouring.

3.3.4.2 Procedure

Sterilize the nichrome straight wire on the flame. Allow the wire to cool for a few

min.

Remove a small amount of the growth with sterilized nichrome straight wire.

Stab the straight wire on to the different plates prepared for testing protease,

amylase and lipase activity.

The plates were incubated at 37 °C for overnight.

After incubation, the colony size and zone of hydrolysis were measured.

The zone of hydrolysis due to amylase was measured by adding iodine.

The plates for testing lipase activity were again incubated at 37 °C for 7 days.

After 7 days colony size and zone of hydrolysis were measured.

The relative protease, amylase and lipase activities were calculated by the given

formula:

Relative activity = Zone of hydrolysis/Colony size


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3.4 Dark fermentative hydrogen production abilities of microbial

isolates Individual abilities of the isolated from divers environmental habitats were tested for

the H2 production under batch culture.

3.4.1 Batch culture conditions and medium

Different bacterial isolates were grown in Himedia nutrient broth (13 g/L DW) and

incubated at 37 oC at 200 rpm for 16-20 h. These actively growing cell cultures were

centrifuged at 6000 rpm for 20 min, washed in 20 mL phosphate buffer saline and

protein content was estimated by Lowry’s method. For batch culture digestion 250

mL of 2% (w/v) glucose in minimal medium (Miller: M-9) composition/L (Na2HPO4,

6 g; KH2PO4, 3 g; NaCl, 0.5 g; NH4Cl, 1 g; MgSO4, 1 mM and CaCl2, 0.1 mM) were

added in 300 mL BOD bottles. These were inoculated individually with different

bacterial strains at the rate of 40 μg cell protein/250 mL glucose solution feed. The pH

of the glucose containing medium was adjusted to 7 The OD660 of the inoculated

medium was measured with spectrophotometer in the beginning and at the end of the

experiment. Incubation bottles were fixed with ground glass necks of B-19 joints. One

of the side arm (outlet) was connected with latex tubing for collecting the gases in

inverted graduated test tube in a beaker (1L) filled with water (600 mL) and another

side arm was provided for flushing gas to create anaerobic condition were made air

tight with stoppers (Figure 3.3).

Figure 3.3: Fermenter set up of 300 mL capacity


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All the bottles were then flushed with Ar gas for 5 min to maintain anaerobic

conditions and incubated at 37 oC. Each day, the pH of the solution was checked by

opening the bottle and readjusted to 7 with 2N NaOH and 2N HCl. After replacing the

stopper, the bottles were reflushed with Ar. The evolved gases were collected by

water displacement method. (Kalia et al., 1994; Porwal et al., 2008; Patel et al., 2010)

(Results pertaining to these experiments have been presented in Table 4.3).

3.4.2 Gas analysis

Evolved gases (a mixture of H2 and CO2) were collected over water (pH 2) in a

graduated gas holder and the volumes calculated at 25 ºC. Gas samples (0.2 mL) were

drawn with 1 mL airtight syringe. The gas composition was determined using gas

chromatograph (GC5700, Nucon Engineers, New Delhi) at ambient temperature by

standard procedures. A stainless steel molecular sieve column (1.8 m long and 2 mm

inner diameter) was used for analyzing H2, O2 and N2 gases, while air, CH4, and CO2

were analyzed by using Porapaq Q column of stainless steel (1.8 m long and 2 mm

inner diameter). The Ar gas was used as a carrier at a flow rate of 30 mL/min. Gas

standards (H2, CH4 and CO2) were run before each set of gas analysis. Although no

CH4 was expected to be evolved in the absence of any added methanogens at any

stage, however, to ensure that no H2 quenching methanogens are present ever as

contaminants; CH4 analysis was done every time gases were analyzed. Gas collection

and analyses were done daily. H2 gas production was calculated from the head space

measurement of gas composition and the total volume of biogas produced, at each

time interval, using the mass balance equation:

V = V0γi + Σ Viγi

where V is the cumulative H2 gas volumes at the current (i); V0 is the volume of

headspace of vials; Vi is the biogas volume discharged from the vials at the time

interval (i); γi is the fraction of H2 gas discharged from the vials at the time interval

(i) (Pan et al., 2008; Porwal et al., 2008).

Hydrogen sulfide (H2S) gas was estimated by passing each gas sample through 10%

lead acetate solution in the airtight column. The volume of the gas absorbed by the

lead acetate solution was used to calculate the quantity of H2S present in the sample

(Kalia et. al., 1992b).


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3.4.3 Gas chromatography system

3.4.3.1 GC switch on sequences

The following sequence must be observed for switching on the GC:

Open the carrier gas supply to the instrument. For this opened the shut on valve

and finally open the cylinder with the help of cylinder key moving anti-clockwise.

Check that the Thermal Conductivity Detector (TCD) power supply is off, current

to minimum position and attenuator to infinity.

Set the column flow rate of 30 mL/min Ar as carrier gas supply and wait for about

10 min.

Switch on the mains of Temperature Controller module and the blower inside the

oven should be on.

Set the oven temperature to 70 oC bring the oven up to the proposed operating

temperature.

Switch on the Injector and Detector heater and set the dial of the Injector and

Detector controller for the required temperature.

The TCD module may now be switched on provided the instrument has been

purged for at least 10 min. Now set the bridge current to the desired value in the

range of 100-110 mA.

Allow the system to stabilize. Bring the attenuator to the lowest setting of 1x

which produces a straight baseline by means of coarse and fine controls. The

Chromatography may be used for an analysis when the baseline is stable. A trial

analysis will indicate the correct setting of the output attenuator for achieving the

desired chromatography, i.e. with all peaks on scale.

Balance the TCD system and adjust.

Sample of 0.2 mL injected by gas tight syringe capacity of 1 mL. The septum is a

self-sealing but after many injections the seal may become imperfect. It is

important to use the correct replacement septum. Incorrect septum material will

block the needle of the syringe, give bleed signal or other problems.

Peaks which go off the scale may be brought on to scale by altering the

attenuator setting and each peak should be marked with the setting on the

attenuator switch used when peak was traced. It is always desirable to record on

chromatogram the instrument’s setting. This permits subsequent comparison

and repetition.


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3.4.3.2 GC switch off sequences

Following sequence should be observed for switching off the instrument:

Switch off the running GC programme and then I/F module.

Reduce the filament current to minimum and then switch off TCD module.

Switch off injector and detector heating.

Lower the oven temperature setting and open the oven door.

Allow the temperature of the oven and detector to come to almost ambient

temperature.

Stop the carrier gas supply to the instrument only when the column and detector

temperature has fallen to near ambient temperature. For this close the shut off

valve and finally close the cylinder with the help of cylinder key moving

clockwise.

3.5 Determination of whole cell protein 3.5.1 Preparation of whole bacterial cell extract for protein

The cell extract was produced as follows. 1 mL cell culture was centrifuged at 14,000

rpm for 5 min to remove supernatant. Suspended the cell pellet in 1 mL DW and

added 2 mL of 2N NaOH solution. Kept for boiling in water bath for 5 min. Solution

was cooled and 2N HCl was added to neutralize the solution. Protein concentration of

the sample was estimated by using Bovine Serum Albumin (BSA) as a standard

protein.

3.5.1.1 Reagents required

Normal saline: 0.9 g sodium chloride dissolved in DW make final volume 100

mL.

2 N NaOH: 8 g sodium hydroxide dissolved in DW makes final volume 100 mL.

2 N HCl: 17.5 mL conc. HCl (35% purity) dissolved in DW make final volume

100 mL.

Cells were lysed by strong alkali treatment before protein estimation as described

below. 1 mL of cell culture was taken and centrifuged to remove the supernatant.

Pellets were resuspended in 1 mL DW and added 2 mL 2N NaOH, then boiled in

water bath for 10 min. This solution was cooled and 2 mL of 2N HCI was added.

Normality of NaOH and HCl should be exactly same. Sample was ready for protein

estimation.


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3.5.2 Estimation of protein by Lowry’s method

The Lowry’s method of protein estimation depends on measuring the color

quantitatively, which is obtained from the reaction of the Folin-Ciocalteu phenol

reagent with the tyrosyl residues of an unknown protein. With the addition of Lowry’s

reagent, which actually contains the alkaline copper tartarate to the entire sample

containing the protein a cupric amino acid complex is formed. The color so formed is

due to the reaction of alkaline copper with the protein and the reduction of

phosphomolybdate by tyrosine and tryptophan present in the protein. The intensity of

the color depends on the amount of these aromatic amino acids present. Protein

absorbs strongly at 280 nm according to their content of the amino acids tyrosine and

tryptophan, and this provides a sensitive form of assay. Protein also absorbs in the far

UV because of the peptide bond. The addition of Folin-Ciocalteu phenol reagent

results in formation of intense blue color whose absorbance can be measured by

spectrophotometer at 750 nm. We can prepare a standard curve by finding the

absorbance of samples containing the known amount of BSA. The concentrations of

standard BSA are in the range of 20-100 µg. The amount of protein in any sample can

be derived from the standard curve.

3.5.2.1 Reagents required

A. Bovine serum albumin (Standard): The BSA was dissolved in DW to get final

concentration of 0.1 mg/mL.

B. Folin-Ciocalteu phenol reagent: The reagent was diluted with distill water in 1:1

ratio.

C. Phosphate buffer saline: Composition/L: NaCl, 8; Na2HPO4, 1.4; KCl, 0.2;

KH2PO4, 0.24.

Table 3.1: Composition of alkaline copper reagent (ACR)

Volume of stock solution to be prepared for samples (mL)

Solution Concentration (w/v)

Stock solution

10 20 30

A 2% Na2CO3a 0.1 N NaOHb 50 100 150

B 2% S.P.T c DW 50 50 50

C 1% CuSO4 DW 50 50 50


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Table 3.2: Preparation of ACR (Alkaline copper reagent) standard

Volume of to be prepared for sample (mL) Solution Volume to be mixed (ratio)

10 20 30

A 98 49 98 147

B 1 0.50 1 1.50

C 1 0.50 1 1.50

Total volume of mixture 50 100 150

Table 3.3: Details of Lowry’s method of protein estimation

Stock solution (BSA) 10 mg/mL

Standard working solution. 0.1 mg/mL (10 μL from stock+990 μL DW)

Testing range 10-100 µg

Sensitivity range 10-1200 µg/mL

3.5.2.2 Procedure for BSA standard preparation

Different concentrations of BSA standard i.e. 100, 200, 400, 600, 800 and 1000

μL were taken from the working solution in separate test tubes.

Total volume was made up to 1000 mL with DW.

5 mL of ACR was added to all the tubes.

Test tubes were incubated at room temperature for 10 min.

500 μL of Follin’s Phenol reagent (dilution ratio is 1:1 with DW) was added to all

the tubes.

Test tubes were incubated at room temperature (in dark) for 30 min.

OD was taken at 750nm immediately.

Table 3.4: Standard for Lowry’s methods

S. No.

Concentration of BSA (µg)

Amt of working solution (µL)

Volume of water (µL)

Volume of ACR (mL)

Volume of Folin’s reagent (µL)

1 10 100 900 5 500

2 20 200 800 5 500

3 40 400 600 5 500

4 60 600 400 5 500

5 80 800 200 5 500

6 100 1000 - 5 500

7 - - 1000 5 500


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3.5.2.3 Protocol for protein estimation of whole cell extract

1 mL sample of whole cell extract was taken and 5 mL ACR (98:1:1) was added.

It was incubated at room temperature for 10 min.

500 μL of diluted Folin’s reagent (1:1 with DW) was added.

Tubes were incubated at room temperature for 30 min.

Sample was read at OD750 nm (immediately).

3.6 Estimation of residual glucose 3,5-Dinitrosalicylic acid (DNSA, IUPAC name 2-hydroxy-3,5-dinitrobenzoic acid) is

an aromatic compound that reacts with reducing sugars and other reducing molecules

to form 3-amino-5-nitrosalicylic acid, which absorbs light strongly at 540 nm (In case

of glucose).

3.6.1 Reagent required

Preparation of DNSA

Reagents: A. Sodium potassium tartarate

B. 2 N sodium hydroxide (2N NaOH)

C. Dinitro salicylic acid (DNSA)

3.6.2 Procedure

Prepared 20 mL of 2N NaOH.

Weighed 1 g DNSA and dissolved in 20 mL NaOH with the help of a magnetic

stirrer for approximately 45 min.

Weighed 30 g of sodium potassium tartarate and dissolved in 50 mL DW.

Slowly poured sodium potassium tartarate solution in the DNSA and NaOH

solution and made the volume up to 100 mL (Note: Wait for the two to mix

properly).

Decanted the contents in a brown bottle. Filter if necessary.

3.6.3 Protocol

Took at least two 20 mL test tubes (i.e. two replicates of each concentration

should be tested) and took an amount of glucose stock solution in each test tube as

per table given below.


46

Prepared a blank, in which case added 500 μL of DW instead of sample.

Added DW as indicated in the table above (preheated to 65 oC).

Incubated precisely at 65 oC for 15 min in a water bath or incubator.

Added 3 mL of DNSA.

Kept tubes (Glucose solution + DW + DNSA) in boiling water-bath for 15 min.

Cooled to room temperature.

Measure the absorbance at 540 nm in a UV-VIS spectrophotometer against a

suitable blank

Table 3.5: Standard for DNSA methods

S. No.

Concentration of glucose (μmol)

Amount of working solution (μL)

Volume of DW (μL)

Amount of DNSA (mL)

1 1 100 1400 3

2 2 200 1300 3

3 3 300 1200 3

4 4 400 1100 3

5 5 500 1000 3

6 - - 1500 3

Note: Test only 1-5 μmol concentration prepared from stock solution D, to study the linear range.

Table 3.6: Details of DNSA method of glucose estimation

Stock solution 180 g/mL

Standard working solution 1.8 mg/mL

Testing range 1-5 μmol

3.7 Determinations of bulk density (BD), pH, total solids (TS),

organic solids (OS) and ash content The total solids and organic solids were estimated according to the method given by

(Kalia et al. 1992b); Bulk density and pH were determined using the method

described by Ng et al. 2002. The samples were weighed in crucible and dried at 110 ±

5 °C for 16-18 h for TS estimation. This dried sample was then ignited, at 600 ± 25

°C for 2 h to determine its ash contents and organic solid.


47

Calculation:

Weight of dry empty crucible = W1g

Weight of empty crucible + Wet sample = W2 g

Weight of empty crucible + Dry sample (110 °C) = W3 g

Weight of empty crucible + Ignited sample (600 °C) sample = W4 g

%TS = W3-W1/ W2-W1x100

%ash = W4-W1/W3-W1x100 w. r. t. %TS = 100

%OS = 100 - % Ash w. r. t. %TS = 100

%TS = %OS + %ash

3.8 Determination of chemical oxygen demand (COD) COD (mg/L) determined by kits were procured from E-Merck. The samples were

digested for 2 h at 148 °C in thermoreactor TR200 (E-Merck), and OD was analyzed

on photometer SQ 118 (E-Merck).

3.8.1 Purpose and scope

The COD determines the amount of O2 required for chemical oxidation of organic

matter. The test is widely used to determine:

i) The degree of pollution load in water bodies and their self-purification capacity.

ii) Efficiency of treatment of plants.

iii) Provides rough idea of biological oxygen demand (BOD), which can be used for

BOD estimation.

COD determination has an advantage over BOD test that the results can be obtained

in less than 5 h whereas, BOD requires three or five days. But the limitation of the test

lies in its inability to differentiate between the biologically oxidizable and biologically

inert material.

3.8.2 Principle

Most of the organic material was oxidized to CO2 and water, when boiled with a

mixture of potassium dichromate in sulfuric acid. A sample is refluxed with a known

amount of potassium dichromate in sulfuric acid medium and excess of potassium

dichromate was titrated against ferrous ammonium sulfate. The amount of dichromate

consumed is proportional to the O2 required to oxidize the organic matter.


48

3.8.3 Reagents

A. Mercuric sulfate (HgSO4)

B. 0.25N potassium dichromate (K2Cr2O7)

Weighed 6.145 g pre dried (at 110 °C for 20-24 h) K2Cr2O7. Transferred to a 500 mL

volumetric flask. Added 100 mL DW to dissolve the salt. The final volume was made

up to 500 mL.

C. Silver sulfate sulfuric acid H2SO4.AgSO4

Weighed 4.4 g silver sulfate (AgSO4) and transferred to a brown bottle (as it is light

sensitive). Measured 500 mL sulfuric acid and transferred it to the bottle containing

AgSO4. The mixture was kept for 16-18 h for proper dissolution. The contents were

shaken well before use.

D. Higher range (1500-10000 mg/L COD): Solution A and B (from MERCK)

E. Lower range (100-1500 mg/L COD): Solution A and B (from MERCK)

3.8.4 Procedure

3.8.4.1 Pre-test for COD estimation

Weighed 20 mg HgSO4 and put in COD cell.

Added 1 mL sample (supernatant).

Added 0.5 mL K2Cr2O7.

Added 1.5 mL H2SO4.AgSO4.

If it appears yellow then expected COD is < 500 mg/L. If brown color appears

then expected COD is more than >1500 mg/L.

Then diluted the solution (sample) accordingly.

3.8.4.2 COD estimation by Merck method

A. For lower range (100-1500 mg/L COD)

Took COD cells.

Added 0.3 mL solution A and 2.3 mL solution B in a cell.

Then added 3 mL sample.

Kept it in thermo-reactor for 2 h.

Then cells were taken out and allow them to cool at room temperature.

OD read at 585 nm (Method no. 237).

Calculation: COD (mg/L) = OD x 1600x dilution


49

B. For higher range (1500-10000 mg/L COD)

Took COD cells.

Added 2.2 mL solution A and 1.8 mL solution B in a cell.

Then added 1 mL sample.

Kept it in thermo-reactor for 2 h.

Then taken out the cells and allow them to cool at room temperature.

OD read at 585 nm (Method no. 237).

Calculation: COD (mg/L) = OD X 4600 X dilution

Precaution: Before taking OD, shake the sample.

3.9 Estimation of total volatile fatty acids (VFA)

3.9.1 Gas liquid chromatography

Sample preparation

1 mL of sample from the reactors withdrawn and centrifuged at 10000 rpm and 4 oC

for 20 min. 200 μL samples were collected and stored in freeze after the addition of

1-2 drops of 25% orthoposphoric acid. The flame ionization detector (FID) mode was

used for the analysis of various volatile fatty acids produced during acidogenic stage

with the gas liquid chromatography (5700, Nucon Engineers). A chromosorb (101),

glass column (length = 4’ (I), O.D.=1/4”, I.D. 3 mm) was used for analyzing the

different fatty volatile fatty acid like acetic acid, propionic acid, butyric acid, valeric

acid, etc. N2 gas was used as the carrier at a flow rate of 30 mL/min. Different

standards were used of known concentration for determining the levels of these in the

sample by comparing the peak areas and retention time.

3.9.2 Volumetric method for estimation of VFA

Diallalo and Albertron method was used for the estimation of VFA.

3.9.2.1 Principle

The pKa value of the VFA lies between 3.3-3.5. These values of VFA samples are

achieve by titrating the sample with 1N H2SO4 solution. Evaporate VFA by lightly

boiling the samples. Neutralized this acidic solution with 0.1N NaOH to give

alkalinity in mg/L.


50

3.9.2.2 Procedure

Adjust your pH meter at 7 using standard buffer.

Check pH of the sample.

Take 50 mL of the decanted sample in a beaker (100 mL) and put a magnetic bar

in it.

Note: In case the quantity of sample available is less than 50 mL, then make up

the final volume to 50 mL by adding DW.

Check pH of the sample.

Switch on magnetic stirrer and put the beaker on it. Insert the pH electrode in to

the sample.

Note the pH and titrate it with 1N H2SO4 till the final pH is 4. Record the volume

of acid consumed for bringing down the pH to 4.

Continue titration till the sample pH gets reduced to approximately 3-3.2 and

again record the volume of the acid consumed.

Warm slightly the sample.

Cool the sample to room temperature.

Record the pH and titrate the sample with 0.1N NaOH to increase the pH up to 4.

Note down the volume of NaOH used.

Continue titration for increasing the pH from 4 to 7.

Record the volume of NaOH used (This value will be used for final calculations).

This step should do very carefully.

Calculation:

Total volatile acids alkanity (TVAA) = mL of 0.1 N NaOH used x 2500 x 2 50 Notes:

If the observed value is greater than 180 mg/L volatile acid alkalinity, then: volatile acid = V.A.A. x 1.5.

If the original sample had been less than 50 mL, then multiply the final volume (after taking into

consideration the value of 1.5) with the dilution factor.

3.10 Microbial consortia preparation for hydrogen production A set of 11 H2 producing bacterial strains: Bacillus sp. strains EGU91 and HPC459,

B. cereus strains EGU41 and EGU43, B. megaterium HPC686, B. pumilus HPC464,

B. thuringiensis EGU45, B. avium EGU31, Enterobacter aerogenes EGU16 and

Proteus mirabilis strains EGU21 and EGU30 were used for preparing MMCs


51

(MMC1-MMC11). These mixed cultures were also designed on the basis of Plackett-

Burman method. The different isolates used for preparing the mixed cultures had

0.85-1.9x106 viable cells/µg protein. Each mixed culture was prepared by mixing

different microbes in equal proportions amounting to a final protein concentration of

40 μg cell protein/mL, unless otherwise mentioned (Results pertaining to these

experiments have been presented in Table 4.4).

3.11 Mixed microbial consortia hydrogen production under batch

culture 3.11.1 Optimization of physiological process parameters

Batch culture experiments were done in 300 mL BOD bottles, with the working

volume of 250 mL M-9 medium using glucose as feed (pH 7), unless otherwise

mentioned. These were inoculated individually with MMCs (MMC4 and MMC6), in

which different bacterial cultures were pooled together in equal proportions 40 µg

protein/mL feed, unless otherwise mentioned. All the batch fermentation experiments

were done in triplicate and incubated at 37 oC, unless otherwise mentioned.

3.11.1.1 Effect of sugars

Different sugars (Glucose, fructose, maltose, lactose, galactose, starch and CM-

cellulose) as carbon sources in the concentration range of 0.5, 1, 2, 3 and 5 % w/v as

feed were tested (Results pertaining to these experiments have been presented in

Table 4.5).

3.11.1.2 Effect of inoculum size

Inoculum sizes were tested in the range of 10, 20, 40, 60, 80, 100 and 120 µg cell

protein/mL feed (Glucose concentration of 0.5% w/v) (Results pertaining to these


3.11.1.3 Effect of medium concentration

Medium (M-9) concentrations used were 0.1, 0.5, 1, 1.5, 2, 2.5, 5 and 10x in glucose

0.5% w/v as feed and inoculum size of 10 μg cell protein/mL feed (Results pertaining

to these experiments have been presented in Table 4.7).


52

3.12 Mixed microbial consortia hydrogen production under

continuous culture

Continuous culture experiments were done in 1 L Aspirator bottles (20x10 cm) with

the working volume of 800 mL (680 mL by medium and 120 mL by ligno-cellulosic

support materials or PVC) M-9 medium (0.05x) using 0.5% glucose as feed (pH 7),

unless otherwise mentioned (Figure 3.4). Inoculum was added individually with

different MMCs (MMC4 and MMC6) in equal proportions amounting to a final of 10

µg cell protein/mL feed. All the continuous fermentation experiments were done in

duplicate and incubation at 37 oC.

Figure 3.4: Fermenter set of 1000 mL capacity

3.12.1 Preparation of immobilized cells for continuous culture digestion

Four lingo-cellulosic materials - Banana leaves (BL), Coconut coir (CC), Groundnut

shells (GS) or Pea-shells (PS) dried (3g each) were packed in to PVC

(Polyvinylchloride) tube (length: 3 cm and diameter: 2 cm) and tied with a 10 cm2

nylon net (Figure 3.5).


53

Figure 3.5: Development of PVC cartridge for immobilization of mixed microbial cultures

The properties of ligno-cellulosic support materials for ash content, pH and bulk

density were given in the Table 3.7.

Table 3.7: Properties of biowaste materials

Type of waste material Ash content (%)

pH Bulk density (g/mL)

Pea-shells (PS) 7.5 5.15 0.444

Banana leave (BL) 10.0 6.63 0.420

Coconut coir (CC) 2.8 6.65 0.062

Groundnut shells (GS) 2.3 5.55 0.400

Values based on two set of experiments and two repetitions. Standard deviation was less than 10%.

3.12.2 Optimization of physiological process parameters

Reactors were run for 16 days of steady state equal to 4 cycles at an HRT of 4 days

under continuous culture, unless otherwise mentioned. Controls with free floating

cells were run in parallel.

PVC ring Dried bio-waste materials (GS, CC, BL and PS)

Nylon net

++++

PVC Cartridge


54

3.12.2.1 Glucose concentration

Glucose concentrations in the range of 0.5, 1 and 2% w/v were used as feed (Results

pertaining to these experiments have been presented in Table 4.9).

3.12.2.2 Medium concentration

Medium M-9 concentrations in the range of 0.05, 0.1 and 0.5x were used. Glucose 0.5%

as feed (Results pertaining to these experiments have been presented in Table 4.10).

3.12.2.3 Effect of HRT

At hydraulic retention time (HRT) of 2 days (or 4 days), 340 mL (or 170 mL) were

drained from each reactor every day, without removing support material and replaced

with 340 mL (or 170 mL) of fresh 0.5% glucose containing M-9 medium (0.05x).

Experiments were run for 41 days of steady state equal to 10 cycles at an HRT of 4

days and for 60 days of steady state equal to 30 cycles at an HRT of 2 days (Results


3.12.2.4 Growth and pH monitoring of microbial population in the reactor

The estimate of the bacterial population retained, within the continuous culture

digestion bottles, due to different support materials was obtained by measuring the

turbidity of the effluent at OD660 and pH on daily basis.

3.13 Reuse of immobilized support material The bioreactors with immobilized support material were washed twice with 0.1N HCl

and then filled with DW and kept at room temperature for 24 h to remove the traces of

the unconsumed feed material and bacterial cell. The washed bioreactors were again

supplemented with different feed material in the same manner as in the beginning.

Thus immobilized support materials were reused for another cycle of

biotransformation (Kalia and Lal, 2006).

3.14 Hydrolytic abilities of microbial isolates

The hydrolytic abilities of the strains were checked enzymes activities for lipase,

amylase and protease at different pH in the range of 5-12 (Results pertaining to these



55

3.14.1 Mixed hydrolytic consortia preparation for hydrolyzing the

biowaste

A set of 11 bacterial strains: B. subtilis EGU475, B. sphaericus strains EGU385 and

EGU542, B. thuringiensis EGU378, Bacillus sp. strains EGU367, EGU85, EGU444

and EGU447, marine bacterium strain EGU409 and P. mirabilis strains EGU32 and

EGU30 were used for the preparation of mixed hydrolytic bacterial cultures (HC1-

HC11). Eleven mixed hydrolytic cultures (HCs) for hydrolysis of biowaste based on

11 different microbial isolates was designed on the basis of Plackett-Burman method.

The different isolates used for preparing the mixed cultures had 0.85-1.9x106 viable

cells/µg protein to achieve a concentration where different isolates were finally

present in equal proportions (Results pertaining to these experiments have been

presented in Table 4.12)..

3.14.2 Hydrogen production abilities of mixed hydrolytic consortium and

mixed microbial cultures strains

For batch-culture digestion, green PS (10% total solids (TS) and 9.5% organic solids)

collected from local market of Delhi (India) were cut into small pieces (1-2 cm). PS

slurry (PSS) (250 mL), 2% TS was made in DW in 300 mL BOD bottles. It was

inoculated individually with HC1-HC11 and MMC1-MMC11 incubated at 37 oC for 2

days and H2 production abilities monitor, unless otherwise mentioned. Each mixed

culture was prepared by mixing different microbes in equal proportions amounting to

a final protein concentration of 10 µg/mL. The pH of the reactors was checked and

adjusted to 7 by 2N NaOH or 2N HCl daily. Anaerobic conditions were maintained in

the reactor by flushing with Ar. All the batch fermentation experiments were done in

duplicate and incubation at 37 oC.

3.14.3 Optimization of process parameters for hydrogen production by

mixed microbial cultures under batch culture from pre-hydrolyzed

pea-shells slurry

3.14.3.1 Effect of media and mixed bacterial cultures

PSS, TS 2% were supplemented with two media (M-9 and GM-2) at a concentration

of 0.05x with during the hydrolysis with 11 HCs. These were then incubated to check

the H2 producing abilities of HCs. These were compared with PSS which was not


56

supplemented with any nutrient medium (Results pertaining to these experiments have

been presented in Table 4.13).

In another setup, PSS without any medium supplement were evaluated for their H2

producing abilities with only mixed microbial cultures - MMC1-MMC11 (Results


Finally a combination of HCs and MMCs were used to evaluate H2 production from PSS

(2% TS) without any nutrient supplement as follows: initial hydrolysis with HC1- HC11

was followed by inoculation with MMC4 and MMC6 i.e. a total of 22 combinations

(Results pertaining to these experiments have been presented in Table 4.15).

3.14.3.2 Effects of inoculum size and fibrous sheath

PSS (2% TS) was hydrolyzed with HCs (HC2, HC5, HC6 and HC8) at two different

inoculum sizes of 10 and 20 µg cell protein/mL feed and incubated period of the 2 days.

The hydrolysed PSS was subdivided into two components: i) with fibrous sheath and ii)

without fibrous sheath. For preparing the PSS without fibrous sheath the following

procedure was adopted: hydrolysed PSS was macerated and sheath-like structures were

separated out by filtration and slurry was centrifuged at 4000 rpm for 20 min at 4 °C. PSS

in 16 different combinations of HCs and MMCs were subjected for H2 production as

follows: (i) HC2 and HC6 with MMC4 and (ii) HC5 and HC8 with MMC6 (Results


3.14.3.3 Effects of incubation period and total solids

PSS (2% TS) were subjected to hydrolysis for periods of the 2 and 4 days. These

hydrolysed feed materials were segregated in to two types: (i) with fibrous sheath and

(ii) without fibrous sheath (By a method described above). These were subjected to

H2 production with MMC4 and MMC6.

PSS (1, 2, 3 and 5% TS) were hydrolyzed with HCs (HC2, HC5, HC6 and HC8) for 2

days. The hydrolysed PSS was subdivided into two components: i) with fibrous

sheath and ii) without fibrous sheath. PSS in 32 different combinations of HCs and

MMCs were subjected for H2 production as follows: (i) HC2 and HC6 with MMC4


57

and (ii) HC5 and HC8 with MMC6 (Results pertaining to these experiments have

been presented in Table 4.17).

3.15 Up-scaling of hydrogen production from biowaste Batch culture scale-up of PSS pre-hydrolyzed with HC2 were done for H2 production

with MMC4 at 0.75, 1.5 and 4 L feed in the reactors of 1, 2 and 5 L capacities,

respectively (Results pertaining to these experiments have been presented in Table 4.18).

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