Chapter-3 Materials and Methods
Chapter-3 Materials and Methods
32
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.
Chapter-3 Materials and Methods
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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.
Chapter-3 Materials and Methods
34
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.
Chapter-3 Materials and Methods
35
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).
Chapter-3 Materials and Methods
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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.
Chapter-3 Materials and Methods
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.
Chapter-3 Materials and Methods
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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
Chapter-3 Materials and Methods
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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
Chapter-3 Materials and Methods
40
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).
Chapter-3 Materials and Methods
41
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.
Chapter-3 Materials and Methods
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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.
Chapter-3 Materials and Methods
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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
Chapter-3 Materials and Methods
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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
Chapter-3 Materials and Methods
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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.
Chapter-3 Materials and Methods
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.
Chapter-3 Materials and Methods
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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.
Chapter-3 Materials and Methods
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
Chapter-3 Materials and Methods
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.
Chapter-3 Materials and Methods
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
Chapter-3 Materials and Methods
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
experiments have been presented in Table 4.6).
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).
Chapter-3 Materials and Methods
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).
Chapter-3 Materials and Methods
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
Chapter-3 Materials and Methods
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
pertaining to these experiments have been presented in Table 4.11).
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
experiments have been presented in Table 4.2).
Chapter-3 Materials and Methods
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
Chapter-3 Materials and Methods
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
pertaining to these experiments have been presented in Table 4.14).
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
pertaining to these experiments have been presented in Table 4.16).
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
Chapter-3 Materials and Methods
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).