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CHAPTER 3
MATERIALS AND METHODS
3.1 GENERAL
This chapter describes the materials and the methodology adopted
in the study on co-digestion of tannery solid wastes. In the materials section,
the substrates selected, criteria for selection of the substrates and inoculum
selected are detailed. The chemicals, buffers and instruments used for
analysis of various characteristics are also discussed. The details of
experiments conducted are presented in section 3.4.
3.2 MATERIALS
3.2.1 Substrates
The substrates selected for the co-digestion studies were (i)
fleshings, (ii) primary sludge and (iii) secondary sludge. Fleshings are the
solid wastes generated during the processing of raw hides/ skins into finished
leather. The primary and secondary sludges used in the study are from a
tannery wastewater treatment plant. Quantity of fleshings generation depends
on type of raw material i.e. hides/skins processed. The quantity of fleshings
generation in turn depends upon the type of animal skin i.e. sheep, goat, cow
calf, buff calf or hide. In Vaniyambadi and Ambur clusters in Tamil Nadu,
India sheep skins are mainly used as raw material for leather processing,
whereas in Ranipet, hides are used. In Pernambut cluster in Tamil Nadu, goat
79
skins are used as raw materials for leather processing. In north and eastern
region i.e. Jalandhar, Kanpur and Kolkata, majority of tanneries use hides as
raw material. Apparently, the usage of raw material depends on type of
leather to be processed and its usage for various applications.
3.2.1.1 Selection of Substrates
Fleshing has 80 – 90 % moisture content and the remaining portion
contains collagen together with fat and lipids. Such lipid-rich waste is
produced in the food processing industry, slaughterhouses, edible oil
processing industry, dairy products industry and from the olive oil mills. In all
the lipid-rich wastes, lipids are one of the main problematic components.
Lipids cause operational problems in anaerobic digesters due to clogging and
also cause mass transfer problems for soluble substrates since they become
adsorbed to the microbial biomass surface (Pereira et al 2004). Nevertheless,
lipids are attractive substrates for anaerobic co-digestion due to the higher
methane yield obtained when compared to proteins or carbohydrates. In this
context, lipid-rich waste can be regarded as a large potential renewable energy
sources (Hansen 1999). In an anaerobic environment, lipids are first
hydrolyzed to glycerol and free long chain fatty acids (LCFAs). This process
is catalyzed by extracellular lipases that are excreted by the acidogenic
bacteria. The further conversion of the hydrolysis products takes place in the
bacterial cells. Glycerol is converted to acetate by acidogenesis, while the
LCFAs are converted to acetate (or propionate in the case of odd-number
carbon LCFAs) and hydrogen through the -oxidation pathway (syntrophic
acetogenesis) (Weng and Jeris 1976). The LCFAs are the key factors in the
inhibition of lipid degradation.
80
The enthalpy is the preferred expression of system energy changes
in many chemical, biological and physical measurements and simplifies the
certain descriptions of energy transfer. An enthalpy change is approximately
equal to the difference between the energy used to break bonds in a chemical
reaction and the energy gained by the formation of new chemical bonds in the
reaction. It describes the energy change of a system at a constant pressure.
Enthalpy change is denoted by H.
H = Hfinal - Hinitial
if the system has a lower enthalpy at the end of the reaction, then it gave off
heat during the reaction (exothermic reaction. ). For exothermic reactions H
is negative (- H).
In most of the biological processes under constant atmospheric
pressure conditions, the heat is absorbed or released in termed enthalpy .The
heat release in this example was calculated based on the reaction enthalpies of
the stoichiometric degradation of reference substances for carbohydrates
(glucose), fats (palmitic acid) and proteins (alanin) and HRº
values were
calculated (D'Ans and Lax 1983).
1 C6H
12O
6 3 CO
2+ 3 CH
4 R° = 138.5 kJ/Mol
2 C3H
7NO
2+ 2 H
2O 3 CO
2+ 3 CH
4+ 2 NH
3 R° = + 198.5
kJ/Mol
2 C16
H12
O6
+ 14 H2O 9 CO2+ 22 CH
4 HR
º = + 544. 5 kJ/Mol
In the anaerobic degradation of fats and proteins, change in
enthalpy is positive indicating that their anaerobic degradation is endothermic.
Treatment of fatty materials by anaerobic digestion is often
hampered because of the inhibitory effect of LCFAs. The LCFAs have been
81
reported to be inhibitory at low concentrations for gram-positive but not for
gram-negative microorganisms (Kabara et al 1977). Methanogens can be
inhibited by LCFAs due to their cell wall, which resembles that of gram-
positive bacteria (Zeikus 1977). The LCFAs show acute toxicity towards
anaerobic consortium by adsorption onto the cell wall/membrane, interference
with the transport or protective function (Rinzema et al 1994). In addition,
sorption of a light layer of LCFAs to biomass leads to the flotation of sludge
and consequent sludge washout (Rinzema et al 1989). In upflow anaerobic
sludge blanket (UASB) reactors, granular sludge flotation sometimes
occurred at concentrations far below the toxicity limit (Hwu et al 1998). The
difficult nature of these wastes could be overcome by co-digestion, which
could be advantageous due to an improved C/N ratio and dilution of the
inhibitory compounds (Tritt 1992).
Fat and proteins, which are biodegradable, yield the highest
percentage of CH4. However, fat and proteins available from industrial wastes
such as slaughter house inhibit the anaerobic digestion process through the
accumulation of volatile fatty acids and long chain fatty acids (Salminen et al
2002 and Broughton et al 1998). In the present study, fleshings are lipid rich
wastes whereas the sludges consist of proteins and carbohydrates. The pH of
fleshings are around 11 to 12, when these sludges are added to the fleshings
first of all it neutralizes the pH. Hence it acts as a buffer and makes the
substrates amenable for anaerobic digestion process. Not only that but also
addition of sludges to the fleshings, the operational problems associated with
the anaerobic digestion of lipid rich wastes, primarily flotation and clogging
of reactors, can be eliminated. Hence these two substrates i.e. primary and
secondary sludges were selected along with fleshings for co-digestion of
tannery solid wastes in the present study.
82
3.2.1.2 Sample Preparation for Fleshings
In the present study, fleshings were collected from a commercial
tannery immediately after fleshing operations are over in the tannery. The
fleshings were arbitrarily cut into small pieces of size 1 x 1 cm with the help
of knife in order to facilitate the feed into reactors with the size of opening of
about 2 cm in diameter.
3.2.1.3 Selection of Mix Proportions
In this study, the experiments were designed based on the amount
of solid wastes generated in the tannery i.e. fleshings, primary and secondary
sludges for processing one tonne of raw hides/ skins into semi-finish leather
or raw to finished leather. Based on waste generation, as detailed in the
chapter 1, section 1.6.2 of Table 1.6 and section 1.7.2 of Table 1.8, the
average quantity of fleshings generation will be 150 kg per tonne of raw
hides/ skins processed. The primary and secondary sludges generation during
treatment of wastewater for processing of one tonne of raw hides/skins into
semi-finish leather and raw hides/skins into to finished leather will be in the
range of 88-123 kg and 179-236 kg respectively. Co-digestion studies were
carried out for various mix proportion of the substrates fleshings (F), primary
sludge (PS) and secondary sludge (SS). The details of substrates and mix
ratios selected are presented in Table 3.1. In order to optimize the mix
proportion i.e. F: PS: SS, the process parameters i.e., biogas generation,
biogas generation per gram of volatile solids added and percentage volatile
solids reductions were taken into consideration.
83
Table 3.1 Substrates and Mix Ratios
Sl.No. Substrates Mix ratio
(F:PS:SS)
PS:SS
ratio
F: S* ratio VS added
(g)
1 F:PS:SS 1.00: 0.25:0.75 0.25:0.75 1.00: 1.00 5 and 8
2 F:PS:SS 1.00:0.30:2.70 0.30:2.70 1.00: 3.00 10 and 12
3 F:PS:SS 1.00:0.50:0.50 0.50:0.50 1.00: 1.00 5 and 8
4 F:PS:SS 1.00:0.50:1.50 0.50:1.50 1.00: 2.00 6 and 7.5
5 F:PS:SS 1.00:0.75:0.25 0.75:0.25 1.00: 1.00 5 and 8
6 F:PS:SS 1.00:1.00:1.00 1.00:1.00 1.00: 2.00 6 and 7.5
7 F:PS:SS 1.00:1.50:0.50 1.50:0.50 1.00: 2.00 6 and 7.5
8 F:PS:SS 1.00:1.50:1.50 1.50:1.50 1.00:3.00 10 and 12
9 F:PS:SS 1.00:1.80:0.20 1.80:0.20 1.00: 2.00 6 and 7.5
10 F:PS:SS 1.00:2.70:0.30 2.70:0.30 1.00: 3.00 10 and 12
Note: *S refers to mixture of PS and SS
Hence the investigations covered 20 experiments (10 x 2). For a
typical tannery, the fleshings generation is same irrespective of type of
process i.e. raw to semi-finished leather or raw to finished leather. The only
variable will be the generation of primary and secondary sludge. Hence in all
the experiments the fleshings proportion was kept constant but PS: SS ratio
was varied. The sludge generation details are presented in section 1.7.2 of
Table 1.7 in chapter 1. Considering this aspect, various mix proportions of F:
PS: SS were tried in the present study. For various mix proportion of
substrates i.e. F:PS:SS, the VS input of 5, 6, 7.5, 8, 10 and 12 grams were
studied with the total solids input for these VS content ranging from 8 to 21
grams. In order to operate the digesters with solids content less than 10% (i.e.
84
low solids system contain less than 10% solids) as reported by
Techobanoglous et al (1993) and also considering the volume of the reactor,
VS input was selected in the present study.
3.2.1.4 Inoculum
The inoculum was collected from an anaerobic digester operating
for the digestion of waste activated sludge (WAS) generated from treatment
of domestic sewage located at Chennai, India.
3.2.2 Chemicals and Buffers
The list of chemicals and the buffers used for characterization of
samples are detailed below:
For measurement of pH in samples, pH electrode was calibrated
using buffers 4,7 and 10 from Merck chemicals. Similarly, oxidation
reduction potential (ORP) electrode was calibrated using buffer 200 m from
HacH.
For analysis of chemical oxygen demand (COD), analytical grade
ammonium ferrous sulphate, concentrated sulphuric acid, silver sulphate,
potassium dichromate, mercuric sulphate and ferroin indicator from Merck
Chemicals were used.
For analysis of total kjeldahl nitrogen (TKN), concentrated
sulphuric acid, catalase indicator (potassium sulphate and copper sulphate),
boric acid, methyl red and methylene blue, isopropyl alcohol and sodium
hydroxide pellets from Merck Chemicals were used.
85
For analysis of ammonia (total and free), boric acid, methyl red,
methylene blue, isopropyl alcohol, sodium hydroxide pellets and sulphuric
acid from Merck Chemicals were used. For analysis of calcium, disodium
salts of ethylene di-amine tetra acetic acid, sodium hydroxide, mixture of
murexide dye and sodium chloride as an indicator were used.
For total chromium analysis, samples were digested using
concentrated sulphuric acid, nitric acid and perchloric acid. The chromium
standard was procured from Merck Chemicals. Sample pellets were prepared
using potassium bromide (KBr) for FT-IR analysis. Commercial grade
steapsin lipase (catalog no. 124549) was procured from Sisco Research
Laboratories, Mumbai, India.
For analytical purpose, laboratory grade chemicals with 99.99 %
purity were used. For reagent preparation, volume make-up, double distilled
water was used. Instruments were calibrated before taking the readings.
3.2.3 Macro and Trace Elements
During the start-up of the experiments, macro nutrients and trace
elements were added in the order of one mL per one liter of reactor volume.
The composition of macro nutrients and trace elements are presented in Table
3.2. The chemicals used for the study were procured from Merck Chemicals.
86
Table 3.2 Composition of Macro Nutrients and Trace Elements
Sl. No Macro Nutrients and Trace Elements Weight Taken
I Macro Nutrients
1 Phosphate Buffer
a NH4Cl 1.4 g*
b KH2PO4 8.5 g*
c K2HPO4 21.75 g*
d Na2 HPO4.7 H2O 33.4 g*
2 CaCl2 27.5 g/L
3 MgSO4.7 H2O 22.5 g/L
4 FeCl3. .6H2O 0.25 g/L
II Trace Elements
a MnCl2. 4H2O 0.18g*
b CoCl2.6H2O 0.2 g*
c CuSO4.5H2O 0.19 g*
d NiSO4.7H2O 0.23 g*
e ZnSO4.7H2O 0.21 g*
f Na2MoO4.2 H2O 0.126 g*
Note : * For preparation of phosphate buffer and trace elements, known quantity of above
referred chemicals were dissolved in one liter of double distilled water.
3.2.4 Instruments Used
pH meter- HacH Sension 378 model equipped with jel filled
electrode
Oxidation Reduction Potential (ORP) - HacH Sension1
model
COD – Thermo reactor for digestion of samples
87
Total Kjeldahl Nitrogen (TKN) - digestion and distillation of
samples were done using Kel Plus – Elite Ex instrument.
Atomic Absorption Spectrophotometer (AAS) from Perkin
Elmer, India was used for total chromium analysis.
Sonication tests were conducted using Digital Sonicator 250
model, Branson, USA.
3.3 ANALYTICAL METHODS
3.3.1 Characterization of Substrates and Inoculum
Fleshings samples were collected from a commercial tannery.
Primary and secondary sludge samples were collected from a common
effluent treatment plant (CETP) exclusively operating for the treatment of
tannery wastewater situated at Chennai, India. Before feeding the substrates
into the digesters, the samples were analyzed in triplicate and the average
values were considered as feed characteristics. In the present study, the
inoculum was obtained from an anaerobic digester operating for the digestion
of waste activated sludge (WAS) generated from treatment of domestic
sewage located at Chennai, India. The inoculum samples were analyzed in
triplicate and the average values were reported. The details of the
characteristics considered and the analytical methods followed are presented
in the Table 3.3.
88
Table 3.3 Characteristics and Analytical Methods
Sl.
No.Characteristics
Analytical Method/
instrument
Applicable to
Reference
Fleshings
Primary,
Secondary Sludge
and Digestate
Inoculum
1 pH Method 4500 – H+ B APHA 1998
2 Alkalinity Method 2320 APHA 1998
3 Total solids and volatile
solids
Method 2540 G APHA 1998
4 Total kjeldahl nitrogen
(TKN)
Method 4500 C APHA 1998
5 Total and Free Ammonia Method 4500 NH3 E APHA 1998
6 Total protein multiplying the TKN
value by the factor of
6.25
7 Fat content Method 5520 E APHA 1998
8 Moisture content CPHEEO
(2000) and
Peavy et al
(1985)
9 Oxidation reduction
potential (ORP)
Using platinum
electrode with HACH
Model 51937
APHA 1998
89
Sl.
No.Characteristics
Analytical Method/
instrument
Applicable to
Reference
Fleshings
Primary,
Secondary Sludge
and Digestate
Inoculum
10 Carbon, hydrogen,
nitrogen and sulphur
content
Elemental Analyzer,
CHNS-O, Model- Euro
EA 3000, Euro Vector
Spa, Via Tortona,
Milan, Italy
11 Volatile fatty acids (VFA) Thermo Scientific
Cerus 800 model gas
chromatography using
flame ionization
detector (FID)
12 Biogas analysis Thermo Scientific
Cerus 800 model gas
chromatography using
Thermal conductivity
detector (TCD)
13 Chemical Oxygen Demand
(COD)
Method 5220 B APHA 1998
14 Soluble COD Method 5220 B APHA 1998
15 Chromium Method 3111 B APHA 1998
16 Calcium Method 3500 – Ca D APHA 1998
Table 3.3 (Contd..)
90
3.3.2 Analysis of Biogas and Volatile Fatty Acids (VFAs)
The composition of biogas and VFA were analyzed with the help of
Thermo Scientific Cerus 800 model gas chromatography (GC). The
composition of biogas i.e CH4, CO2 and H2 was analyzed with the help of the
GC fitted with a thermal conductivity detector (TCD) and a 1.83 m x 3.18 mm
ID stainless steel packed column with a molecular sieve of 5A. The oven,
injector and detector temperatures were kept at 50ºC, 70ºC and 200ºC
respectively. Helium was used as carrier gas at a flow rate of 2 mL /minute.
After biogas generation ceased, the digestate samples were centrifuged at
10000 rpm for 15 minutes. Centrifuged samples were acidified using
concentrated formic acid to a pH of 2 to 3 for VFA analysis. Mixed standard
contains acetic, propionic, isobutyric, butyric, isovaleric, valeric, isocaproic
heptonic and hexonic acids were used for calibration of GC to analyze VFA
composition. The standard was procured from Supelco, Balgalore, India.
The VFA was measured with the help of GC fitted with flame
ionization detector (FID) and capillary column of 0.32 mm ID and 60 m
length. The oven, injector and detector temperatures were kept at 110ºC,
180ºC and 220ºC respectively. Helium was used as carrier gas at a flow rate
of 2 mL per minute with split ratio of 1:10. One µL of acidified samples were
injected to GC to analyze VFA composition for acetic, propionic, isobutyric,
butyric, isovaleric, valeric, isocaproic, heptonic and hexonic acids
individually. Acetic acid equivalent of VFA was used to calculate the ratio of
VFA to alkalinity ratio.
3.3.3 Characterization of Digestate
3.3.3.1 Elemental Analysis
The digestate at the end of co-digestion process was characterized
in order to ascertain how the substrates were transformed into the end
91
products. Also this will help in deciding the whether the digestate can be used
as a manure. At the end digestion i.e. after 50 days, the digestate samples
were air dried to remove moisture. Carbon, nitrogen, hydrogen, sulphur
content present in the digestate were analyzed using Elemental Analyzer,
CHNS-O (Model- Euro EA 3000).
3.3.3.2 Fourier Transform Infrared Spectrometry (FT-IR) Analysis
Substrates (i.e. fleshings, primary sludge, and secondary sludge),
inoculum and digestate samples (after 10 days of digestion and at the end of
digestion process) were air dried to remove moisture. Pellets of sample with
potassium bromide (KBr) were made in the ratio of 5: 1. The pellets were
subjected to FT-IR analysis using transmission mode. The measurements
were carried out in the mid-infrared range from 4000 to 500 cm–1
with ABB
MB 3000 FT-IR spectrometer.
3.3.3.3 Thermogravimetric Analysis (TGA)
Digestate at the end of co-digestion process was air dried. Thermal
profiles were taken from 0 to 800ºC using thermogravimetric analyzer (TGA,
Model Q50) to assess the weight changes in digestate as a function of
temperature.
3.3.3.4 Differential Scanning Calorimeter (DSC) Analysis
Digestate at the end of co-digestion process was air dried. Air dried
digestate sample was taken for DSC studies. Thermal profiles were taken
from 0 to 300ºC using DSC (Model Q200), in an inert atmosphere of nitrogen
to assess thermal changes as a function of input temperature.
92
3.3.3.5 Scanning Electron Microscopy (SEM) Analysis
Fleshings and digestate was fixed in 2% glutaraldehyde (w/v) 2 h
independently. After washing with saline solution, the samples were
dehydrated in 30 – 100% water ethanol ratios. The air-dried particles were
coated with 120– 130 µm gold in argon medium. Scanning electron
microscopy (SEM) observations were performed on a scanning device
attached to a JEOL JM-5600 electron microscope at 20 kV accelerating with
an electron beam of voltage 5–6 nm. The SEM images were taken to identify
and to confirm the stages of hydrolysis and digestion of substrates during co-
digestion process.
3.3.3.6 Particle Size Analysis
At the end of co-digestion process the digestate was analyzed for
particle size distribution using laser scattering particle size distribution
analyzer using Horiba LA-950 model. Digestate sample was directly taken for
particle size distribution after co-digestion process.
3.4 EXPERIMENTAL SETUP
In the present study, first the characterization of substrates and
inoculum, optimization studies i.e effect of mix proportion of substrates on
co-digestion, effect of residence time on co-digestion, effect of inoculum to
substrate ratio, detailed co-digestion studies and characterization of digestate
were carried out. The effect of pretreatment of primary and secondary sludges
on co-digestion process for enhancement of biogas generation and the effect
of lipase addition on digestion process were investigated next.
To conduct the experiments, tee actors were fabricated in 3 sizes
i.e. (i) 0.65 L (ii) 2 L and (iii) 5 L. The reactors were made up of glass.
93
Similarly for biogas collection, glass bottles were used. Altogether two
experimental setups were used to cover the studies planned. The details are
presented in Table 3.4.
Table 3.4 Reactor Details
Sl.No. Experimental
Setup
Number
Experiments
Conducted
Reactor
Volume (L)
Working
Volume (L)
1 I (i) optimization studies,
(ii) pretreatment and
(iii) lipase studies
0.65 0.50
2 II (i) co-digestion studies 2 or 5 1.5 or 3.0
The main criteria for deciding the size of laboratory scale reactors
is the ease with which operating parameters could be altered and the results
could be monitored. The size of reactors varied depending upon the
requirement, but for experimental work, an upper limit of 5 L capacity was
used (Stafford et al 1980; Hobson and Wheatley 1992).
3.4.1 Experimental Setup I
To measure biogas, 650 mL glass bottle was filled with water and
closed with a rubber cap and aluminum seal to make it air tight. The biogas
collection bottle was kept in an inverted position. Both reactor and biogas
collection bottles were connected by a flexible rubber tube. Biogas generation
from the reactors was monitored by means of a water displacement method
based on mariotte principle i.e. the volume of water displaced is equivalent to
volume of biogas generated (Itodo et al 1992). The schematic diagram of the
experimental setup I is shown in Figure 3.1.
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Figure 3.1 Experimental Setup I
3.4.2 Experimental Setup II
After optimization of mix proportion of substrates, residence time
and inoculum to substrate ratio, co-digestion studies were carried out using 2
L or 5L capacity reactors. Rectors with 3 wide ports were fabricated using
the Borosil glass. One port used for feed purpose was fitted with rubber cork
to make it air tight, the second port was fitted with rubber cork with gas
collection system and third port was made as dummy. The schematic diagram
of the experimental setup II is shown in Figure 3.2.
1
1
2
3
4
4
5
8
7
4
6
LEGEND
1 Reactor
2 Feed
3 Rubber tubing
4 Needle
5 Funnel
6 Water
7 Support stand
8 Water displaced by biogas
95
Figure 3.2 Experimental Setup II
The reactors were provided with an outlet in order to withdraw a
part of digested sludge at the end of the digestion process. The volume of
biogas generated was measured using another glass bottle either 2 L or 5 L
capacity filled with water having an out let at the bottom. A conical flask with
funnel was placed below the out let of glass bottle filled with water to
measure volume of biogas. Both the reactor and gas collection system were
connected with flexible rubber tube. Biogas generation from the reactors was
monitored by means of a water displacement method.
RUBBER TUBING
BIOGAS
FEED
INLET
DIGESTER GAS
HOLDER
BIOGAS
WATER
GRADUATED
BEAKER
OUTLET
SUBSTRATES
96
3.4.3 Conditions Maintained
After adding the substrates and inoculum, the reactors were closed
with a rubber cap and an aluminum seal to make them air tight. The head
space of the reactors was purged with nitrogen gas at the rate of 15 mL per
second for 25 minutes into the reactors to remove oxygen and to maintain
anaerobic conditions. Daily the reactors were gently agitated twice to avoid
stratification. All the experiments were conducted in the ambient temperature
(i.e 28 ± 50
C).
3.5. PHYSICO-CHEMICAL CHARACTERISTICS OF
SUBSTRATES AND INOCULUM
Characterization of substrates and inoculum were carried out as per
Standard Methods, 19th
Edition (APHA 1998). The details of the
characteristics and analytical methods followed are present in Table 3.3 of
section 3.3.1.
3.6 ELEMENTAL ANALYSIS
The elemental analysis of substrates and inoculum in terms of
carbon, hydrogen, nitrogen, sulphur on dry weight basis was done using the
elemental analyzer, CHNS-O (Model- Euro EA 3000).
3.7 OPTIMIZATION STUDIES
Studies were carried out to optimize the (i) mix proportion of
substrates, (ii) residence time and (iii) inoculum to substrate ratio.
97
3.7.1 Effect of Mix Proportion of Substrates on Co-Digestion
Experiments were conducted by varying the mix proportions of
fleshings, primary sludge and secondary sludge to evaluate the effect of mix
proportion of substrates with the volatile solids input of 5, 6, 7.5, 8, 10 and 12
grams. The details of combination of mix proportions of substrates are
presented in Table 3.1 of section 3.2.1.3. The experiments were carried out
for a residence time of 50 days. Daily biogas generation was monitored. For
mix proportion of substrates each, duplicate reactors were operated and the
mean values are reported. The biogas generation and VS reduction were
observed for all the mix ratios of the substrates.
The best mix proportion leading to the maximum biogas generation
was selected to further study the (i) effect of inoculum to substrate ratio (ii)
co-digestion studies (iii) effect of pretreatment of sludges on co-digestion and
(iv) effect of application of lipase on digestion process for a residence time of
50 days.
3.7.2 Effect of Residence Time on Co-Digestion
For various mix proportions as detailed in section 3.2.1.3 of Table
3.1, the optimum residence time required for co-digestion of tannery solid
wastes was ascertained.
3.7.3 Effect of Inoculum to Substrate (I/S) Ratio
The seven reactors were called R1, R2, R3, R4, R5, R6 and R7 of
650 mL capacity was used to study the effect of I/S ratio on co-digestion. The
substrate refers to the combination of fleshings, the primary sludge and the
secondary sludge. To study the effect of inoculum to substrate (I/S) ratio, the
I/S ratios of 0.25, 0.50, 0.67, 1.00, 1.50, 2.00 and 2.30 were used for the
98
constant substrate VS input of 7.5 grams. The details of quantity of inoculum
added are presented in Table 3.5.
Table 3.5 Details of Inoculum to Substrate Ratio
Reactor
Inoculum to
Substrate (I/S)
ratio
Inoculum (I)
added
(grams on VS
basis)
Substrate (S)
added ( grams
on VS basis)
Ratio of gCOD
substrate to
gCODinoculum
R1 0.25 1.87 7.5 4.26
R2 0.50 3.75 7.5 2.12
R3 0.67 5.00 7.5 1.59
R4 1. 00 7.50 7.5 1.06
R5 1.50 11.22 7.5 0.71
R6 2.00 14.96 7.5 0.53
R7 2.30 17.28 7.5 0.46
For each I/S ratio, duplicate reactors were operated to find the
repeatability and the performance is reported for the observed mean values.
The process was evaluated for biogas generation, pH, oxidation reduction
potential (ORP) and specific methane production rate.
3.8 DETAILED CO-DIGESTION STUDIES
This is the main part of the research work. In this part, it was
proposed to conduct a detailed investigation into the co-digestion process to
arrive at the process parameters which will be useful for design purposes. The
study involved (i) use of optimum conditions already decided in the previous
parts of the study, (ii) applying various organic loading to study its effect on
99
performance of the co-digestion process and (iii) simulating the effect of the
semi-continuous feeding of the organic matter on the co-digestion process.
For the optimum conditions obtained from mix proportion of
substrates and I/S ratio (section 3.7.1 and section 3.7.3), the reactors were
operated with VS input of 38, 45, 53, 60, 68 and 145 grams in 6 feeds to
study the effect of multiple feeds on co-digestion process. At the end of
digestion of the first feed (first cycle), fresh feed was added and part of
digestate was wasted. The study involved 6 feeds (6 cycles) for a total study
period of 300 days. The feed details of the reactors for various volatile solids
loads are presented in the Table 3.6. The photo of the experimental setup is
depicted in Figure 3.3.
Table 3.6 Feed Details of the Reactors for Various Volatile Solids
Loads
Sl.No. Reactor
VS added
for each
feed
(grams)
Maximum
Residence time
for each cycle of
digestion (days)
No. of
Cycles
Total No. of
Days of
Operation of
Reactors
1 ROL(38) 38 50 6 300
2 ROL(45) 45 50 6 300
3 ROL(53) 53 50 6 300
4 ROL(60) 60 50 6 300
5 ROL(68) 68 50 6 300
6 ROL(145) 145 50 6 300
100
Figure 3.3 Photo of the Experimental Set-Up for the Co-Digestion
Studies
The performance of the co-digestion process at the end of each feed
was evaluated for biogas generation and other process parameters such as pH,
ORP, methane yield, VFA, alkalinity, VFA to alkalinity ratio, TKN, total
ammonia, calcium and total chromium.
3.9 EFFECT OF PRETREATMENT OF SLUDGES
The sludges primarily consist of extra-cellular polymeric
substances (EPS) and takes longer time for degradation. In the present co-
digestion studies both the sludges i.e. primary and secondary, were taken into
consideration. The secondary sludge is more difficult to degrade than primary
sludge. For rupturing of microbial cell wall in order to release soluble COD,
various pretreatment processes have been investigated by the researchers on
WAS. Limited studies have been carried out for sludges generated from
tanneries.
Reactors
Biogas
collection
arrangement
101
In order to increase SCOD in sludge samples, various
pretreatment processes viz., ozonation, ultrasonication, peroxide treatment,
alkaline treatment and alkaline thermal treatments were investigated in the
present study. The primary and the secondary sludge samples were
centrifuged at 10000 rpm for 15 minutes then supernatant was filtered through
0.45 µm and analyzed for SCOD as per standard methods ( Method 5220 B).
The increase in SCOD was assessed by application of various pretreatment
methods on primary and secondary sludge samples individually. The increase
in SCOD was calculated based the formula given below:
SCOD ( % ) = x 100 Eqn (3.1)
All the pretreatment experiments were carried out in triplicate with
initial SCOD of 1200 –1300 mg/L for the primary sludge and 1050 – 1200
mg/L for the secondary sludge. The details are presented in the following
sections.
3.9.1 Evaluation of Pretreatments
3.9.1.1 Effect of Pretreatment using Ozone
Ozonation studies were carried out using Fera LG, India. A
laboratory ozone generator ( Model SA001) was used to produce 3 g/h of
ozone from ambient air as inlet gas of the ozone generator. Ozonation studies
were carried out in a cylindrical glass reactor of one liter capacity by bubbling
ozone. The primary and the secondary sludge samples were subjected to
pretreatment using ozone to enhance soluble chemical oxygen demand
(SCOD). The volume of sludge taken was 250 mL. Ozone was purged
through sludge samples at various doses of 0.15, 0.18 and 0.20 g O3/g TS for
a contact time of 60 minutes. As the ozone dose increased from 0.15 g/g TS to
0.18 g /g TS, an increase in SCOD was observed. When the ozone dose was
further increased to 0.2 g /g TS, 20 percent reduction in SCOD was observed.
102
This is due to the mineralization of SCOD. Hence, in the present study, an
ozone dose of 0.18 g O3/g TS yielded an optimum condition for rupturing cell
membranes. At different time intervals, sludge samples were collected, i.e.
after 10, 15, 30, 45 and 60 minutes and analyzed for SCOD. In these
experiments the contact time and the comparative percent increases in SCOD
were ascertained.
3.9.1.2 Effect of Pretreatment using Ultrasonication
Sonication tests were conducted with a Digital Sonicator 250
model, Branson, USA. The sonicator is equipped with a tuning feature to
precisely maintain a frequency of 20 kHz in order to ascertain the constant
horn amplitude. Tiehm et al (2001) demonstrated that the degradation of
excess waste activated sludge is more efficient when low frequencies are
used. Hence in the present study, 20 kHz frequency with a power input of
230 volts was selected. Two hundred and fifty milliliters of the primary
sludge and secondary sludge sample were taken separately and was placed in
a 500 mL beaker with a standard disruptor horn allocated for disruption of
cells. The disruptor was placed 2 cm above the beaker bottom. The same
procedure was followed for secondary sludge samples also. The tests were
conducted for 30 seconds followed by a break for 1 second to control the heat
released during ultrasonication of sludge. Temperature was measured with the
help of a built-in temperature probe equipped with the sonicator. Samples
were collected after the sonication periods of 0.5, 1, 2 and 5 minutes for the
primary sludge and 0.5, 1 and 2 for the secondary sludge samples. After
sonication the sludge samples were analyzed for SCOD individually. In these
experiments, the contact time and comparing the percent increase in SCOD
were ascertained.
103
3.9.1.3 Effect of Peroxide Pretreatment
Advanced Oxidation Processes (AOPs) are related to the
production of highly oxidizing agents (hydroxyl radicals) which enhance and
accelerate the decoloration, detoxification, degradation and biodegradability
of the toxic, inhibitory and bio-recalcitrant wastes. When a solution mainly
undergoes indirect reactions with OH-radicals for instance in a solution with a
high pH value or an AOP-process, the presence of scavengers is undesired.
The scavengers react very fast with OH-radicals and lower the oxidation
capacity. For this kind of processes a low scavenging capacity is required.
Reaction between carbonate radical and hydroxyl radicals is roughly 45 times
faster than that that of hydroxyl radical and bicarbonate radical. This process
is more renounced as pH increases to the alkaline range.
HCO3- + OH CO3 + H2O
CO3 + OH CO3 +
OH-
In addition to scavenging of hydroxyl radicals the carbonate
radicals react with hydrogen peroxide at alkaline pH. Crittenden et al (1999)
reported that the dissociated form of hydrogen peroxide in alkaline media
reacts with hydroxyl radicals more than two orders of magnitude faster than
hydrogen peroxide and decreases the oxidation efficiency by consuming these
hydroxyl radicals. Considering the problems associated with the scavenging
effects of carbonate radicals and bicarbonate radicals, pH of 3.0 was selected
in the present study.
In the present study, primary and secondary sludge samples were
subjected to peroxide pretreatment to increase SCOD. The addition of
hydrogen peroxide (H2O2) to the sludge samples is an effective method to
increase the soluble COD. The pH of sludge samples was adjusted to 3.0 by
104
using H2SO4. After adjustment of pH, peroxide dose was varied to 0.03, 0.06,
0.09 and 0.15 g of H2O2 / gram of TS in the case of primary sludge samples
and the dose was varied to 0.06, 0.12, 0.18, 0.3 H2O2 / gram of TS in the case
of secondary sludge samples. In these experiments the peroxide dose was
optimized for primary and secondary sludge samples and the percent increase
in SCOD was determined. Un-reacted hydrogen peroxide was measured
using iodometry. In all the pre-treatment experiments with hydrogen
peroxide, no residual peroxide was found. Hence the SCOD measured was
only from the organic matter.
3.9.1.4 Effect of Alkaline Pretreatment
The primary and the secondary sludge samples were subjected to
alkaline pretreatment in order to enhance the soluble chemical oxygen
demand (SCOD). The pH of sludge samples was adjusted to 9, 10, 11 and 12
by addition of (1N) NaOH as alkali, which helps the cell wall to rupture and
to release the intracellular material. In these experiments the pH corresponds
to the maximum percent increase in SCOD was noted.
3.9.1.5 Effect of Alkaline Thermal Pretreatment
To study the effect of alkaline thermal treatment, the pH of the
sludge samples were first adjusted to a value identified in alkaline
pretreatment (section 3.9.1.4) and then thermally treated at temperatures of
40, 50 and 60ºC. Samples were collected after alkaline thermal treatment and
soluble COD was analyzed individually. With these experiments the
temperature at which the maximum percent increase in SCOD found was
selected.
105
3.9.1.6 Selection of Appropriate Pretreatment Processes
For the above discussed pretreatment processes, a comparative
analysis of increase in SCOD was done and two best processes were selected.
These two processes were adopted in the further co-digestion studies.
3.9.2 Co-Digestion Studies Without and With Pretreated Sludges
In the evaluation of pretreatments, two best pretreatment processes
were proposed to be selected. These two processes were used in the co-
digestion studies to evaluate the actual enhancement of the biogas generation
by both the processes. To evaluate the enhancement in biogas generation a
control reactor was also maintained. The control reactor and the performance
of study reactors were designed using the conditions arrived from the
optimization studies. The control reactor was used for the purpose of co-
digestion without pretreated sludges as used in the optimization studies.
Duplicate reactors were operated to find the repeatability and the performance
was reported for the observed mean values.
,3.10 EFFECT OF LIPASE ADDITION ON DIGESTION
PROCESS
The biotechnological approach of application of enzymes in waste
treatment is a new area of research. Enzyme application is an option to hasten
the digestion process and the present study attempts to use one such enzyme
namely steapsin, a commercial grade lipase. In the present study, commercial
grade steapsin lipase (catalog no. 124549) was procured from Sisco Research
laboratories, Mumbai, India. The steapsin is a digestive lipase found in the
pancreatic juice which catalyzes the hydrolysis of triglycerides from vegetable
oils, animal fatty acids and glycerol. Steapsin lipase is water soluble, stable at
neutral to alkaline pH conditions with a minimum activity of 40-70 units per
106
mg of protein. The COD of lipase was 300 mg/g and contains carbon content
of 52 %, nitrogen content of 26 % and phosphorus content of 0.01 %. The
details of lipase doses added in different reactors are presented in Table 3.7.
Table 3.7 Details of Lipase Added
Reactor Lipase dose (g / 7.5 g of VS)
R1 (control) Nil
R2 0.25
R3 0.50
R4 0.75
R5 1.00
R6 Only Inoculum plus 0.75 g of
Lipase
The co-digestion studies were carried out with a volatile solids
input of 7.5 grams on dry weight basis in 5 reactors i.e. R1, R2, R3, R4 and
R5. The reactor R1 is the control reactor without lipase addition. In reactors
R2, R3, R4 and R5, the lipase dose as presented in Table 3.7 was added.
After feeding the substrates, seed and lipase into the reactors, the reactors
were closed with a rubber cap and an aluminum seal to make them air tight.
To maintain anaerobic conditions nitrogen gas was purged. Duplicate reactors
were operated to find the repeatability and the performance was reported for
the observed mean values. The photo of the experimental setup for one set of
reactors is depicted in Figure 3.4.
107
Figure 3.4 Photo of the Experimental Set-up for the Lipase Studies
The optimum lipase dose was ascertained based on the volume of
biogas generated. The co-digestion process was evaluated without and with
addition of lipase. To confirm whether the added lipase is a potential carbon
source and contributes to biogas generation the biogas generated by the
inoculum against inoculum plus lipase addition was monitored. The studies
were carried out for the substrate pH range of 7. 5 to 8.0 at an ambient
temperature i.e 28 ± 5 º C.
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