CHAPTER 3 MATERIALS AND METHODS - …shodhganga.inflibnet.ac.in/bitstream/10603/9622/2/11...78 CHAPTER 3 MATERIALS AND METHODS 3.1 GENERAL This chapter describes the materials and

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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

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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.

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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

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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.

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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.

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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.

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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.

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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.

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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

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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.

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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..)

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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

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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.

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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.

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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

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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

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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.

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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

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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

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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.