Accepted Manuscript

Title: COMPARISON OF BIOCHEMICAL METHANE

POTENTIAL AND METHANOGEN MORPHOLOGY OF
DIFFERENT ORGANIC SOLID WASTES CO-DIGESTED
ANAEROBICALLY WITH TREATMENT PLANT SLUDGE

Authors: Helen Adetoun Lawal-Akinlami, Shanmugam

Palaniyandi

PII: S0957-5820(17)30033-2
DOI: http://dx.doi.org/doi:10.1016/j.psep.2017.02.001
Reference: PSEP 968

To appear in: Process Safety and Environment Protection

Received date: 27-7-2016

Revised date: 30-1-2017
Accepted date: 1-2-2017

Please cite this article as: Lawal-Akinlami, Helen Adetoun, Palaniyandi,

Shanmugam, COMPARISON OF BIOCHEMICAL METHANE
POTENTIAL AND METHANOGEN MORPHOLOGY OF DIFFERENT
ORGANIC SOLID WASTES CO-DIGESTED ANAEROBICALLY WITH
TREATMENT PLANT SLUDGE.Process Safety and Environment Protection
http://dx.doi.org/10.1016/j.psep.2017.02.001

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COMPARISON OF BIOCHEMICAL METHANE POTENTIAL AND METHANOGEN
MORPHOLOGY OF DIFFERENT ORGANIC SOLID WASTES CO-DIGESTED
ANAEROBICALLY WITH TREATMENT PLANT SLUDGE.

Author names and affiliations

Lawal-Akinlami, Helen Adetouna,b and Shanmugam, Palaniyandia,*
a
Environmental Science and Engineering Division, CSIR-Central Leather Research Institute, Adyar,

Chennai 600020, Tami Nadu, India.

b
Academy of Scientific and Innovative Research (AcSIR), Anusandhan Bhawan, 2, Rafi Marg,

New-Delhi 110 001, India.

*Corresponding author

Name : Shanmugam Palaniyandi

Telephone : 0091-44-24437211

Fax : 0091-44-24911589

Email address: pashanmugam@yahoo.com

Postal address: Senior Principal Scientist, Environmental Science and Engineering Division,

CSIR-Central Leather Research Institute, Adyar,

Chennai 600020, Tami Nadu, India.

1
Research Highlights

 Organic solid wastes were subjected to anaerobic digestion at mesophilic temperature.

 Differences in peak time depend on the biodegradability of the substrate composition.

 Mixture of the wastes produced more biogas yield compared to the mono-digestion.

 Efficiency of the sCODr and VSr showed significant differences in the wastes.

Abstract

This paper describes biomethanation of tannery limed fleshing (TLF), fruits and vegetables (FVW),

and sugarcane juice residue (SJR) with pre-fermented tannery-based common effluent treatment plant

sludge (CETP), as an inoculum. The BMP, microbial morphology and other bioprocess efficiency

parameters were evaluated using a laboratory scale batch reactor, with a substrate/inoculum ratio of

under a mesophilic anaerobic (35± 2°C) condition and mixed at 100 rpm in orbit shaker. Maximum

cumulative biogas yield was observed in FVW (2468.50 ± 0.44 Nm.mL.d-1) followed by TLF and

comparatively to the inoculum. The specific methane yield observed ranged from 0.169 ± 0.05 to

± 0.00 Nm.L.g-1VSr for the wastes, against 0.204 ± 0.01 Nm.L.g-1VSr for control treatment. The

mixture of all the three wastes (CLFS) benefitted to yield more biogas compared to the

The highest VSr and sCODr efficiency of 62.44 ± 0.32 and 93.46 ± 1.04 % obtained in FVW

to other wastes, coincides with the methanogenic microbial count and densities. A good correlation

= 0.82) was also observed between the experimental BMP and stoichiometric methane potential of

various organic solid wastes.

Keywords: Organic solid wastes; Common effluent treatment plant sludge; Anaerobic digestion;
Biochemical methane potential; Methanogens

2
1. Introduction

Leather manufacturing processes contribute to tremendous pollution of ground water and soil

contamination due to the release and percolation of toxic chemicals from the solid and liquid wastes. The

environmental impacts of tanneries are the major concerns which have become a problem to the tanning

industry in the recent past. This has spawned unhygienic environmental and safety conditions in the

leather manufacturing areas. The solid wastes such as raw trimmings, liquors, shavings, fleshings,

sludge, and buffing dust are inevitable by-products of the leather manufacturing processes (Sundar et al.,

2010) that cause pollution and pose a serious disposal problem.

Each tonne of raw hide processed produces 800 kg of solid wastes in leather processing (Salman, 2015).

About 150 kg of tannery wastes composed of raw hide trimmings (5- 7 %), tannery fleshings (56 - 60 %),

hide splits and chrome shavings (35- 40 %) are generated (Rao et al., 1994; Salman, 2015) which are not

utilized while, about 1.67×109 m2 of leather is estimated to being made annually in the world (FAO

2001). This creates disposal problem in tanneries and, thus causes environmental pollution. The existing

solid waste management techniques such as landfilling, incineration, and composting trigger a primary,

secondary and tertiary environmental impact that cause groundwater contamination, odour, pests

infestation, greenhouse gases emission, climate change and epidemiological problems. Alternatively,

the recycling of the sludge as inoculum and generation of biogas with these tannery solid wastes in

anaerobic digestion process has been experimented and evaluated in this study.

Anaerobic digestion (AD) process decomposes organic matter by diverse microbial consortia in an

oxygen-free environment, using series of metabolic reactions such as hydrolysis, acidogenesis,

acetogenesis and methanogenesis for biogas production with an energy-rich methane as an end product

3
(Lastella et al., 2002). Anaerobic digestion has many environmental and energy benefits coupled with

environmental protection. AD is widely used as a viable technology for the treatment of organic solid

and liquid waste, because of its reduction in waste volumes, carbon offsettings, carbon footprint coupled

with renewable energy generation. AD has been demonstrated for a wide range of feedstocks including

industrial and municipal organic solid wastes, agricultural wastes, food industry wastes, slaughterhouse

wastes etc. However, the efficiency of biogas and power generation depends on the waste composition

and process efficiency during AD (Lin et al., 1997; Navia et al., 2002).

Ward et al. (2008) described the benefits of AD to reduce environmental pollution in two ways: Firstly,

the sealed environment of the process prevents the release of methane into the atmosphere and secondly,

the methane produced serves as fuel for human utilization (in vehicles, electricity and cooking purposes).

Nevertheless, AD process has a number of limitations that includes slow reaction rates, long retention

times, complex operation, sensitivity to waste loads improper mixing and toxic materials (Li et al.,

2011). There is also the danger of fire outbreak if the gas leaks due to carelessness.

Similar to the huge tannery limed fleshing wastes generated in tanneries, fruits and vegetable wastes and

sugarcane juice residue accounted for the largest portion of wastage in the agricultural sector. Molasses,

a typical liquid waste from sugarcane processing contain high carbon with 52% of fermentable sugars

and, with relatively high values of chemical oxygen demand (COD) of more than 100,000 mg.L-1

(Lutoslawki et al., 2011) that affect the environment when it is directly discharged onto land. Processing

of fruits and vegetables produces two kinds of wastes: the solid waste (peel/skin, seeds and stones) and

liquid waste (juice and wash-water), which contain highly biodegradable organic matter. Fruits and

vegetable wastes are generated during selecting, sorting and boiling processes. Thus, the improper

4
disposal of these fruits and vegetable wastes leads to breeding of flies, rodents infestation, dysentery and

other epidemiological problems.

The suitability of these wastes and their conversion rates to biogas have been proven by the biochemical

methane potential (BMP) test, which is a relatively economical and reliable method (Owen et al., 1979;

Angelidaki et al., 2009; Li et al., 2011) that can pave a path for the designing of continuous or

semi-continuous AD systems. BMP measures the biomethane or biogas produced by a known quantity

of waste, and also estimates the degree of hydrolysis with the retention time required for the complete

conversion of the mono-substrate or co-digested solid wastes into soluble volatile fatty acids (VFAs)

through hydrolysis and further into methane by methanogenesis processes. A number of studies have

established and demonstrated BMP test with the use of solid wastes substrates like organic fraction of

municipal solid waste (OFMSW) (Neilfa et al., 2015), slaughterhouse waste (Flores-Juarez et al., 2014),

fats, oil and grease (FOG) (Li et al., 2011), leather fleshing waste with different sludge (Shanmugam and

Horan, 2008) etc.; with cow dung, pig manure (Alvarez et al., 2010) and, fermented sewage sludge as

inoculum. In addition to this, Erguder et al. (2001) digested cheese whey using upflow anaerobic sludge

blanket (UASB) reactors after the batch BMP experiments provided fundamental information on

substrate characteristics, loading rate ranges and biogas yield. But, little research was focused on BMP

testing of tannery wastes, sugarcane juice residue, fruits and vegetable wastes, using a tannery-based

common effluent treatment plant (CETP) sludge. Therefore, this present study has been attempted to

determine the BMP, process efficiency and the substrate to inoculum ratio in a laboratory scale batch

anaerobic reactor and, to characterize the morphology of the methanogens responsible for gas

production.

5
2. Materials and Methods
2.1. Collection and preservation of organic solid wastes

In this study, three waste materials were used namely: tannery limed fleshing (TLF), fruits and vegetable

(FVW) wastes, and sugarcane juice residue (SJR) together with the Common Effluent Treatment Plant

(CETP) sludge. The TLF was collected from the Pilot Tannery of Central Leather Research Institute,

Chennai, Tamil Nadu, India. Crushed fruits and vegetable wastes were collected at the milling stage

from Koyambedu Biogas plant, Chennai, while the sugarcane juice residue was collected from a

roadside sugarcane spot near Anna University Chennai, India. The TLF and crushed FVW were chopped

and minced with a blender (Jai Lakshmi, model CM/L 3713860) at 15,000 rpm for 10 minutes in order to

have increased surface area. The sugarcane juice residue was properly sieved using 0.5mm sieve to

remove debris. Then, the samples were preserved in the refrigerator at 4°C prior to analysis.

2.2. Preparation of anaerobic seed-inoculum

The samples of biological secondary sludge was obtained from the secondary clarifier tank and it was

used as the base material for preparing the seed inoculum, collected from the tannery based Common

Effluent Treatment Plant (CETP) in Pallavaram, Chennai, Tamil Nadu, India. The collected sludge was

anaerobically activated by adding stock mineral solutions of freshly prepared defined nutrients (Owen et

al., (1979). The pH of the seed inoculum prepared was adjusted to 6.5 and kept at room temperature for

five days under an anaerobic condition. It was then pre-fermented for acclimatization before the

substrates were being added. An appropriate substrate was added to the acclimatized anaerobic seed

sludge, which was used in the batch BMP experiment.

6
2.3. Characterization of solid waste feedstock

The organic solid wastes used as substrates were characterized according to standard methods for the

examination of water and wastewater (APHA, 1998) procedure 2540G, in determining the total solids

(TS), volatile solids (VS) and fixed solids (FS). The pH was determined immediately upon collection of

samples. The ratio of the organic and inorganic (O/I) contents was calculated.

2.3.1. Elemental composition analysis

The elemental compositions [carbon(C), hydrogen (H), nitrogen (N) and oxygen (O)] of each organic

waste were determined using CHNS Analyzer Model Euro vector 3000 series, Italy (available in CSIR-

CLRI, Chennai, India) according to ASTM D-5291 method based on dry weight. The oxygen content

was calculated by difference. An aliquot of the sample was oven dried at 105°C and kept in a desiccator

prior to analysis. 0.5-1mg of the dried sample was taken into an aluminium boat. The initial weights

were noted and the boats were loaded in the CHNS analyzer. The CHNO and ash data were used to

estimate the calorific energy value (CEV) using Eq. (1) proposed by Channiwala and Parikh, (2002):

where CHNO and A represent the mass of carbon, hydrogen, nitrogen, oxygen and ash on dry weight

basis. Based on the elemental composition, the empirical formula, COD equivalent (COD´/weight) and

theoretical equation on the atomic composition basis of the waste materials were used to calculate the

stoichiometric methane potential (SMP), and compared with experimental BMP (BMPexp) as presented

in Eqs. 2, 3 and 4. The theoretical amount of the gases produced and COD´/weight were calculated

according to Rittmann and McCarty, (2012) in Eqs. (2) and (3) respectively. The presence of proteins

and ammonia are considered in Boyles Eq. (4) (Neilfa et al., 2015).

and

7
where,

2.3.2. Experimental set-up for biochemical methane potential (BMP)

Each of the minced TLF, FVW and SJR was mixed with a substrate to seed inoculum ratio of 3:1 (wet

weight), by adding 300ml of minced waste materials to 100ml of the seed inoculum as the working

volume in a 500 ml capacity batch reactor (borosilicate glass serum bottles) while, the remaining 100 ml

was left as headspace for biogas collection. These borosil bottles were sealed hermetically with butyl

rubber stoppers and aluminium crimps. The control experiment which contained the seed inoculum

alone was set-up along with the combination of all the solid wastes (CLFS) and operated simultaneously.

The reactors were kept in the orbital shaker at a constant mesophilic temperature of 35± 2°C for 42 days

and continuously stirred at 100 rpm for thorough mixing with a periodical waiting time during the

experimental period.

2.4. Daily measurement of gas production

The daily methane production from the batch experiment was obtained through alkaline-water

displacement method (Fig.1) and calculated as Nm.mL of CH4.d-1 at normal temperature and pressure

(NTP). In this method, the gas line from the reactor was connected to an aspirator bottle with the same

headspace as the BMP bottle. A graduated measuring cylinder was placed at the end of the tap to

8
measure the amount of alkaline-water displaced (in millilitres) as an equivalent of methane gas produced

from the BMP bottle.

2.4.1. Biogas composition analysis

Biogas sample was collected from the headspace of the batch reactors with a gas-tight syringe and a

sample volume of 500 µL was injected into the gas chromatograph (GC) for analysis. The methane

(CH4) content (in percent) was determined using GC (CHEMITO GC-7610) equipped with a thermal

conductivity detector (TCD) and a capillary column (Poropak Q). The carrier gas used was nitrogen

(N2) while, the analysis was carried out at a carrier gas flow rate of 30 mL.min-1 with the injector,

column, and detector temperatures at 120, 90, and 120 ºC respectively.

2.5. Bioprocess performance evaluation of the solid wastes during AD

2.5.1. Physicochemical analysis

The BMP, specific methane yield (SMY) and efficiency of VS removed (VSr ) of each solid waste

experimented were calculated (Angeldaki et al., 2009; Alvarez et al., 2010; Kaosol and Sohgrathok,

2012). The SMY and VSr were calculated using the equations as follows:

where sub-indexes refer to the removed, initial and final volatile solids. The efficiency of VSr (%) of the

waste materials was obtained by using Eq. (7):

The initial and final bioprocessing impact of the fermentation in the BMP bottle were evaluated with

5-10 ml of the supernatant (centrifuged at 5800xg for 10mins) to determine the soluble chemical oxygen

9
demand (CODs), volatile fatty acids (VFA by distillation method), free ammonia (NH3), alkalinity

(ALK) and the total anaerobic microbial count according to standard methods. The efficiency of the

soluble chemical oxygen demand removal (sCODr) was also calculated using the same formula as VSr.

2.5.2. Scanning Electron Microscopy (SEM)

An aliquot of granules from each waste in the batch reactor was collected and prepared for SEM analysis

as described by Angenent et al., 2000. The morphology of the microbial population at high gas

production in each reactor during the anaerobic digestion was observed using High-Resolution Scanning

Electron Microscopy (HR-SEM) examination (model: FEI Quant FEG 200). The specific

microorganisms in the anaerobic reactors were identified based on the morphological characteristics

reported in the past research investigations (Gonzalez-gil et al., 2001; Liu and Tay, 2004; Pandey et al.,

2011).

2.6. Statistical analysis

Data obtained during the experiments were analyzed statistically by determining the standard deviation

of the means and analysis of variance (ANOVA) using IBM SPSS Statistics version 20. Duncan’s New

Multiple Range test was used to separate the means at significance level of P ≤ 0.05.

3. Results and Discussion

3.1. Characterization of the organic solid wastes

The characterization of the organic solid wastes such as colour, pH, TS (%), VS (% of TS), FS (% of TS)

and O/I ratio are presented in Table 1. The pH of the different waste materials used as substrates ranged

from 5.70 (± 0.18) to 12.60 (± 0.32), while the control (anaerobic seed inoculum) had pH of 7.20 (±

0.20). The TS obtained for TLF, FVW, SJR and the inoculum ranged from 4.60 ± 0.10 to 9.72 ± 0.11 %,

10
whereas the VS values ranged between 51.38 ± 0.50 and 96.27 ± 0.25 % of TS respectively. The FS,

which is the inorganic portion of the wastes ranged from 3.73 ± 0.06 to 46.59 ± 0.11 % of TS,

with the O/I ratio ranging from 1.10 ± 0.13 to 25.81 ± 0.31 respectively. The C/N ratios (Table 2) were

found to be in the range of 4.41 ± 0.03 and 29.39 ± 0.41. Fruits and vegetable wastes had the lowest total

solids (less than 8-18%) with high volatile solids (80-90%) which were confirmed as easily degradable

material in the anaerobic reactors. However, the substrate-SJR had the highest VS and C/N ratio (96.27 ±

0.25 %, 29.39 ± 0.41) while, the seed inoculum had the lowest VS with 51.38 ± 0.50 % and C/N of 8.48

± 0.22. Basically, TLF contains a high amount of fat and protein; FVW organic fraction includes sugars,

pectin, hemicellulose, cellulose and lignin while, SJR contains mainly fermentable sugars (such as

sucrose), mineral salts and vitamins (Faria et al., 2011). The TLF waste had the highest value of

nitrogen which suggests that substrate-TLF has nitrogen-rich content similar to slaughterhouse waste

(Flores-Juarez et al., 2014) compared to the rest of the waste materials. Even though, Bouallagui et al.

(2009a) suggested that a C/N ratio between 22 and 25 seemed to be optimum for anaerobic digestion of

fruits and vegetable wastes; Lee et al. (2009b) reported that the optimal C/N ratio for anaerobic

degradation of organic waste was 20–35 which is similar in SJR. The physico-chemical compositions of

the organic waste materials were significantly different from each other.

3.2. Biochemical Methane Potential (BMP)

3.2.1. Daily and cumulative gas production of the different substrates in the BMP bottle

The daily gas production of the different organic solid wastes used as substrates for AD was shown in

Figure 2a. A slow start-up of AD was observed for substrate-FVW and the inoculum whereas, a quick

gas production was observed in the rest. This could be attributed to an initial adaptation of

microorganisms to the nutrients available in preparation for AD process. The peak daily gas production

11
was observed in substrate-FVW with 327.4 Nm.mL.d-1 on 17th day whereas the lowest gas peak (110

Nm.mL.d-1) was observed in the inoculum alone on the 10th day. Substrates- TLF, SJR and CLFS (a

mixture of all the wastes) had a peak daily gas production of 302, 271.6 and 187.9 Nm.mL.d-1 on day

21st, 17th and 17th respectively. This indicates that the difference in peak time range of the organic wastes

depends on the biodegradability of the substrate composition and its readily available nutrients for

microbial utilization. From the 23rd day, the daily gas production showed a decreasing trend in both the

substrates and inoculum. The cumulative biomethane produced from different organic solid wastes is

illustrated in Figure 2b. FVW had the highest cumulative biomethane (2468.50 ± 0.44 Nm.mL.d-1)

production followed by TLF (2016.30 ± 2.11 Nm.mL.d-1), while the control gave the lowest value of

475.40 ± 1.45 Nm.mL.d-1. From the FVW, a steep increase in the CH4 production was observed until 7

days, which is attributed to the presence of readily available soluble carbon source (after the breakdown

of organic macromolecules to free fatty acids (FFA) for energy). This was attributed to the high C/N

ratio and soluble (liquid) nature of the waste that paves a path for rapid substrate degradation in

anaerobic condition for gas production and a similar result was also reported by Wang, (2008). This

study clearly indicates that the fruits and vegetable wastes were easily biodegradable due to the high

moisture content as confirmed by the initial peak of biogas yield.

On the contrary, a lower gas production (lag phase) was initially observed in TLF, which could be

attributed to poor degradation of proteins and fats, as the degradation depends on the subcutaneous fat

and adhering protein body (N2 source) only. The degradation of these macromolecules into consistent

FFA or peptide/amino acids in a soluble form, delays or slows down methane production. However, once

the required nutrients/substrates are available, the onset of methanogenesis commences gradually. Since

the substrate (TLF) is proteinous in nature, ammonia (NH3) is liberated as co-product in this anaerobic

method (Khalid et al., 2011). Thus, the produced ammonia accumulates at high pH due to the reduction

in VFA. This could further result in lowering methanogenesis as well as the sodium sulphide (Na2S)

12
inhibition in the substrate (fleshings) from beam-house operations. Therefore, the lower cumulative

methane (2016.30 ± 2.11 Nm.mL.d-1) observed for TLF compared to that of FVW substrate could be

attributed to the presence of NH3 and Na2S which are considered to pose a threat to the survival and

activities of the methane producing archaea (Sakar et al., 2009; Khalid et al., 2011). This was in line with

a low C/N ratio that could have contributed to the accumulation of ammonia to have become toxic for

methanogenic bacteria; a cause for reduced gas yield (Palatsi et al., 2010). Although the poor C/N ratio

of a highly proteinaceous waste such as leather and slaughterhouse wastes become detrimental to

methanogenesis (Shanmugam and Horan, 2008; Palatsi et al., 2010), the biogas yield could possibly be

enhanced through an increased C/N ratio. In spite of the highest C/N ratio observed in substrate-SJR, a

lowest cumulative gas production of 1625.4 ± 2.34 Nm.mL.d-1 was obtained compared to other

substrates used, which was attributed to the rapid hydrolysis to VFA production and saccharification of

the carbon sources (to ethanol) as depicted by high VFA and an acidic pH (5 - 6) could have led to

fermentative solventogenesis instead of methanogenesis. However, the mixture of all the three waste

materials (CLFS) resulted in lower methane yield (1853.8 ± 1.04 Nm.mL.d-1) as a result of the waste

mixing ratio of 1:1:1 used which could have caused pH variation and possibly lead to a shift in microbial

population for biogas production. The control experiment gave the lowest methane production (475.40 ±

1.45 Nm.mL.d-1) out of all which was attributed to lack of substrate addition and nutrients depletion.

3.2.2. Removal efficiency of the crude organic matter ( VSr and CODr)

The crude organic matter present in these solid wastes materials are in the form of VS and COD. The

efficiency of VSr of the entire BMP experiment is presented in Figure 3. Besides the part of the substrate

been gasified, there were significant differences in the VSr observed among TLF, FVW, SJR and the

control as the rates of biogas production differed appreciably according to the TS concentrations. A

13
maximum percentage of VSr was observed in the BMP bottle containing the FVW (62.44 ± 0.32d %

VSr) followed by TLF (56.69 ± 0.50c % VSr) and SJR (43.57 ± 0.34b % VSr) as compared to the

control BMP bottle with a minimal value of 12.97 ± 0.41a % VSr. The lower biogas yields could be

attributed possibly to the TS concentrations of the feedstocks and inhibition of methanogenic bacteria, an

observation which correlates with the substrates degradation that took place between the 11th and 23rd

day of digestion (Fig. 2a). Thus, the inhibitory substances present during the anaerobic fermentation

process of various solid waste materials could be neutralized to a considerably reduced level and thus

enhance the biogas production by co-digestion (Bouallagui et al., 2009a). Nevertheless, the result of

this study showed a higher range of TS concentrations between 15.03 ± 0.03a - 35.44 ± 0.43d g (initial

TS) and 9.88 ± 0.29a - 28.52 ± 0.47d g (final TS), which is similar to the values of high-solids sludge

reported by Lay et al., 1997 and Liao et al., 2014. The efficiency of the sCODr showed significant

different values of 72.88 ± 0.93a, 73.94 ± 0.26a, 93.46 ± 1.04c, 73.42 ± 0.82a and 79.36 ± 1.73b for the

control, TLF, FVW, SJR and CLFS, respectively.

3.2.3. Specific methane yields of the organic wastes

The specific methane yield (SMY) per gram of VSr obtained from different waste materials is presented

in Figure 4. The SMY for substrates- FVW, SJR and TLF were 0.478 ± 0.00, 0.184 ± 0.10 and 0.169 ±

0.05 Nm.L.g-1VSr respectively, compared to the inoculum alone (0.204 ± 0.01 Nm.L.g-1VSr). The

highest methane yield obtained from FVW reactor could be as a result of enhanced growth and rapid

proliferation of microorganisms, and production of all necessary metabolizing enzymes to degrade the

substrate. This coincides with the anaerobic microbial count (1.89 ± 0.00 ×104 CFU/mL) obtained that

could have accelerated the process of biodegradation in FVW during anaerobic digestion than the others

(Table 3). In contrast, SJR and TLF had a lower SMY of 0.184 ± 0.10 and 0.169 ± 0.05 Nm. L.g-1VSr

14
respectively, as against the highest yield observed in FVW. The lower yield in TLF waste could mostly

be due to the long-chain fatty acids (LCFA) and ammonia produced during lipid and protein degradation,

which is inimical to methanogenesis. This suggests a blocked mass transfer that could have resulted

from the build-up of some inhibitory products, whose effects may be an impending factor according to

the report of Baskar et al., 2014. However, the lower yield in SJR could be attributed to the increased

carbohydrate and simple sugar contents that lead to lesser energetic end products, with higher energy

content per electron equivalent than acetate or other organic molecules during AD processes (Rittmann

and McCarty, 2012). Besides, the initial VFA value of 21,089 ± 10.63 mg.L-1 obtained indicates a rapid

accumulation of VFA and prevention of methanogenesis that further leads to the pH drop (from pH 6.54

to 4.06) observed at the end of digestion; with further gradual reduction confirmed (2155.47 ± 2.25

mg.L-1) on the final day of the experiment attributed to the conversion of VFA to gas. Thus, a metabolic

shift was observed from methanogenesis to solventogenesis by a rapid change in pH. Therefore, the

result obtained in this study clearly demonstrated that the FVW serves not only as a better nutrient for

anaerobes but also, as a superior co-substrate for methanogenesis with TLF in a co-digestion system.

This was in agreement with the study reported by Gomez et al., 2006 that the codigestion of FVW with

primary sludge yielded more biogas than the sludge alone. The methane content generated from the

fermentation of studied substrates ranged from 31.75 - 62.73 %.

3.3. Bioprocess evaluation

The anaerobic bioprocess evaluation of the solid waste materials before (initial) and after (final) the

fermentation was shown in Table 3. The parameters employed such as soluble chemical oxygen demand

(CODs), VFA, NH4, ALK, pH and an anaerobic microbial count showed a significant reduction from the

initial values to the final values of digestion. The initial CODs from the reactor ranged from 4,870 ±

399.62 (inoculum) to 63,998.33 ± 227.51 mg.L-1 (CLFS), while the final CODs showed values between

15
4,870 ± 399.62 (inoculum) and 13,210 ± 1,065.04 mg.L-1 (CLFS) respectively, with the percentage of

CODs removal ranging from 72.88 ± 0.93 - 93.46 ± 1.04 %. SJR and TLF had the highest VFA values of

21, 089 ± 10.63 and 1322.77 ± 2.75 mg.L-1 respectively, compared to the control value of 4,263.84 ±

1.24 mg.L-1 at the initial stage. The final VFA of the substrates decreased, ranging between 314.76 ±

0.26 and 2,155.47 ± 2.25 mg.L-1 as the same. During the SJR fermentation process, a larger

concentration of VFAs (21,089 mg.L-1) accumulation could lead to the drop in pH as observed by

Sumardiono et al., 2013. They reported that carbohydrate-rich substrates are rapid producers of VFAs

and protein-rich substrates with moderate ammonia act as a good buffering agent due to the production

of ammonia, even at a higher accumulation of VFA. The higher content of NH3 obtained in TLF

(21,10.92 ± 1.01 mg.L-1) confirmed inhibition in the digestion process as compared to the control

treatment (184.55 ± 0.58 mg.L-1) and other waste materials used. This finding was supported by Duan et

al. (2012) that reported an excess production of ammonia at the methanogenesis stage attributed to an

inhibition and elevated pH by rapid conversion of VFA to CH4, and eventually ceased the biogas yield

as observed in TLF. Compared to the initial pH of the waste materials in the reactor which was adjusted

to pH 6.50 ± 0.01- 6.67± 0.03, the final pH of TLF and FVW showed a higher pH of 8.11 ± 0.02 and 6.85

± 0.04 respectively. pH value is an important parameter that affects the growth of microbes and, the

amount of CO2 and VFA produced during the anaerobic fermentation. Hence, the pH showed a stable

condition within the optimum pH range (5.5– 8.5) as reported by Sung and Lui (2003) and, Zupancic and

Grilic (2012). An increased alkalinity in all the reactors coincided with increased pH and a decreased

VFA and vice versa. The ALK values ranged from 11,566.67 ± 251.66 (inoculum) to 36,060.67 ± 115.47

(CLFS) mg CaCO3.L-1 for the initial digestion whereas, the final values observed ranged between 9800 ±

16
147.99 and 25, 233.33 ± 152.75 mg CaCO3.L-1, respectively. Since alkalinity is based on carbonate

(CO32-) in equilibrium with the dissolved CO2, a higher alkalinity signifies a higher buffering capacity

that could possibly be achieved at stable pH (Sakar et al., 2009). However, the TLF (being a

protein-rich substrate) would have liberated ammonia when degrading which could have further

contributed to the high alkalinity observed beside rapid conversion of VFA at the end of its digestion.

The values observed conforms within the range of 2,000 to 18,000 mg CaCO3.L-1 (Cuetos et al., 2008)

as reported from the previous investigation. The initial anaerobic microbial count ranged from 1.10 ±

0.21×103 to 5.5± 0.54×106 CFU/mL, whereas the final microbial count ranged between 4.80 ± 0.50

×103 and 3 ± 0.20 ×106 CFU/mL for the wastes respectively. This data could be attributed to the

microbial adaptation and interactions in the AD system, to degrade the pollutant compounds for growth,

which was later on dominated by the methanogens in the reactor for gasification. Microorganisms

proliferate in wastes due to the available nutrients that support their growth (Giller and Cadisch, 1997),

which could be associated with the pattern of the microbial growth, competition between

microorganisms and varying abilities to break down the different available substances for growth, cell

synthesis and transformation in the system. Even though the microbial plate count (in this study)

showed a low plating efficiency, the SEM was further conducted to determine the structures of the

anaerobic organisms present.

3.4. Stoichiometric methane potential through empirical formulae of wastes

The empirical formula and stoichiometric or theoretical methane potential (SMP) equations of the

feedstocks were calculated based on the elemental composition of the solid waste materials (Table 2).

The calculated empirical formula was used to determine the theoretical COD equivalent (COD´/weight)

17
of each organic waste and expressed in terms of the measured volatile solids. The empirical formula for

the seed inoculum, TLF, FVW and SJR were C9.9H21.83O6.18N, C5.15H10.09O2.28N, C10.82H17.57O6.53N and

C34.29H50.26O16.3N, respectively while, the calculated COD equivalent from the elemental composition

were 1.45, 1.51, 1.21 and 1.65 gCOD´/gVS for the same, which agreed with the result published

elsewhere (Rittmann and McCarty, 2012). The gross calorific energy value (CEV) gives the tendency to

quantify the total energy introduced into the BMP bottle (reactor) that can be obtained in the form of

biogas and digestate. Thus, the maximal GCEV obtained was found in SJR (22.89 MJ/kg) and the

minimal with 10.97MJ/kg for seed inoculum, respectively. In this study, the empirical formula obtained

for TLF seems higher (Table 2) compared to the reported formulation of Shanmugam and Horan (2008)

for leather fleshing as C4H13O2N with an average value of 1.4 gCOD.g-1VS. Zuo et al. (2014) reported an

empirical formula of C12.74H18.79O9.12N with a theoretical methane production estimated as about 0.41 L

CH4. g-1VS for raw vegetable waste alone. Moreover, the SMP gave an indication of the estimated

maximum methane production expected from a specific waste. The SMP values obtained were 0.43, 0.53

and 0.58 Nm.L.g-1VSadded for FVW, TLF and SJR, respectively, relative to the seed inoculum (0.51

Nm.L.g-1VSadded) according to the formula of Neilfa et al. (2015); Fig. 5). However, the BMPexp yields

obtained were substantially different, ranging from 0.16 to 0.48 Nm.L.g-1VSr for the mixture of the three

wastes and FVW, respectively. Nevertheless, the BMPexp values obtained in this study (Fig. 5) were

much lower than theoretical yield in accordance with Mussoline et al. (2013). This could be attributed to

some difficulties in degrading tightly lignocellulosic and recalcitrant materials as well as a need of wider

range C/N ratios for optimum digestion performance as suggested by Zhang et al. (2008). A good

correlation (R2 = 0.82) was observed between the experimental BMP and COD equivalent determined

through the empirical equations.

18
3.5. Microbial morphology by SEM analysis in the BMP

The HR-SEM micrographs of the different feedstocks are presented in Figure 6. The HR-SEM showed

different bacterial structures, densities and populations present in each reactor containing different waste

materials. It was evident from the micrographs that the anaerobic digestion mixture had rod-shaped

(bacilli) and spherical shaped (cocci) as predominant microbes. There was no clear image of

microorganisms in the reactor containing substrate-SJR (data not presented), whereas there were

structures of microorganisms being observed in the remaining TLF and FVW reactors. A greater

diversified population of bacilli and cocci organisms of various sizes were observed in the reactor

containing the mixture of the three wastes materials-TLF, FVW and SJR (Fig. 6b) as compared to the

control experiment (Fig. 6a). The mixture of the substrates used could be responsible for the increased

microbial population influenced by the amount of biomethane production in the reactor. Presumably, the

observed rod-shaped bacteria could be a Methanobrevibacter-like while, the cocci could be

Methanococcus-like (Pandey et al., 2011).

In TLF-reactor, a bamboo-shaped rod with flat ends (Fig. 6c) was observed which was distinctly

different from the microbial morphology observed in SJR and FVW. This was similar to the images

obtained from the previous studies reported by Gonzalez-gil et al. (2001), Liu and Tay (2004) and

Pillay et al. (2004), that operated an anaerobic baffled reactor (ABR), up flow anaerobic sludge blanket

(UASB) granules of a bench-scale reactor and full-scale expanded granular sludge bed reactor. (Fig. 6c)

revealed a typical morphology of Methanosaeta methanogen with a length of 234.37 micronmeter (µm)

and 23.74 µm in diameter. It also resembled a filament-type granule with a predominantly long

rod-shaped organism which could be a Methanothrix-like (Liu and Tay, 2004). Methanosaeta is

represented by only one genus Methanosaeta belonging to the family Methanosaetaceae, with their cells

producing methane by splitting acetate (Hassan et al., 2015) which is an acetoclastic organism that has a

higher affinity for acetate solely as substrate (Pandey et al., 2011). They are either present as

19
a filament (Liu and Tay, 2004) or clusters of rod-shaped (Gonzalez-gil et al., 2001) or single-rod shaped

microorganisms (Pillay et al., 2004 and Gonzalez-gil et al., 2001). This organism could have

predominated as a result of the substrate composition that significantly influenced the microstructure

formation of the granule.

In contrast to the organism present in the substrate-TLF reactor, there was a greater prevalence of

cocci-shaped methanogens in the reactor containing fruits and vegetable wastes (Fig. 6d). These

resembled Methanococcus-like micro-organisms which could also resemble Methanosarcina or

Methanocorpusculum species (Pillay et al., 2004). The cells observed were irregular cocci-shaped with

a diameter range of 190.4 and 229.3 nanometers (nm). The cocci-like methanogen observed shared

similarity with the micrographs reported by Pillay et al. (2004) and Gonzalez-gil et al. (2001) from fruits

and vegetable wastes. The faster growth rate and lower affinity for acetate in Methanosarcina could

have been the reason for the larger population density and highest gas production that was recorded for

substrate-FVW.

4. Conclusion

The study demonstrated vastly significant differences in the compositions of the wastes and bioprocess

evaluation parameters. FVW had a higher C/N ratio, highest CH4, SMY and microbial count that

distinctively confirmed a promising co-digesting source of high bioenergy production comparatively to

TLF and SJR. The gas production efficiency was consistent with the microbial density and populations

of Methanothrix compared to the prevalent Methanococcus observed in TLF and FVW respectively.

Therefore, FVW serves as a better bioenergy substrate to enhance gas production for co-digestion

purposes particularly with TLF. This paper recommends a further study be conducted on the mixture(s)

of these substrates in continuous plant operations. The ANOVA results showed that the organic solid

wastes were significantly different (P≤ 0.05) in all characteristics of the AD process to each other.

20
5. Acknowlegdements

The authors express their profound gratitude to Central Leather Research Institute, Adyar, Chennai,

India for providing the laboratory for carrying out this study and Indian Institute of Technology, Madras

(SEMJUL/02/2014S13) for conducting the HR-SEM analysis. This work was supported under the

Department of Biotechnology (DBT), Government of India and The World Academy of Sciences

(TWAS) Predoctoral Fellowship (FR 3240255094). We are grateful to them all.

21
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Fig. 1. Schematic diagram of the laboratory scale anaerobic batch BMP reactor.

28
(2a)

(2b)

Fig. 2. Gas production of TLF, FVW, SJR, CLFS and the inoculum (a) daily (b) cumulative.

Legend:

TLF: Tannery limed fleshing; FVW: Fruits and vegetable wastes; SJR: Sugarcane juice residue;

CLFS: Mixture of the three wastes; c: cumulative.

29
Fig. 3. Removal efficiency of VS from different organic solid waste materials.

Legend:

TLF: Tannery limed fleshing; FVW: Fruits and vegetable wastes; SJR: Sugarcane juice residue;

CLFS: Mixture of the three wastes.

30
Fig. 4. Specific methane yields of the different organic solid wastes during anaerobic digestion.

Legend:

TLF: Tannery limed fleshing; FVW: Fruits and vegetable wastes; SJR: Sugarcane juice residue;

CLFS: Mixture of the three wastes.

31
Fig. 5. Experimental and stoichiometric methane potentials of the different organic solid waste

materials.

32
 a b

cocci

rod

c d

Fig. 6. Methanogens from the different solid waste materials and control treatment subjected to

anaerobic digestion in a batch reactor.

(a) Micrograph of seed inoculum in the reactor (control), (b) micrograph of three substrates with seed

inoculum (CLFS), (c) micrograph of TLF with the seed inoculum, (d) micrograph of FVW with the

seed inoculum.

33
Table 1: Characterization of the organic solid wastes (substrates).

Organic solid wastes used

Physicochemical Seed inoculum Tannery limed Fruits and vegetable Sugarcane juice

parameters (Control) fleshing (TLF) wastes (FVW) residue (SJR)

Sample colour Black Dirty white Greenish-yellow Army green

pH 7.20 ± 0.20c 12.60 ± 0.32d 6.46 ± 0.04b 5.70 ± 0.18a

TS (%) 4.60 ± 0.10a 9.13 ± 0.12c 4.92 ± 0.03b 9.72 ± 0.11d

VS (% TS) 51.38 ± 0.50a 64.40 ± 0.40b 86.90 ± 0.10c 96.27 ± 0.25d

FS (% TS) 46.59 ± 0.11d 35.56 ± 0.11c 13.85 ± 0.13b 3.73 ± 0.06a

O/I ratio 1.10 ± 0.13a 1.81 ± 0.31b 6.27 ± 0.06c 25.81 ± 0.31d

Data are presented as Mean ± SD. Values followed by similar alphabets along the same column are not

significantly different at P≤ 0.05.

34
Table 2: Elemental composition and empirical formula-related analyses.

Seed inoculum Tannery limed Fruits and vegetable Sugarcane juice

Elements Units
(Control) fleshing (TLF) wastes (FVW) residue (SJR)

Carbon, C (%TS) 25.02 ± 0.41a 32.53 ± 0.05b 39.69 ± 0.24c 53.78 ± 0.11d

Hydrogen, H (%TS) 4.61 ± 0.09a 5.32 ± 0.08b 5.37 ± 0.11b 6.57 ± 0.07c

Nitrogen, N (%TS) 2.94 ± 0.12b 7.38 ± 0.05d 4.28 ± 0.07c 1.84 ± 0.06a

Oxygen, O (%TS) 20.84 ± 0.15b 19.23 ± 0.09a 36.81 ± 0.08d 34.09 ± 0.32c

C/N ratio 8.48 ± 0.22b 4.41 ± 0.03a 9.28 ± 0.17b 29.39 ± 0.41c

Calorific Energy
MJ/kg 10.97 ± 0.12a 14.77 ± 0.10b 16.03 ± 0.18c 22.90 ± 0.11d
Value (CEV)

Empirical
C9.9H21.83O6.18N C5.15H10.09O2.28N C10.82H17.57O6.53N C34.29H50.26O16.3N
formula

COD equivalent
gCOD´/g VS 1.45 1.51 1.21 1.65
(COD´/weight)

Methane (CH4)
(%) 31.75 46.05 62.73 36.68
content

Data are presented as Mean ± SD. Values followed by similar alphabets along the same column are not
significantly different at P≤ 0.05.

35
Table 3: Bioprocess evaluation of the organic solid wastes during and after anaerobic digestion.

Organic CODs VFA Free NH3 ALK Anaerobic count

Time pH
solid wastes (mg.L-1) (mg.L-1) (mg.L-1) (mg.L-1) (CFU/mL)

Seed
INITIAL 18000 ± 2000 4263.84 ± 1.24 105.10 ± 2.33 6.5± 0.01 11566.67 ± 251.66 1.10 ± 0.21×102
inoculum

(Control) FINAL 4870 ± 399.62 314.70 ± 0.26 184.55 ± 0.58 6.47 ± 0.05 9800 ± 147.99 4.80 ± 0.50 ×103

Tannery

limed INITIAL 41627.67 ± 668.93 10263.23 ± 7.09 453.58 ± 0.38 6.53 ± 0.01 15700 ± 700.00 1.67 ± 0.32 ×102

fleshing
FINAL 10848 ± 221.71 1322.77 ± 2.75 2110.92 ± 1.01 8.11± 0.02 25233.33 ± 152.75 5.02 ± 0.55 ×104
(TLF)

Fruits and

vegetable INITIAL 56400.33 ± 100 9294.50 ± 25.02 252 ± 2.00 6.52 ± 0.01 17000 ± 435.90 1.87 ± 0.25 ×103

wastes
FINAL 3687.33 ± 585.389 1068.37 ± 1.48 818.67 ± 2.52 6.81 ± 0.04 20500 ± 100.00 3.30 ± 0.47 ×105
(FVW)

Sugarcane

juice residue INITIAL 38413.33 ± 574.92 21088.81 ± 10.63 294.69 ± 0.32 6.54 ± 0.02 28013.33 ± 32.15 2.51 ± 0.24 ×103

(SJR) FINAL 10206.66 ± 205.99 2155.47 ± 2.25 784.67 ± 1.53 4.06 ± 0.04 10333.33 ± 577.35 6.50 ± 0.58 ×104

Mixture of

the 3 wastes INITIAL 63998.33 ± 227.51 8067.00 ± 7.02 315.17 ± 1.76 6.67 ± 0.03 36060.67 ± 115.47 5.50 ± 0.54 ×104

(CLFS) FINAL 13210 ± 1065.04 1027.17 ± 0.20 1097.33 ± 2.52 6.07 ± 0.20 11700 ± 200.00 3.00 ± 0.20 ×106

Data are presented as Mean ± SD.

36