Journal of Biotechnology 253 (2017) 14–22

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Journal of Biotechnology
journal homepage: www.elsevier.com/locate/jbiotec

Biochemical Engineering/Bioprocess Engineering

Potential of fecal waste for the production of biomethane, bioethanol and MARK
biodiesel
⁎
Mohamed A. Gomaa, Raeid M.M. Abed
Biology Department, College of Science, Sultan Qaboos University, P. O. Box: 36, PC 123, Al Khoud, Oman

A R T I C L E I N F O A B S T R A C T

Keywords: Fecal waste is an environmental burden that requires proper disposal, which ultimately becomes also an
Biofuels economic burden. Because fecal waste is nutrient-rich and contains a diverse methanogenic community, it has
Biomethane been utilized to produce biomethane via anaerobic digestion. Carbohydrates and lipids in fecal waste could
Bioethanol reach up to 50% of the dry weight, which also suggests a potential as a feedstock for bioethanol and biodiesel
Biodiesel
production. We measured biomethane production from fecal waste of cows, chickens, goats and humans and
Manure
compared the microbial community composition before and after anaerobic digestion. We also compared the
Sludge
fecal waste for cellulase production, saccharification and fermentation to produce bioethanol and for lipid
content and fatty acid profiles to produce biodiesel. All fecal waste produced biomethane, with the highest yield
of 433.4 ± 77.1 ml CH4/g VS from cow fecal waste. Production of bioethanol was achieved from all samples,
with chicken fecal waste yielding as high as 1.6 ± 0.25 g/l. Sludge samples exhibited the highest extractable
portion of lipids (20.9 ± 0.08 wt%) and conversion to fatty acid methyl esters (11.94 wt%). Utilization of fecal
waste for the production of biofuels is environmentally and economically beneficial.

1. Introduction waste could be achieved through several processes, including gasifica-

Every year, millions of kilograms of fecal waste are being produced
by dairy cattle and goats, broiler chickens and humans; estimated at
2372 kg per cattle per year (Chastain and Camberato, 2004), 48 kg per
chicken per year (Chastain et al., 2001) and 127 kg per average person
per year (Asl and Hosseini, 2000). This amount of waste poses an
environmental threat concerning greenhouse gas (GHG) emissions and
water pollution (Miller et al., 2011; Davis et al., 2012). Manures are
known to produce about 10% of agricultural GHG emissions in the
environment (Steinfeld et al., 2006). Fecal waste has long been used as
a fertilizer and agricultural land conditioner (Hansen, 2006). Yet, it
may also contain antibiotics and zoonotic pathogens that limits its use
as a fertilizer (Venglovsky et al., 2009). With concerns for this
detrimental effect on the environment, an efficient utilization of fecal
waste could be a useful alternative.
With the rising demand for energy supply, there is a dire need for an
environmentally benign and sustainable energy source. Previous studies
have demonstrated the potential of fecal waste in the production of
bioenergy (Cantrell et al., 2008; Champagne, 2008; Kargbo, 2010; Kim
et al., 2014b). Production of methane containing biogas from fecal
tion, pyrolysis and anaerobic digestion (Demirbaş, 2001). Biogas yields
differ from one source of fecal waste to another based on the
composition of the waste and its microbial community composition
(Farnworth et al., 1995; Wang et al., 2014; Li et al., 2015). Several
reports have demonstrated the potential use of fecal waste for the
generation of biomethane with a yield range of 75–850 ml CH4/g VS
(Hill, 1984; Wen et al., 2007; Kim et al., 2014b; Noorollahi et al., 2015).
While most of these reports focused on a single fecal waste source, few
studies have compared the yields from different waste materials under
the same conditions (Li et al., 2015; Noorollahi et al., 2015). Further-
more, the microbial communities, including methanogens, in different
fecal waste have been rarely identified and compared using next
generation sequencing (Wirth et al., 2012; Li et al., 2013).
Fecal waste contains around 30–50% carbohydrates of its dry
weight, of which between 50–84% could be converted to simple sugars
(Wen, 2004; Chen et al., 2005; Liao et al., 2006). This suggests a
potential use of fecal waste hydrolysate as a feedstock for the produc-
tion of bioethanol. The technologies involved in pretreatment and
extraction of cellulose have been well established (Sun and Cheng,
2002; Taherzadeh and Karimi, 2008; Sarkar et al., 2012). Research was

Abbreviations: GHG, Greenhouse gases; NGS, Next generation sequencing; CMC, Carboxy methyl cellulose; CMCase, Carboxy methyl cellulase; SSF, Solid-substrate fermentation; FAME,
Fatty acid methyl esters; TSS, Total suspended solids; COD, Chemical oxygen demand; TP, Total phosphorous; TS, Total solids; VS, Volatile solids; GC, Gas chromatography; FID, Flame
ionization detector; OTUs, Operational taxonomic units; DNS, Dinitrosalicylic acid; GC–MS, Gas chromatography–mass spectrometry
⁎
Corresponding author.
E-mail address: rabed@mpi-bremen.de (R.M.M. Abed).

http://dx.doi.org/10.1016/j.jbiotec.2017.05.013
Received 16 February 2017; Received in revised form 23 April 2017; Accepted 19 May 2017
Available online 22 May 2017
0168-1656/ © 2017 Elsevier B.V. All rights reserved.
M.A. Gomaa, R.M.M. Abed
conducted on the use of cattle and chicken fecal waste for the
production of bioethanol by acid hydrolysis or enzyme saccharification
(Champagne, 2008; Woldesenbet et al., 2013; Vancov et al., 2015; Yang
et al., 2015). Recently, cow manure was fed to Bacillus subtilis to
produce biologically active carboxy methyl cellulase (CMCase) in a
solid-substrate fermentation (SSF) (Vijayaraghavan et al., 2016).
Although this study provided a cheap source of CMCase, it has yet to
demonstrate the potential use of this biologically produced enzyme in
the cellulose-to-ethanol conversion process. There has been no reports
that utilized fecal waste for enzyme generation and saccharification in a
single process. Besides their richness in carbohydrates, fecal waste
contains between 7 and 36% lipids that can be utilized for biodiesel
production, also referred to as fatty acid methyl esters (FAME), through
lipid extraction and transesterification (Kargbo, 2010; Kwon et al.,
2012). Research has focused on sewage sludge as an economically
promising source of lipids for biodiesel production (Siddiquee and
Rohani, 2011; Kwon et al., 2012). The cost of biodiesel production from
sewage sludge was estimated to be 0.03 USD per liter using current
technology (Kwon et al., 2012). Apart from analyzing gut enzymes of
farm animals for biofuel production (Solomon et al., 2016) and the
reported conversion of manure to biodiesel by housefly larvae (Yang
et al., 2014), little has been done at exploring other fecal waste sources
for biodiesel production.
Here, we compared fecal waste from four sources (i.e. cow, goat,
chicken and human) for their potential use in the production of
biomethane, bioethanol and biodiesel. Biomethane production was
measured after 49 days of anaerobic incubation and the microbial
communities were compared before and after in order to identify key
methanogens. For bioethanol production, fecal waste was first used to
produce the CMCase enzyme, which was then used for saccharification
of fresh fecal waste. Bioethanol was produced by fermentation of the
reducing sugars using an indigenous Clostridium strain. Biodiesel was
also produced by simultaneous extraction and transesterification of the
lipid fraction from the fecal waste.
2. Materials and methods
2.1. Sample collection
Fresh cow, goat and chicken excrement samples were collected from
the Sultan Qaboos University agricultural farm, while dewatered
primary sewage sludge and sewage water samples were collected from
a municipal sewage treatment plant in Muscat, Oman. Total suspended
solids (TSS), alkalinity, chemical oxygen demand (COD), total phos-
phorous (TP), total solids (TS), volatile solids (VS), moisture content,
and pH in all samples were measured (Supplementary file 1) according
to previously described methods (Association et al., 1915; Jirka and
Carter, 1975; Hamilton and Zhang, 2011). The differences in microbial
communities, amounts and composition of fermentable components
due to their different diets between the fecal waste samples were
purposefully ignored to explore the potential of these fecal wastes
without alteration of their natural states.
2.2. Biomethane production from fecal waste
The production of biomethane from fecal waste was tested in
triplicates in closed glass bottles under strictly anaerobic conditions
as previously described in (Lay et al., 1997). Five grams of fresh fecal
waste biomass was added into 165 ml pre-autoclaved serum glass
bottles inside an anaerobic chamber (Forma Anaerobic System
Model 1025/1029, Thermo Scientific), where the atmosphere was
composed of 5% H2, 10% CO2, and 85% N2. Sewage water (pH 7)
was used as a cultivation medium after autoclaving and removal of
all dissolved oxygen. Ten ml of the autoclaved sewage water was
added onto the biomass and the vials were sealed air tight with a
20 mm butyl septum and aluminum seal caps. The vials were incubated
15
Journal of Biotechnology 253 (2017) 14–22
in the dark at 37 °C in a shaking incubator with a mixing speed of
80 rpm for 49 days.
The amount of methane produced in the headspace of each vial was
analyzed using a gas chromatograph (GC) equipped with a flame
ionization detector (FID). A linear calibration curve using 6 different
methane concentrations (2.5–100% ± 5%) was used. A 0.2 ml sample
of the headspace was obtained using a 0.1–0.5 ml gas-tight glass syringe
(Hamilton GASTIGHT ® Model 1725 SL SYR, Large Removable NDL,
22 s ga, 2 in, point style 2). The sample was injected manually into a
GC-FID (Agilent 6890N, Agilent Technologies) equipped with an HP-
PLOT/Q megabore column (30 m × 0.53 mm × 40 μm) with Helium
as the carrier gas at 45 ml/min. A 10:1 split injection mode was used
with the following GC temperatures: injection port, 150 °C; initial
column temperature, 35 °C; initial hold time, 3 min; temperature ramp,
20 °C; final column temperature, 150 °C; final hold time, 5 min. The
total GC-FID run time was 15 min.
2.3. Microbial community analysis
DNA was extracted from the original fecal biomass and at the end of
the previous experiment to compare the microbial communities before
and after the incubation and to identify the methanogens using MiSeq
sequencing of 16S rRNA genes. The biomass was removed from the
vials and the DNA was extracted using MOBIO PowerSoil ® DNA
Isolation Kit following the manufacturer’s instructions. Purified DNA
extracts from the fecal biomass before and after the incubation were
then submitted to Molecular Research MR DNA laboratory (www.
mrdnalab.com, Shallowater, TX, USA) for Illumina MiSeq sequencing
of the 16S rRNA genes using the primers whoi341 (CCTACGG
GNGGCWGCAG) and whoi785 (GACTACHVGGGTATCTAATCC), which
are known to be universal primers for both archaea and bacteria, with
barcode on the forward primer (Klindworth et al., 2013). Six samples
were pooled together in equal proportions based on their molecular
weight and DNA concentrations. Pooled samples were purified using
calibrated AMPure XP beads. The pooled and purified PCR products
were used to prepare a DNA library by following Illumina TruSeq DNA
library preparation protocol. Sequence analysis was carried out using
the Mothur MiSeq SOP pipeline (Schloss et al., 2009). Briefly, barcodes
were removed and sequences < 200 bp and sequences with ambiguous
base calls were eliminated. Sequences were denoised, operational
taxonomic units (OTUs) generated and chimeras removed. OTUs were
defined by clustering at 3% divergence (97% similarity). Final OTUs
were taxonomically classified using BLASTn against a curated Green-
Genes database (DeSantis et al., 2006). Rarefaction curves and diversity
indices (OTU richness, Chao-1 and ACE) were calculated using the
Mothur software.
2.4. Bioethanol production from fecal waste
Bioethanol production from the fecal waste involved three stages
namely (1) production of CMCase enzyme, (2) fecal biomass sacchar-
ification using CMCase and (3) fermentation using the obtained
reducing sugars. The fecal waste biomass was dried in the sun for
3 days and homogenized into coarse powder prior to use. For the
production of CMCase, 7 g of the homogenized biomass was mixed
gently with 0.1 M Tris-HCl buffer (pH 8.0), to maintain moisture
content of 90% (w/v), and the mixture was then autoclaved. The fecal
waste was incubated with 10% (v/v) inoculum (OD600 of 0.85) of an
indigenous cellulase-producing B. licheniformis BW-24 (hereafter re-
ferred to as BW-24) to obtain 100% moisture content. The inoculum of
this strain was prepared by growing on LB broth at 37 °C for 24 h. The
bacterial cells were harvested by centrifugation and washed using
sterile distilled water to remove all medium residues. The inoculum was
re-suspended in Tris-HCl buffer (pH 8.0) and added directly to the fecal
biomass. The incubation was carried out at 37 °C for 72 h, which is the
optimal temperature and time for CMCase production (unpublished,
M.A. Gomaa, R.M.M. Abed Journal of Biotechnology 253 (2017) 14–22

personal communication). The production of the CMCase in the fecal

waste samples was compared with a positive control containing 1.25 g
CMC, 0.25 g NaNO3, 0.25 g K2HPO4, 0.25 g KCl, 0.125 g MgSO4,
0.125 g yeast extract dissolved in 250 ml distilled water and a negative
control containing the same medium but without CMC. Crude enzyme
was extracted using distilled water. The CMCase activity was measured
using dinitrosalicylic acid (DNS) assay (Wood and Bhat, 1988). One
unit of CMCase activity is defined as the amount of enzyme needed
to liberate 1 μmol of reducing sugars per minute (Vijayaraghavan
et al., 2016).
Saccharification was performed by adding the extracted CMCase to
7 g of homogenized fecal biomass. The mixture was incubated for
10 days at 37 °C. To compare the efficiency of the biologically extracted
CMCase with the commercially available CMCase, the same amount of
autoclaved fecal biomass was additionally hydrolyzed with similar
concentrations of CMCase isolated from Aspergillus niger with activity of
300 U/g solid (Sigma-Aldrich®, Germany). After 10 days, the treated Fig 1. The methane yield of fecal waste from four sources over a period of 49 days of
anaerobic digestion. The scale on the y-axis has a break (dotted line) to accurately show
biomass and reducing sugars were aseptically transferred to a 50 ml
the production of the lower yielding samples.
tube and placed in the anaerobic chamber for 24 h prior to fermenta-
tion. A native Clostridium strain AK-1 (referred hereafter as AK-1) was
of methane after 49 and 21 days of incubation with maximum amounts
used for the fermentation of cellulase-treated biomass to produce
of 433 ± 77 and 208 ± 89 ml CH4/g volatile solids (VS), respectively
bioethanol. AK-1 was grown in TYG broth for 24 h (OD600 of 0.85)
(Fig. 1). While the maximum amount of produced methane in the case
under anaerobic conditions and this was the inoculum for the
of sludge was detected after 21 days of incubation, this amount
fermentation experiment. The fermentation medium (containing
continued to increase in the Cow fecal waste to reach its maximum
0.75 g K2HPO4, 0.75 g KH2PO4, 0.7 g MgSO4·7H2O, 0.017 g
after 49 days. Chicken and goat fecal waste yielded lower amounts of
MnSO4·5H2O, 0.01 g FeSO4·7H2O, 2 g (NH4)2SO4, 1 g NaCl, 2 g
methane reaching a maximum of ≤17 ± 6 ml CH4/g VS after 49 and
Asparagine, 0.004 g p-aminobenzoic acid, 5 g yeast extract, 4.08 g
21 days, respectively. Analysis of the methane production rate from the
CH3COONa·3H2O in 1 l of distilled water) was prepared with less water
sludge sample showed the highest productivity from day 3 until day 21
to account for the water containing the extracted CMCase.
at a rate of 13 ml CH4/g VS/day. Cow fecal waste resulted in the
Fermentation medium was added to the tubes to a final volume of
highest methane yields of 433 ml CH4/g VS due to the steady rate
25 ml, then 3% AK-1 inoculum (8.6 × 106 cells/ml) was added. As an
(10 ml CH4/g VS/day) observed after the lag phase. Both sludge and
added control, autoclaved but untreated homogenized biomass was
cow samples had a 3 day lag phase, yet there was a difference in their
prepared for fermentation using the same conditions to check if CMCase
rates after day 3.
is being produced by the AK-1 itself. The tubes were closed tight and
A total of 302,395 and 260,920 16S rRNA archaeal and bacterial
mixed gently before incubation at 35 °C under anaerobic conditions for
sequences were obtained by MiSeq from the fecal waste, respectively
21 days. Every 2 days, 1 ml was taken from each sample to check for
(Table 1). Rarefaction curves showed good coverage of the bacterial
bioethanol production using gas chromatography-mass spectrometry
sequences whereas more sequences were needed to cover the whole
(GC–MS).
archaeal diversity since the curves did not level off at the 97% cut-off
(Fig. 2). OTU richness, Chao-1 and ACE diversity indices showed higher
2.5. Biodiesel production from fecal waste
values in the bacterial than in the archaeal communities (Table 1).
These values decreased after incubation under methanogenic condi-
The dried and homogenized fecal biomass was used for simulta-
tions, except in the case of chicken and goat wastes (Table 1). Cluster
neous extraction of total lipids and transesterification to produce
analysis showed that the archaeal and bacterial communities were
biodiesel (Folch et al., 1957). One gram of biomass was suspended in
different before and after incubation (Fig. 2). The archaeal communities
20 ml of chloroform/methanol (2:1) mixture and incubated at 50 °C for
of chicken waste after incubation was the most distant from others
24 h at 120 rpm. The supernatant was recovered by centrifugation, and
(Fig. 2).
the process was repeated 2 times but for 1 h each under the same
In all original fecal waste, ≥97% of archaeal sequences belonged to
conditions. The supernatants from the 3 steps were pooled in a fresh
Methanobacteria, whereas the sludge archaeal community contained
tube, then the phases were separated by adding 0.9% NaCl solution
46% Methanomicrobia, 32% Methanobacteria and 21% Thaumarchaeota
and centrifuging for 5 min at 1200 x g. The chloroform layer was
(Fig. 3). The majority of the archaeal sequences belonged to Methano-
recovered and placed in a pre-weighed glass tube. The aqueous
brevibacter (92–96% of total sequences). In sludge, Methanobrevibacter
layer was washed once with 2 ml chloroform and the chloroform was
constituted only 25% of total sequences, but Methanosaeta was most
then added to the same pre-weighed tube. The chloroform was
abundant (43% of total sequences). At the end of incubation, the
evaporated under a stream of nitrogen and the total lipids were
abundance of Methanobacteria decreased to 38–46% of total sequences
determined by the weight difference. Further analysis of fatty acid
in all samples (Fig. 3). Sequences belonging to Methanobrevibacter
methyl esters was done by re-suspending the lipids in chloroform and
decreased in all samples, but sequences belonging to Methanobacterium
injecting it in a GC–MS.
increased in the cow and goat samples (Fig. 3). Unlike Methanobacteria,
Methanomicrobia increased after incubation from < 1% to 19–39% of
3. Results
total sequences. Most of these sequences were affiliated to Methano-
corpusculum (17% of total sequences in the chicken sample) and
3.1. Biomethane production and methanogens
Methanosarcina (34–37% in the cow and goat samples). The relative
abundance of Thermoplasmata sequences reached 34% of total se-
Biomethane production by different fecal waste exhibited clear
quences at the end of incubation in the chicken sample, but remained <
variations. Cow fecal waste and sludge showed the highest production

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Table 1
MiSeq sequencing and archaeal and bacterial diversity estimators for the sludge and manure samples before and after incubation under anaerobic conditions.

Fecal waste source Archaeal communities

Total No. of sequences No. of OTUs1 Chao-1 ACE Total No. of sequences No. of OTUs1 Chao-1 ACE

Before incubation After incubation

Sludge 20000 145 193 192 79107 116 146 143
Chicken 8095 69 90 91 13881 65 86 83
Goat 14273 100 142 135 100208 78 102 102
Cow 14729 105 160 163 52102 75 94 92

Bacterial communities
Sludge 50127 281 375 379 18895 184 257 260
Chicken 39716 124 196 199 18372 167 235 248
Goat 29485 141 208 212 7308 198 242 244
Cow 41059 139 208 208 55958 78 134 139

1
Operational taxonomic unit at 3% sequence dissimilarity based on equal subsets of sequences for all samples, Chao-1 is based on rare OTUs in a given sample and ACE is abundance-
based coverage.

4% in the cow and goat samples. The archaeal groups in the sludge
samples remained more or less at the same relative abundance, except
Thermoplasmata, which increased to 11% of total sequences. Sequences
belonging to Methanosaeta, Methanospirillum and Thermogymnomonas
were only detected in the sludge sample at the end of incubation.
The bacterial communities showed clear differences in the original
fecal waste, and underwent shifts by the end of the incubation (Fig. 3).
While Verrucomicrobiae constituted ≥94% of total sequences in the
original cow and goat samples, it made up only 28% and 34% of the
sequences in the original chicken fecal waste and sludge, respectively
(Fig. 3). These sequences were affiliated to Akkermansia. In the sludge
sample, sequences belonging to Clostridia and Betaproteobacteria were
additionally encountered and in chicken fecal waste, > 52% of total
sequences belonged to Planctomycetia. Among the detected genera in
the sludge and chicken fecal waste were Pelotomaculum, Nitrosovibrio,
Prosthecobacter, Pirellula, Luteolibacter and Chthoniobacter (Fig. 3). At the

Fig. 2. Rarefaction curves of archaea and bacteria (top panel) and cluster analysis depicting the similarity in archaeal and bacterial communities (bottom panel) of fecal waste from four
sources before and after 49 days of anaerobic digestion. The legend displayed in the (top panel) is valid for the entire figure. The asterisk denotes the community before incubation.

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M.A. Gomaa, R.M.M. Abed Journal of Biotechnology 253 (2017) 14–22

Fig. 3. The relative abundance (%) of bacteria versus archaea (top panel) before and after 49 days of anaerobic digestion. The relative abundance (%) of the major classes are depicted for
archaea (middle panel) and bacteria (bottom panel) in fecal waste from four sources.

end of the incubation, sequences of Cloacimonetes dominated the sludge
and cow samples (55% and 83% of total sequences, respectively),
Betaproteobacteria dominated cow, sludge and goat samples (13–39%)
and Planctomycetia and Verrucomicrobiae dominated chicken and goat
samples (13–44%). Similar genera were detected at the end of incuba-
tion but at different relative abundances (Fig. 3).
3.2. CMCase and bioethanol production
Using the native strain BW-24, saccharification of different fecal
waste biomass revealed different CMCase enzyme activities (Fig. 4A).
The highest CMCase activity was obtained by hydrolyzing the chicken
and the goat fecal waste and were estimated to be 390 ± 90 and

Fig. 4. (A) Enzymatic activity of the biologically produced CMCase from fecal waste along with the activity of the commercially obtained CMCase. (B) The resulting sugars measured after
saccharification of fecal waste from four sources using the produced CMCase compared to commercially obtained CMCase.

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M.A. Gomaa, R.M.M. Abed Journal of Biotechnology 253 (2017) 14–22

Fig. 5. Bioethanol production from AK-1 over a period of 8 days. (A) the bioethanol yields of the positive control using glucose, negative control using no sugar and untreated fecal waste
biomass. (B) the bioethanol yields of fecal waste from four sources hydrolyzed with biologically produced CMCase. (C) the bioethanol yields of fecal waste from four sources hydrolyzed
with commercially obtained CMCase.

262 ± 34 U/g, respectively (P < 0.05). Sludge and cow fecal waste
yielded less active enzymes at 126 ± 33 and 77 ± 21 U/g, respec-
tively (P < 0.05). Hydrolysis of fresh fecal waste using the extracted
CMCase showed that the amounts of produced reducing sugars were
positively correlated with the obtained biological CMCase activity
(Fig. 4B), where the highest CMCase activity in the case of chicken
fecal waste produced the highest reducing sugars concentration. While
the concentrations of reducing sugars were higher (P < 0.005) in the
commercial CMCase compared with the biologically produced CMCase,
when both used at the same concentrations, these values were lower in
the case of sludge and cow fecal waste (Fig. 4B).
Fermentation of reducing sugars using AK-1 showed an increased
bioethanol production compared with fermentation using untreated
fecal waste biomass (Fig. 5A). This increase was continuous until day 8
(Fig. 5B and C), after which the concentration remained unchanged
until 21 days of incubation (data not shown). Bioethanol yields
increased with increasing initial concentrations of reducing sugars
(Fig. 4B), regardless whether biological or commercial CMCase enzyme
was used (Fig. 5B and C). The amounts of bioethanol produced were
similar or higher in the case of commercial CMCase. While the highest
bioethanol yield of 1.6 g/l was obtained by fermenting chicken fecal
waste treated with commercial CMCase, fermentation of the same
biomass treated with biological CMCase resulted in 38% less ethanol.
Fermentation of other fecal waste treated with commercial and
biological CMCase yielded bioethanol of < 1 g/l.
3.3. Biodiesel yield
The total extracted lipids from all fecal waste materials ranged
between 11 and 21 wt%, with the highest value from the sludge sample
(Table 2). The conversion of lipids to FAME was highest in sludge and
reached 12 wt% (Table 2). The FAME profiles obtained from chicken,
goat and cow fecal waste were comparable, while myristoleic (C14:1)
and palmetoleic (C16:1) acids were only detected in the profile
obtained from sludge. The FAME amounts reached 40–119 mg when
calculated per g of dry biomass, with the highest value detected in the
sludge sample.
4. Discussion
4.1. Biomethane and methanogenic community analysis
Our anaerobic incubations revealed the potential of using fecal
waste for biomethane production. By comparing total biomethane
yields and archaeal communities, it was evident that cow fecal waste
had the most productive methanogenic community. The setup of the
incubations favored a more dominant and less diverse archaeal com-
munity as indicated by the change in the relative abundance of bacteria
versus archaea and the reduction in archaeal OTU richness. The highest
biomethane yield obtained in our study was 433 ml CH4/g VS, which
was higher than previously reported yields from different fecal waste
(140–270 ml CH4/g VS) when converted to the same units (Noorollahi
et al., 2015). Nevertheless, when compared with yields from larger
scale (up to 50000 liters) bioreactors with optimized conditions and
pretreated fecal waste, our results show room for improvement (Kim
et al., 2014a; Li et al., 2015). The differences in the total amounts and
rates of produced methane could be attibuted to the differences in
methangenic communities as well as the nature of the used fecal waste.
Although the archaeal community composition from cow, goat and
chicken fecal waste were comparable, a higher methanogenic activity
was observed in cow fecal waste. This supports previous findings that
cow waste enhances methanogen diversity and methane emission (Kim
et al., 2014b). Co-digestion of sludge with cow fecal waste could be a
potential next step to optimize biomethane yields.

Table 2
FAME profile (in wt%) for the produced biodiesel from the studied fecal waste compared to produced biodiesel from other sources.

FAME Sludge Chicken Goat Cow Sludge + H2SO41 Activated Sludge2 Chlorella protothecoides3

Lauric (C12:0) 2.3 3.8 6.4 11.8 1.2 0.6 –

Myristoleic (C14:1) 2.2 – – – 3.6 5.8 1.3
Palmetic (C16:0) 38.5 4.1 53.2 7 39.7 73.3 13
Palmetoleic (C16:1) 3.7 – – – 1.6 – 0.9
Stearic (C18:0) 11.3 11.1 8 19.4 15.3 10.2 2.8
Oleic (C18:1) 21.6 50 21.5 29.7 27.4 2.9 60.8
Linoleic (C18:2) 7.5 24.7 5.5 9.3 3.8 0.6 17.3
Linolenic (C18:3) 12.9 6.5 5.3 22.7 – – –

Lipid (wt%) 20.9 ± 0.1 11.3 ± 0.4 11 ± 0.9 13.8 ± 0.2

FAME (wt%) 12 4 6 6.5
FAME (mg/g) 119.4 40.3 59.5 65

1
Huynh et al. (2012); 2Olkiewicz et al. (2016); 3Xu et al. (2006).

19
M.A. Gomaa, R.M.M. Abed
The production of biomethane from chicken and goat fecal waste,
although with low yields, was supported by the enrichment of
methanogenic community at the end of incubation. The high solid
concentration (TS) in the chicken and goat samples (25% and 26%,
respectively vs. 9% and 7% for sludge and cow samples, respectively)
and the low agitation rate of the setup could be major causes in the
reduced methane yields (Karthikeyan and Visvanathan, 2012; Liao
et al., 2014). The deterioration of high TS anaerobic digestion could
also result in increased accumulation of ammonia (Karthikeyan and
Visvanathan, 2012), which inhibits methane production (Hansen et al.,
1998). Although we have not measured ammonia in the samples, our
bacterial community analysis showed an increased number of known
nitrogen fixing bacteria (such as Paenibacillus and Bacteroides sp.).
Decreased methane production could also be due to the presence of
phosphate, which has been shown to inhibit the activity of acetoclastic
methanogens (Conrad et al., 2000).
4.2. Bacterial and archaeal communities
The bacterial and archaeal genera in the original sludge sample
were different from all other samples, which could be due to physio-
logical and/or diet differences between humans and herbivorous
animals. The used experimental setup apparently favored the archaeal
community, as could be inferred from the dramatic shift in the relative
abundance of archaea from 18 to 33% before incubation to 42–95%
after incubation in all samples. Dominance of the genus
Methanobrevibacter (class Methanobacteria), which includes mainly
hydrogenotrophic methanogen, in all fecal waste (except sludge) has
been previously reported in cow waste slurry and in goat rumen
(Wright et al., 2008; Walter et al., 2015). At the end of our incubations,
there was a clear shift towards an acetoclastic dominant methanogenic
community, favoring Methanosarcina (from 0.07–12% to 16–35%) in all
samples except chicken. Previous studies have related the increase of
Methanosarcina to an increase in acetate levels (Padmasiri et al., 2007;
Walter et al., 2015).
Unlike in our study, long term anaerobic digestion was reported to
cause a shift towards a hydrogenotrophic dominant community (Guo
et al., 2014; Jang et al., 2014). It is likely that this shift was not
observed in our study due to the relatively short incubation period
(49 days). The decrease in hydrogenotrophic methanogens was further
supported by the concomitant decrease in the syntrophic fermentative
bacteria, like Clostridia, whose activity provides H2 to these methano-
gens (Guo et al., 2014). Fermentative bacteria are capable of producing
acetate from CO2, which if accumulated in the medium along with H2,
will reduce the pH (Li et al., 2011). Since acidic pH values inhibit
methane production (Debeer et al., 1992; Kim, 2003), the observed
decrease in pH of our sludge (6.2) and chicken (5.1) samples, unlike
cow (7.3) and goat (7.5) samples, along with the increase of Thermo-
plasmata indicate that acidity increased over time.
4.3. Bioethanol through CMCase digestion of biomass
Unlike other processes that use commercial CMCase for sacchar-
ification of biomass for bioethanol production (Puspawati et al., 2015;
Viola et al., 2016), we used a previously described 3-stage process
(Sukumaran et al., 2009) that combines CMCase production, sacchar-
ification using produced CMCase and then fermentation of the resulting
sugars to produce bioethanol. This approach was successful in convert-
ing fecal waste into bioethanol and had several advantages. Firstly, the
cost of commercial CMCase (ranging between $102–2537 for 100 g,
Sigma-Aldrich® and Thermo Fisher Scientific) could be saved by
producing the enzyme within the same process. Secondly, fecal waste
was used in the entire process, making the process more economical,
and will significantly reduce their negative impact on the environment.
This approach also utilizes indigenous organisms (i.e. BW-24 and AK-1)
for CMCase production and fermentation of reducing sugars to
20
Journal of Biotechnology 253 (2017) 14–22
bioethanol. Activities of CMCase produced from BW-24 (77–390 U/g)
were comparable with that of Bacillus subtilis IND19, which was used
to produce CMCase from cow fecal waste (41–497 U/g), and
that of commercially obtained CMCase from A. niger (300 U/g)
(Vijayaraghavan et al., 2016).
Reducing sugars produced from fecal waste hydrolysis with CMCase
were consistent with the activities obtained from the different types of
fecal waste, although some amounts obtained were lower than expected
when comparing commercial and biological CMCase. This deviation
could be because the activities were tested using CMC, while the
reducing sugars were generated from digesting different fecal waste. It
could also be attributed to the different composition of fecal waste,
where the relative CMCase activity could be affected by the presence of
condensed tannins or high glucose concentrations (Benoit and Starkey,
1968; Xiao et al., 2004). Nevertheless, reducing sugar values were still
consistent with the types of fecal waste, where chicken samples that
gave the highest CMCase activity also gave the most reducing sugars.
Bioethanol yields obtained were consistent with the amounts of
reducing sugars measured for all samples, including the positive and
negative controls. The bioethanol yields of 0.17-0.40 ml/g that resulted
from the controls of all fecal waste samples, with no enzymatic
treatment, could be attributed to residual reducing sugars present after
autoclaving the fecal waste. Chicken fecal waste resulted in the highest
reducing sugar values, thus produced the highest bioethanol yield of 1.6
and 1.05 ml/g biomass from commercial and biological CMCase treat-
ments, respectively. This yield is higher than some reported results
(0.06 ml/g biomass) using swine fecal waste and corn Stover in an
anaerobic digestion (Maclellan et al., 2013). Considering the amount of
used fecal waste, our bioethanol yields were also comparable to
fermentation processes using Saccharomyces cereviciae, which reported
yields as high as 1.3 ml/g biomass (Vancov et al., 2015). Therefore,
production of bioethanol using this approach is promising and econom-
ically feasible, however, further research is required for scale-up and
optimization.
4.4. Biodiesel
The extractable lipids differed for all fecal waste, yet the simulta-
neous lipid extraction and transesterification resulted in a conversion
range of 4–12% FAME of dry weight. Sludge has resulted in the highest
FAME yield of 119 mg/g dry weight, which was almost double that of
the other samples and close to the theoretical maximum found in
previous reports (Kargbo, 2010). Although optimization is not the scope
of this study, yet the yield obtained has potential to exceed this
theoretical maximum by utilizing a tubular reactor, activated alumina
and optimized temperature and solvent-to-sludge ratio (Kwon et al.,
2012). Several reports have highlighted the potential of sewage sludge
as an economically cheap alternative feedstock for biodiesel produc-
tion, with reported costs of 0.03-0.85 USD per liter oil (Mondala et al.,
2009; Siddiquee and Rohani, 2011; Kwon et al., 2012). The FAME
profile of all samples contained the most common fatty acids found in
biodiesel, being palmitic, stearic, oleic, linoleic and linolenic acids
(Knothe, 2008). Similar profiles were detected in previous studies using
sewage sludge and microalgae (Xu et al., 2006; Huynh et al., 2012;
Olkiewicz et al., 2016) (Table 2).
5. Conclusions
Utilization of fecal waste for the production of biomethane,
bioethanol and biodiesel is feasible and environmentally benign.
Efficiency of waste-to-bioenergy conversion depends on the source of
fecal waste and type of bioenergy to be produced. For example, cow
fecal waste was superior to other sources in biomethane production.
The 3-stage process previously described has economical benefits on
bioethanol production, due to the enzyme generation stage and nature
of biomass used in the process. Since little research has been performed
M.A. Gomaa, R.M.M. Abed
in this region on the use of fecal waste for biofuel production, further
research is highly encouraged to optimize production and minimize
costs.
Conflict of interest
There is no conflict of interest declared for any of the authors of this
manuscript.
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Availability of data and materials
The datasets used and/or analysed during the current study are
available from the corresponding author on reasonable request.
Competing interests
The authors declare that they have no competing interests.
Funding
This research was funded by The Research Council (TRC) of Oman
(grant RC/ENG/PCED/14/01).
Authors' contributions
MAG and RMMA designed the study. MAG performed the experi-
ments. MAG and RMMA analyzed and interpreted the data. MAG and
RMMA wrote and formatted the manuscript.
Acknowledgment
The authors would like to thank Asma Al-Kindi for providing the
strains used to produce bioethanol.
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