Sewage sludge rich in carbohydrates and other nutrients could be a good feedstock for fuel/chemical production. In this study, fungal and engineered bacterial cultivations were integrated with a modified anaerobic digestion to accumulate fatty acids on sewage sludge. The anaerobic digestion was first adjusted to enable acetogenic bacteria to accumulate acetate. A fungus (Mortierella isabellina) and an engineered bacterium (Escherichia colt created by optimizing acetate utilization and fatty acid biosynthesis as well as overexpressing a regulatory transcription factor fadR) were then cultured on the acetate solution to accumulate fatty acids. The engineered bacterium had higher fatty acid yield and titer than the fungus. Both medium- and long-chain fatty acids (C12:0-C18:0) were produced by the engineered bacterium, while the fungus mainly synthesized long-chain fatty acids (C16:0-C18:3). This study demonstrated a potential path that combines fungus or engineered bacterium with anaerobic digestion to achieve simultaneous organic waste treatment and advanced biofuel production.
Integration of sewage sludge digestion with advanced biofuel synthesis
Bioresource Technology 132 (2013) 166–170
Contents lists available at SciVerse ScienceDirect
journal homepage: www.elsevier.com/locate/biortech
Integration of sewage sludge digestion with advanced biofuel synthesis
Zhiguo Liu a, Zhenhua Ruan a, Yi Xiao b, Yu Yi b,c, Yinjie J. Tang b, Wei Liao a, Yan Liu a,⇑
Department of Biosystems and Agricultural Engineering, Michigan State University, East Lansing, MI 48824, USA
Department of Energy, Environmental and Chemical Engineering, Washington University, St. Louis, MO 63130, USA
Key Laboratory of Combinatory Biosynthesis and Drug Discovery (Ministry of Education), School of Pharmaceutical Sciences, Wuhan University,
185 East Lake Road, Wuhan 430071, PR China
h i g h l i g h t s
" Combining anaerobic digestion and pure culture presents a waste-to-biofuel solution.
" Both engineered E. coli and fungus can utilize acetate in the digestion efﬂuent.
" The newly engineered E. coli had higher fatty acid yield than wild-type fungus.
a r t i c l e
i n f o
Received 12 November 2012
Received in revised form 3 January 2013
Accepted 4 January 2013
Available online 16 January 2013
a b s t r a c t
Sewage sludge rich in carbohydrates and other nutrients could be a good feedstock for fuel/chemical production. In this study, fungal and engineered bacterial cultivations were integrated with a modiﬁed
anaerobic digestion to accumulate fatty acids on sewage sludge. The anaerobic digestion was ﬁrst
adjusted to enable acetogenic bacteria to accumulate acetate. A fungus (Mortierella isabellina) and an
engineered bacterium (Escherichia coli created by optimizing acetate utilization and fatty acid biosynthesis as well as overexpressing a regulatory transcription factor fadR) were then cultured on the acetate
solution to accumulate fatty acids. The engineered bacterium had higher fatty acid yield and titer than
the fungus. Both medium- and long-chain fatty acids (C12:0–C18:0) were produced by the engineered
bacterium, while the fungus mainly synthesized long-chain fatty acids (C16:0–C18:3). This study demonstrated a potential path that combines fungus or engineered bacterium with anaerobic digestion to
achieve simultaneous organic waste treatment and advanced biofuel production.
Ó 2013 Elsevier Ltd. All rights reserved.
Sewage sludge is a complex mixture that contains organic, inorganic, and biological residues from municipal wastewater treatment. National Research Council (NRC) estimated that
approximately 5.6 million dry tons of sewage sludge is generated
annually from wastewater treatment operations in the US (Committee on Toxicants and Pathogens in Biosolids Applied to Land
and N.R.C., 2002). Due to the concern of public health, the sewage
sludge must be treated to eliminate human pathogens before its
land application and public distribution. On the other hand, the
sludge is rich in organic nutrients such as carbohydrates and proteins. It has potential to be utilized by various biological processes
to produce value-added products.
⇑ Corresponding author. Address: Department of Biosystems and Agricultural
Engineering, Michigan State University, 203 Farrall Hall, East Lansing, MI 48824,
USA. Tel.: +1 517 432 7387; fax: +1 517 432 2892.
E-mail address: firstname.lastname@example.org (Y. Liu).
0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
Anaerobic digestion (AD) is a process that is able to simultaneously stabilize sewage sludge (eliminate human pathogens)
and convert the sludge to bioenergy and fertilizer (Chen et al.,
2008). AD includes three major biological steps: microbial hydrolysis of organic polymers (proteins and carbohydrates) into monomers (sugars, amino acids); acidogenesis and acetogenesis to
convert sugars and amino acids into acetic acid and other organic
acids; and methanogesis to generate methane and carbon dioxide
from organic acids. Methane as the main product of anaerobic
digestion of the sewage sludge can be used for electricity
generation. However, relatively low electricity buy-back rates
(the primary revenue from methane of AD) limit biosolids applications, and relatively high capital costs for AD electricity generation
system (electricity for the grid) challenge the economic feasibility
of AD technology for various scale operations, particularly medium
and small sewage sludge operations.
To make AD more suitable for a wide range of applications and
more economical accessible, this study investigated an integrated
bioprocess that combines a modiﬁed AD process with fungal and
bacterial fermentations to accumulate fatty acids for advanced fuel
Z. Liu et al. / Bioresource Technology 132 (2013) 166–170
production. It has been reported that AD process under unfavorable digestion conditions such as low pH and existence of inhibitors was capable of degrading organic matters into acetic acid
and other organic acids instead of methane (Rughoonundun
et al., 2010). Acetic acid as an important industrial intermediate
can be used as a carbon source to support a variety of microbes
for fuel/chemical production. Immelman discovered a fungus, Mucor circinelloides, that is able to accumulate linolenic acid from acetate as the sole carbon source (Immelman et al., 1997). Christophe
et al. have reported that an oleaginous yeast, Cryptococcus curvatus,
was able to sequentially utilize glucose and acetic acid to accumulate lipid (Christophe et al., 2012). Lee et al. co-cultured two bacteria of Clostridium butyricum and Rhodobacter sphaeroides on acetic
acid to produce hydrogen (Lee et al., 2012). While, no studies have
been reported to apply the oleaginous fungus Mortierella isabellina
and engineered Escherichia coli on acetate from AD for advanced
In this study, a lipid accumulation fungus, M. isabellina, and an
engineered bacterium, E. coli were tested to utilize the acetate from
AD treated sewage sludge to produce fatty acids. Correspondingly,
a stepwise strategy was designed to fulﬁll the investigation (Fig. 1):
(1) Modify AD to convert sewage sludge to acetate; (2) Apply M.
isabelina and engineered E. coli for conversion of acetate to microbial fatty acids. The results of this study provide a new strategy to
utilize sewage sludge for advanced biofuel production.
2.1. Anaerobic treatment of sewage sludge
The efﬂuent from aeration pond was obtained from East Lansing
Waste Water Treatment Plant (East Lansing, MI, USA). The efﬂuent
was centrifuged at 2851 g for 20 min to separate sludge from the
efﬂuent. The sludge was then pretreated at 100 °C for 1 h (Rughoonundun et al., 2010). A total solid of 5% of the sludge was used for
acetic acid production. The anaerobic digestion was carried out
using 500 mL anaerobic bottles with 400 mL pretreated sludge
medium. An anaerobic seed from Michigan State University pilot
anaerobic digester was added into the culture at a ratio of 12.5%
(v/v) at the beginning of the culture. Two treatments including
chemical inhibition (using iodoform to inhibit methanogens and
pH around 7.0) and pH adjustment (pH adjusted to 5.0) were carried out. Iodoform solution was prepared using pure ethanol to dissolve iodoform and make 20 g/L of iodoform solution. 0.4 mL/L of
iodoform solution was added into the culture every 48 h, and pH
was controlled around 7 using 30% (w/w) NaOH. For pH adjustment treatment, 10% (v/v) hydrochloric acid was used to control
Fig. 1. Lipid accumulation on acetate from anaerobic digestion.
the pH at 5 (Rughoonundun et al., 2012). Feeding and sampling
during the anaerobic digestion were conducted under anaerobic
environment created by Simplicity 888 Automatic Atmosphere
Chamber (PLAS & LABS, Lansing, MI).
2.2. Microbial fermentation for fatty acids production
M. isabellina ATCC 42613 was obtained from the American Type
Culture Collection (Manassas, VA). The culture conditions were
previously reported with slight modiﬁcation (Ruan et al., 2012).
2 g/L yeast extract was used as the nitrogen source. M. isabellina
ATCC 42613 was cultured in nitrogen-limited medium at three initial acetate concentrations (2.55, 4.85, 7.21 g/L). The growth medium (pure acetate medium or medium from anaerobic digestion)
was autoclaved and inoculated with a 10% (v/v) seed culture and
cultivated at 25 ± 1 °C on a rotary shaker (Thermal Scientiﬁc) with
a speed of 180 rpm.
To engineer E. coli strain for efﬁciently producing fatty acids
from acetic acid, acs, fadR, and tesA genes were over-expressed in
a fatty acid degradation deﬁcient mutant BL21(DfadE). The acs
gene (acetyl CoA syntheatase, for acetic acid assimilation) (Lin
et al., 2006) was cloned into pUC19 K (ColE1 ori, kanr) via SphI/
XbaI, resulting in pYX30 (unpublished data). The tesA gene encoding acyl-ACP thioesterase (Lu et al., 2008) and the fadR gene encoding fatty acid metabolism regulator proteins (Zhang et al., 2012)
was then introduced into a BglBrick vector (p15A ori, cmr) through
EcoRI/XhoI and BglII/BamHI respectively to construct pA58c-TR
(unpublished data). Finally, the mutant E. coli strain BL21
(DfadE)/pYX30 + pA58c-TR was generated to utilize acetate for
fatty acid production. The engineered strain was pre-cultured on
a M9 medium (containing 33.9 g/L disodium phosphate, 15.0 g/L
monopotassium phosphate, 2.5 g/L sodium chloride, 5.0 g/L ammonium chloride, 0.1 mM CaCl2, 2 mM MgSO4, 4 g sodium acetate,
25 lg/ml kanamycin, 0.5% yeast extract, and 12.5 mg/L chloramphenicol to hold the other plasmid) for 13 h. All E. coli cultures
were on a rotary shaker with a speed of 200 rpm at 37 °C. When
OD600 reached $3.0 (late log phase, acetate was mostly used up),
isopropyl beta-D-1 thiogalactopyranoside (IPTG) and fresh acetate
stocks (pure acetate or AD acetate) were added to induce free fatty
acid production. For fatty acid production using pure acetic acid,
the E. coli was inoculated into a medium containing 0.2 mM IPTG
and $4.8 g/L acetic acid; after 12 h culture, acetic acid ($5 g/L in
the culture) was supplemented, and the culture was continued
for another 12 h. For fatty acid production using AD acetate, the
E. coli was cultured for 24 h on the medium containing 0.2 mM
IPTG and the sterilized AD acetate (3.1 g/L).
2.3. Analytical methods
Acetic acid concentration was detected following instructions of
Megazyme Acetic Acid Kit assay procedure (www.megazyme.com).
HPLC with Aminex HPX-87H column (Bio-Rad Lab, Hercules, CA,
USA), 65 °C, 0.6 ml/min, 25 min, RID, was also used to analyze acetate and other organic compounds (Ruan et al., 2012). Fungal cell
mass was collected by ﬁltration and washed twice with deionized
water. The cell mass was determined by drying under 105 ± 1 °C
overnight to obtain a constant weight. Dried fungal cells were then
ground in a mortar and used for lipid extraction according to Bligh
and Dyer method (Bligh and Dyer, 1959). Fatty acids in the fungal
lipid were analyzed via a modiﬁed method (Ruan et al., 2012) that
fatty acids were measured by their methyl ester.
Fatty acids produced by engineered E. coli were measured
through a modiﬁed method (Aldai et al., 2006; Lu et al., 2008;
Steen et al., 2010; Voelker and Davies, 1994). The culture was extracted by methanol–chloroform. The organic layer was transferred to a new tube and used vacuum to remove the solvent.
Z. Liu et al. / Bioresource Technology 132 (2013) 166–170
Methyl derivation of fatty acids was performed at 40 °C (Steen
et al., 2010). The samples were then extracted by ethyl acetate before GC–MS analysis. The methyl esters were analyzed using GC
(Hewlett Packard model 7890A, Agilent Technologies, equipped
with a DB5-MS column, J&W Scientiﬁc) and a mass spectrometer
(5975C, Agilent Technologies). The fatty acid methyl esters were
quantiﬁed based on the standards, including methyl ester of
dodecenoic acid (C12:1), the F.A.M.E. Mix (C8–C24), methyl oleate,
methyl myristoleate, and methyl pentadecanoate (purchased from
3. Results and discussion
3.1. Optimization of anaerobic digestion for acetic acid production
from sewage sludge
During AD process, acetic acid and other organic acids were produced as intermediates by bacteria in the stages of hydrolysis, acidogenesis, and acetogenesis (Yue et al., 2010). In regular digestion
processes, these organic acids are quickly metabolized by methanogens to produce methane and carbon dioxide (Gavala et al.,
2003). It has been reported that pH is one of the most important
factors to inﬂuence the AD process through controlling populations
of acidogenic bacteria and methanogens in the microbial communities. Low pH was reported to have a signiﬁcant negative impact
on methanogens and a minor impact on acidogenic bacteria, which
consequently leads the anaerobic digestion to accumulating acetic
acid and other organic acids etc (Zoetemeyer et al., 1982). Therefore, reducing pH of the digestion can be an easy strategy to modify
the anaerobic digestion to accumulate acetic acid. In addition, it
has also been studied that using methanogen inhibitors such as
iodoform is another effective way to adjust the digestion to accumulate organic acids (Aiello-Mazzarri et al., 2006; Rughoonundun
et al., 2010; Rughoonundun et al., 2012). Thus, a comparison between two different AD control strategies was fulﬁlled to produce
acetic acid from sewage sludge.
The preliminary digestion under different pH values presented
that the cultures under pH lower than 6 signiﬁcantly reduced
methane production (data not shown). Correspondingly, a pH of
5 was selected to evaluate the efﬁciency of acetic acid production.
In comparison, the chemical inhibitor was applied to the digestion
to improve acetic acid production from the sewage sludge at a neutral pH condition. As shown in Fig. 2, both digestions showed acetic
acid accumulation. The digestion with pH control accumulated
1.64 g/L acetic acid in 11 days of the culture, and further increasing
Fig. 2. Acetate accumulation between pH control strategy and inhibitor control
strategy⁄. ⁄The error bars represent standard errors (n = 2).
culture time did not contribute to the acetic acid accumulation.
While, inhibitor control strategy demonstrated a slow start-up that
the digestion only produced 0.7 g/L of acetic acid in the ﬁrst
14 days of the culture. A fast acetate accumulation started after
20 days. The concentration of acetic acid reached the highest of
4.34 g/L that was approximately three times higher than that from
low pH one. Thus, the chemical inhibitor approach was selected to
prepare AD acetate for following experiments of fungal and bacterial fatty acid accumulation.
3.2. Fatty acids accumulation from M. isabellina and engineered E. coli
with acetic acid as sole carbon source
A fungus (M. isabellina) and an engineered bacterium (E. coli)
were cultured on pure acetate and the acetate from the modiﬁed
AD to accumulate fatty acids. As presented in Fig. 3, M. isabellina
can efﬁciently consume acetate from pure acetate solution. At
low acetate concentration of 2.55 g/L, acetate was consumed within 2 days, while it took four days to consume acetate at high acetate concentrations of 4.85 and 7.21 g/L. Even though the culture
at the higher concentration needed 2 more days to uptake the acetate, the acetate consumption rates at the acetate concentrations of
4.85 and 7.21 g/L in the ﬁrst two days culture were 1.86 and 2.64 g/
L/day, which were signiﬁcantly higher than 1.28 g/L/day of the acetate concentration of 2.55 g/L. Meanwhile, the fatty acid contents
in fungal biomass demonstrated that higher acetate concentration
of 7.21 g/L had the highest fatty acid concentration of 0.173 g/L
compared to 0.127 and 0.166 g/L from acetate concentrations of
2.55 and 4.85 g/L, respectively (Table 1). The results of acetate consumption and fatty acid accumulation on pure acetate solution elucidated that increasing acetate concentration within the
experimental range beneﬁted the fungal growth and fatty acid
Meanwhile, fungal culture on AD acetate solution showed a
slow consumption of acetate. There was still 1.86 g/L acetate remained in the fermentation broth after 5 days culture (Fig. 3).
The fatty acid concentration at the end of the fermentation was
0.06 g/L, and corresponding fatty acid yield was 6.9% of the theoretical yield (assuming 0.29 g fatty acid/g acetate). Compared with
the cultures on pure acetate, consumption of acetate from AD was
much slower (Fig. 3), and the fatty acid yield and concentration
were signiﬁcantly lower (Table 1). It is apparent that some inhibitory compounds in the AD waste caused the inferior performance
of M. isabellina on the AD acetate solution. Thorough investigation
is needed to further analyze AD efﬂuent compositions and optimize the fungal fermentation medium.
Fig. 4 presented acetate utilization between wild-type E. coli
and engineered E. coli. Wild-type E. coli had limited capability to
utilize acetate ($6 g/L) to grow because of the inhibitory effect of
Fig. 3. M. isabellina culture on pure acetate and acetate from AD efﬂuent⁄. ⁄The error
bars represent standard errors (n = 2).
Z. Liu et al. / Bioresource Technology 132 (2013) 166–170
Fatty acids produced from pure acetic acid and AD efﬂuenta.
From pure acetic acid
From AD efﬂuent
Initial acetate concentration (g/L)
Acetate consumption (g/L)
Composition of fatty acids
Total fatty acid (mg/L)
Fatty acid conversion (g fatty acid produced/g acetate consumed
Fatty acid yield (% of theoretical yield)b
Data are the average of two replicates.
The theoretical yield of fatty acid from acetate was assumed to be 0.29 g fatty acids/g acetic acid.
into shorter chain fatty acids with C12:0, C14:0, C16:0 and C16:1,
occupying over 80% of the total fatty acids in weight from pure acetic acid, and over 70% from AD acetate solution. Meanwhile, fungal
fermentation accumulated more long chain fatty acids such as
C16:0, C18:0, C18:1 and C18:2, over 77% of total fungal fatty acids
were C16:0 and C18:1 (Table 1). This signiﬁcant difference indicates that although both wild M. isabellina and engineered E. coli
are capable of utilizing acetate as solo carbon source and accumulating fatty acids simultaneously, the carbon ﬂows of these two
strains are signiﬁcantly different.
Fig. 4. Acetic acid utilization by wild-type E. coli and engineered E. coli with acs
gene (overexpress of acetyl-CoA synthase) in aerobic culture (37 °C)a,b. aThe culture
medium contained M9 salts, pure acetate and 2 g/L yeast extract. bThe error bars
represent standard errors (n = 2).
acetate to the strain. With expressing acs gene, the engineered
E. coli signiﬁcantly improved the efﬁciency of acetate utilization
to accumulate biomass. In 14 h culture, 3.4 g/L acetate has been
consumed to accumulate approximate 0.9 g/L bacterial biomass
(Fig. 4). Table. 1 showed that the engineered E. coli with acs, fadR
and tesA genes accumulated 0.07 g fatty acids/g acetate from the
culture on 9.5 g/L of pure acetate. In contrast, the same strain utilized the AD acetate solution, and produced 0.09 g fatty acid/g acetate. The yields of the cultures on pure acetate and AD acetate were
$24% and $30% of the theoretical yield, respectively (Table 1).
Compared to fungal fatty acid accumulation from acetate, engineered E. coli demonstrated a superior performance on acetate utilization efﬁciency. The yield on AD acetate solution was
approximately four times higher than corresponding fungal culture
(Table 1). Interestingly, during fatty acid accumulation, the engineered bacterium had higher yield on AD acetate than on pure acetate (Table 1), which indicated that the mutant strain utilized other
nutrients (such as organic acids) in AD efﬂuent for product synthesis. In-depth investigations are also needed to further explore the
relationship between the engineered strain and other unidentiﬁed
compounds in the AD acetate solution.
Fatty acid composition further demonstrated the differences on
fatty acid distribution between these two strains. In fermentation
of engineered E. coli, more carbon resources were observed to ﬂow
This study utilized sewage sludge to produce fatty acids for biofuels production. AD was modiﬁed to treat sewage sludge and
accumulate acetate. Chemical control was found to be a more
favorable strategy than pH control for acetate accumulation during
the AD. An engineered E. coli was created and showed signiﬁcant
higher yield and productivity in fatty acid production than that
of wild-type M. isabellina, while both strains demonstrated strong
capabilities to utilize acetate from the AD for fatty acid accumulation. This study demonstrates a potential solution to select proper
microbial hosts for utilization of sewage sludge for advanced biofuel production.
This paper is based on research funded by the Bill & Melinda
Gates Foundation. The ﬁndings and conclusions contained within
are those of the authors and do not necessarily reﬂect positions
or policies of the Bill & Melinda Gates Foundation. The authors also
acknowledge the Mass Spectrometry Facility at Michigan State
University for providing the fatty acid composition analysis, and
I-CARES center at Washington University for providing lab supplies. The authors also thank Dr. Fuzhong Zhang at Washington
University in St. Louis for his help to develop the engineered
E. coli strain.
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