Bioresource Technology 132 (2013) 166170

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Bioresource Technology
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,
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 c 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
b

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 efuent. " 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

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 modied anaerobic digestion to accumulate fatty acids on sewage sludge. The anaerobic digestion was rst 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 (C12:0C18:0) were produced by the engineered bacterium, while the fungus mainly synthesized long-chain fatty acids (C16:0C18:3). 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. 2013 Elsevier Ltd. All rights reserved.

Article history: Received 12 November 2012 Received in revised form 3 January 2013 Accepted 4 January 2013 Available online 16 January 2013 Keywords: Anaerobic digestion Fatty acid Mortierella isabellina Escherichia coli Sewage sludge

1. Introduction 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: liuyan6@msu.edu (Y. Liu).
0960-8524/$ - see front matter 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.01.017

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 modied AD process with fungal and bacterial fermentations to accumulate fatty acids for advanced fuel

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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 fuel production. 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 fulll 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. Methods 2.1. Anaerobic treatment of sewage sludge The efuent from aeration pond was obtained from East Lansing Waste Water Treatment Plant (East Lansing, MI, USA). The efuent was centrifuged at 2851 g for 20 min to separate sludge from the efuent. 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

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 modication (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 Scientic) with a speed of 180 rpm. To engineer E. coli strain for efciently producing fatty acids from acetic acid, acs, fadR, and tesA genes were over-expressed in a fatty acid degradation decient 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 ltration 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 modied 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 modied method (Aldai et al., 2006; Lu et al., 2008; Steen et al., 2010; Voelker and Davies, 1994). The culture was extracted by methanolchloroform. The organic layer was transferred to a new tube and used vacuum to remove the solvent.

Fig. 1. Lipid accumulation on acetate from anaerobic digestion.

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Methyl derivation of fatty acids was performed at 40 C (Steen et al., 2010). The samples were then extracted by ethyl acetate before GCMS analysis. The methyl esters were analyzed using GC (Hewlett Packard model 7890A, Agilent Technologies, equipped with a DB5-MS column, J&W Scientic) and a mass spectrometer (5975C, Agilent Technologies). The fatty acid methyl esters were quantied based on the standards, including methyl ester of dodecenoic acid (C12:1), the F.A.M.E. Mix (C8C24), methyl oleate, methyl myristoleate, and methyl pentadecanoate (purchased from Sigma).

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 rst 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 modied AD to accumulate fatty acids. As presented in Fig. 3, M. isabellina can efciently 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 rst two days culture were 1.86 and 2.64 g/ L/day, which were signicantly 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 beneted the fungal growth and fatty acid accumulation. 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 signicantly 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 efuent 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

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 inuence the AD process through controlling populations of acidogenic bacteria and methanogens in the microbial communities. Low pH was reported to have a signicant 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 fullled to produce acetic acid from sewage sludge. The preliminary digestion under different pH values presented that the cultures under pH lower than 6 signicantly reduced methane production (data not shown). Correspondingly, a pH of 5 was selected to evaluate the efciency 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).

Fig. 3. M. isabellina culture on pure acetate and acetate from AD efuent. The error bars represent standard errors (n = 2).

Z. Liu et al. / Bioresource Technology 132 (2013) 166170 Table 1 Fatty acids produced from pure acetic acid and AD efuenta. From pure acetic acid M. isabellina Initial acetate concentration (g/L) Acetate consumption (g/L) Composition of fatty acids C12:0 (mg/L) C13:0 (mg/L) C14:0 (mg/L) C14:1 (mg/L) C16:0 (mg/L) C16:1 (mg/L) C17:1 (mg/L)c C18:0 (mg/L) C18:1 (mg/L) C18:2 (mg/L) C18:3 (mg/L) Total fatty acid (mg/L) Fatty acid conversion (g fatty acid produced/g acetate consumed Fatty acid yield (% of theoretical yield)b
a b c

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From AD efuent E. coli M. isabellina 4.96 3.10 22 2 5 27 5 3 64 0.02 6.9 E. coli 3.1 3.1 18 10 81 60 38 13 28 21 267 0.086 29.7

2.55 2.55 42 4 8 48 14 10 126 0.05 17.2

4.85 4.35 53 5 11 65 20 12 166 0.04 13.8

7.21 7.1 55 5 11 69 21 11 172 0.02 6.9

9.80 9.55 38 250 14 157 130 28 34 36 687 0.07 24.1

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. 2-Hexyl-cyclopropaneoctanoic 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 signicant 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 ows of these two strains are signicantly different. 4. Conclusion This study utilized sewage sludge to produce fatty acids for biofuels production. AD was modied 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 signicant 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. Acknowledgements This paper is based on research funded by the Bill & Melinda Gates Foundation. The ndings and conclusions contained within are those of the authors and do not necessarily reect 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. References
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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 signicantly improved the efciency 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 efciency. 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 efuent for product synthesis. In-depth investigations are also needed to further explore the relationship between the engineered strain and other unidentied 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 ow

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