Bioresource Technology 132 (2013) 166–170

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Bioresource Technology

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Integration of sewage sludge digestion with advanced biofuel synthesis

0960-8524/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.biortech.2013.01.017

⇑ Corresponding author. Address: Department of Biosystems and AgriculturalEngineering, 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).

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, USAb Department of Energy, Environmental and Chemical Engineering, Washington University, St. Louis, MO 63130, USAc 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

Article history:Received 12 November 2012Received in revised form 3 January 2013Accepted 4 January 2013Available online 16 January 2013

Keywords:Anaerobic digestionFatty acidMortierella isabellinaEscherichia coliSewage sludge

a b s t r a c t

Sewage sludge rich in carbohydrates and other nutrients could be a good feedstock for fuel/chemical pro-duction. In this study, fungal and engineered bacterial cultivations were integrated with a modifiedanaerobic digestion to accumulate fatty acids on sewage sludge. The anaerobic digestion was firstadjusted to enable acetogenic bacteria to accumulate acetate. A fungus (Mortierella isabellina) and anengineered bacterium (Escherichia coli created by optimizing acetate utilization and fatty acid biosynthe-sis as well as overexpressing a regulatory transcription factor fadR) were then cultured on the acetatesolution to accumulate fatty acids. The engineered bacterium had higher fatty acid yield and titer thanthe fungus. Both medium- and long-chain fatty acids (C12:0–C18:0) were produced by the engineeredbacterium, while the fungus mainly synthesized long-chain fatty acids (C16:0–C18:3). This study demon-strated a potential path that combines fungus or engineered bacterium with anaerobic digestion toachieve simultaneous organic waste treatment and advanced biofuel production.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Sewage sludge is a complex mixture that contains organic, inor-ganic, and biological residues from municipal wastewater treat-ment. National Research Council (NRC) estimated thatapproximately 5.6 million dry tons of sewage sludge is generatedannually from wastewater treatment operations in the US (Com-mittee on Toxicants and Pathogens in Biosolids Applied to Landand N.R.C., 2002). Due to the concern of public health, the sewagesludge must be treated to eliminate human pathogens before itsland application and public distribution. On the other hand, thesludge is rich in organic nutrients such as carbohydrates and pro-teins. It has potential to be utilized by various biological processesto produce value-added products.

Anaerobic digestion (AD) is a process that is able to simulta-neously 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 hydroly-sis of organic polymers (proteins and carbohydrates) into mono-mers (sugars, amino acids); acidogenesis and acetogenesis toconvert sugars and amino acids into acetic acid and other organicacids; and methanogesis to generate methane and carbon dioxidefrom organic acids. Methane as the main product of anaerobicdigestion of the sewage sludge can be used for electricitygeneration. However, relatively low electricity buy-back rates(the primary revenue from methane of AD) limit biosolids applica-tions, and relatively high capital costs for AD electricity generationsystem (electricity for the grid) challenge the economic feasibilityof AD technology for various scale operations, particularly mediumand small sewage sludge operations.

To make AD more suitable for a wide range of applications andmore economical accessible, this study investigated an integratedbioprocess that combines a modified AD process with fungal andbacterial fermentations to accumulate fatty acids for advanced fuel

Z. Liu et al. / Bioresource Technology 132 (2013) 166–170 167

production. It has been reported that AD process under unfavor-able digestion conditions such as low pH and existence of inhibi-tors was capable of degrading organic matters into acetic acidand other organic acids instead of methane (Rughoonundunet al., 2010). Acetic acid as an important industrial intermediatecan be used as a carbon source to support a variety of microbesfor fuel/chemical production. Immelman discovered a fungus, Mu-cor circinelloides, that is able to accumulate linolenic acid from ace-tate as the sole carbon source (Immelman et al., 1997). Christopheet al. have reported that an oleaginous yeast, Cryptococcus curvatus,was able to sequentially utilize glucose and acetic acid to accumu-late lipid (Christophe et al., 2012). Lee et al. co-cultured two bacte-ria of Clostridium butyricum and Rhodobacter sphaeroides on aceticacid to produce hydrogen (Lee et al., 2012). While, no studies havebeen reported to apply the oleaginous fungus Mortierella isabellinaand engineered Escherichia coli on acetate from AD for advancedfuel production.

In this study, a lipid accumulation fungus, M. isabellina, and anengineered bacterium, E. coli were tested to utilize the acetate fromAD 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 micro-bial fatty acids. The results of this study provide a new strategy toutilize sewage sludge for advanced biofuel production.

2. Methods

2.1. Anaerobic treatment of sewage sludge

The effluent from aeration pond was obtained from East LansingWaste Water Treatment Plant (East Lansing, MI, USA). The effluentwas centrifuged at 2851 g for 20 min to separate sludge from theeffluent. The sludge was then pretreated at 100 �C for 1 h (Rughoo-nundun et al., 2010). A total solid of 5% of the sludge was used foracetic acid production. The anaerobic digestion was carried outusing 500 mL anaerobic bottles with 400 mL pretreated sludgemedium. An anaerobic seed from Michigan State University pilotanaerobic digester was added into the culture at a ratio of 12.5%(v/v) at the beginning of the culture. Two treatments includingchemical inhibition (using iodoform to inhibit methanogens andpH around 7.0) and pH adjustment (pH adjusted to 5.0) were car-ried out. Iodoform solution was prepared using pure ethanol to dis-solve iodoform and make 20 g/L of iodoform solution. 0.4 mL/L ofiodoform solution was added into the culture every 48 h, and pHwas controlled around 7 using 30% (w/w) NaOH. For pH adjust-ment 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 samplingduring the anaerobic digestion were conducted under anaerobicenvironment created by Simplicity 888 Automatic AtmosphereChamber (PLAS & LABS, Lansing, MI).

2.2. Microbial fermentation for fatty acids production

M. isabellina ATCC 42613 was obtained from the American TypeCulture Collection (Manassas, VA). The culture conditions werepreviously reported with slight modification (Ruan et al., 2012).2 g/L yeast extract was used as the nitrogen source. M. isabellinaATCC 42613 was cultured in nitrogen-limited medium at three ini-tial acetate concentrations (2.55, 4.85, 7.21 g/L). The growth med-ium (pure acetate medium or medium from anaerobic digestion)was autoclaved and inoculated with a 10% (v/v) seed culture andcultivated at 25 ± 1 �C on a rotary shaker (Thermal Scientific) witha speed of 180 rpm.

To engineer E. coli strain for efficiently producing fatty acidsfrom acetic acid, acs, fadR, and tesA genes were over-expressed ina fatty acid degradation deficient mutant BL21(DfadE). The acsgene (acetyl CoA syntheatase, for acetic acid assimilation) (Linet al., 2006) was cloned into pUC19 K (ColE1 ori, kanr) via SphI/XbaI, resulting in pYX30 (unpublished data). The tesA gene encod-ing acyl-ACP thioesterase (Lu et al., 2008) and the fadR gene encod-ing fatty acid metabolism regulator proteins (Zhang et al., 2012)was then introduced into a BglBrick vector (p15A ori, cmr) throughEcoRI/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 forfatty acid production. The engineered strain was pre-cultured ona M9 medium (containing 33.9 g/L disodium phosphate, 15.0 g/Lmonopotassium phosphate, 2.5 g/L sodium chloride, 5.0 g/L ammo-nium 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 chloram-phenicol to hold the other plasmid) for 13 h. All E. coli cultureswere on a rotary shaker with a speed of 200 rpm at 37 �C. WhenOD600 reached �3.0 (late log phase, acetate was mostly used up),isopropyl beta-D-1 thiogalactopyranoside (IPTG) and fresh acetatestocks (pure acetate or AD acetate) were added to induce free fattyacid production. For fatty acid production using pure acetic acid,the E. coli was inoculated into a medium containing 0.2 mM IPTGand �4.8 g/L acetic acid; after 12 h culture, acetic acid (�5 g/L inthe culture) was supplemented, and the culture was continuedfor another 12 h. For fatty acid production using AD acetate, theE. coli was cultured for 24 h on the medium containing 0.2 mMIPTG and the sterilized AD acetate (3.1 g/L).

2.3. Analytical methods

Acetic acid concentration was detected following instructions ofMegazyme 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 ace-tate and other organic compounds (Ruan et al., 2012). Fungal cellmass was collected by filtration and washed twice with deionizedwater. The cell mass was determined by drying under 105 ± 1 �Covernight to obtain a constant weight. Dried fungal cells were thenground in a mortar and used for lipid extraction according to Blighand Dyer method (Bligh and Dyer, 1959). Fatty acids in the fungallipid were analyzed via a modified method (Ruan et al., 2012) thatfatty acids were measured by their methyl ester.

Fatty acids produced by engineered E. coli were measuredthrough a modified method (Aldai et al., 2006; Lu et al., 2008;Steen et al., 2010; Voelker and Davies, 1994). The culture was ex-tracted by methanol–chloroform. The organic layer was trans-ferred to a new tube and used vacuum to remove the solvent.

168 Z. Liu et al. / Bioresource Technology 132 (2013) 166–170

Methyl derivation of fatty acids was performed at 40 �C (Steenet al., 2010). The samples were then extracted by ethyl acetate be-fore GC–MS analysis. The methyl esters were analyzed using GC(Hewlett Packard model 7890A, Agilent Technologies, equippedwith a DB5-MS column, J&W Scientific) and a mass spectrometer(5975C, Agilent Technologies). The fatty acid methyl esters werequantified based on the standards, including methyl ester ofdodecenoic acid (C12:1), the F.A.M.E. Mix (C8–C24), methyl oleate,methyl myristoleate, and methyl pentadecanoate (purchased fromSigma).

3. Results and discussion

3.1. Optimization of anaerobic digestion for acetic acid productionfrom sewage sludge

During AD process, acetic acid and other organic acids were pro-duced as intermediates by bacteria in the stages of hydrolysis, aci-dogenesis, and acetogenesis (Yue et al., 2010). In regular digestionprocesses, these organic acids are quickly metabolized by metha-nogens to produce methane and carbon dioxide (Gavala et al.,2003). It has been reported that pH is one of the most importantfactors to influence the AD process through controlling populationsof acidogenic bacteria and methanogens in the microbial commu-nities. Low pH was reported to have a significant negative impacton methanogens and a minor impact on acidogenic bacteria, whichconsequently leads the anaerobic digestion to accumulating aceticacid and other organic acids etc (Zoetemeyer et al., 1982). There-fore, reducing pH of the digestion can be an easy strategy to modifythe anaerobic digestion to accumulate acetic acid. In addition, ithas also been studied that using methanogen inhibitors such asiodoform is another effective way to adjust the digestion to accu-mulate organic acids (Aiello-Mazzarri et al., 2006; Rughoonundunet al., 2010; Rughoonundun et al., 2012). Thus, a comparison be-tween two different AD control strategies was fulfilled to produceacetic acid from sewage sludge.

The preliminary digestion under different pH values presentedthat the cultures under pH lower than 6 significantly reducedmethane production (data not shown). Correspondingly, a pH of5 was selected to evaluate the efficiency of acetic acid production.In comparison, the chemical inhibitor was applied to the digestionto improve acetic acid production from the sewage sludge at a neu-tral pH condition. As shown in Fig. 2, both digestions showed aceticacid accumulation. The digestion with pH control accumulated1.64 g/L acetic acid in 11 days of the culture, and further increasing

Fig. 2. Acetate accumulation between pH control strategy and inhibitor controlstrategy⁄. ⁄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 thatthe digestion only produced 0.7 g/L of acetic acid in the first14 days of the culture. A fast acetate accumulation started after20 days. The concentration of acetic acid reached the highest of4.34 g/L that was approximately three times higher than that fromlow pH one. Thus, the chemical inhibitor approach was selected toprepare AD acetate for following experiments of fungal and bacte-rial fatty acid accumulation.

3.2. Fatty acids accumulation from M. isabellina and engineered E. coliwith 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 modifiedAD to accumulate fatty acids. As presented in Fig. 3, M. isabellinacan efficiently consume acetate from pure acetate solution. Atlow acetate concentration of 2.55 g/L, acetate was consumed with-in 2 days, while it took four days to consume acetate at high ace-tate concentrations of 4.85 and 7.21 g/L. Even though the cultureat the higher concentration needed 2 more days to uptake the ace-tate, the acetate consumption rates at the acetate concentrations of4.85 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 ace-tate concentration of 2.55 g/L. Meanwhile, the fatty acid contentsin fungal biomass demonstrated that higher acetate concentrationof 7.21 g/L had the highest fatty acid concentration of 0.173 g/Lcompared to 0.127 and 0.166 g/L from acetate concentrations of2.55 and 4.85 g/L, respectively (Table 1). The results of acetate con-sumption and fatty acid accumulation on pure acetate solution elu-cidated that increasing acetate concentration within theexperimental range benefited the fungal growth and fatty acidaccumulation.

Meanwhile, fungal culture on AD acetate solution showed aslow consumption of acetate. There was still 1.86 g/L acetate re-mained in the fermentation broth after 5 days culture (Fig. 3).The fatty acid concentration at the end of the fermentation was0.06 g/L, and corresponding fatty acid yield was 6.9% of the theo-retical yield (assuming 0.29 g fatty acid/g acetate). Compared withthe cultures on pure acetate, consumption of acetate from AD wasmuch slower (Fig. 3), and the fatty acid yield and concentrationwere significantly lower (Table 1). It is apparent that some inhibi-tory compounds in the AD waste caused the inferior performanceof M. isabellina on the AD acetate solution. Thorough investigationis needed to further analyze AD effluent compositions and opti-mize the fungal fermentation medium.

Fig. 4 presented acetate utilization between wild-type E. coliand engineered E. coli. Wild-type E. coli had limited capability toutilize 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 errorbars represent standard errors (n = 2).

Table 1Fatty acids produced from pure acetic acid and AD effluenta.

From pure acetic acid From AD effluent

M. isabellina E. coli M. isabellina E. coli

Initial acetate concentration (g/L) 2.55 4.85 7.21 9.80 4.96 3.1Acetate consumption (g/L) 2.55 4.35 7.1 9.55 3.10 3.1

Composition of fatty acidsC12:0 (mg/L) – – – 38 – 18C13:0 (mg/L) – – – – – 10C14:0 (mg/L) – – – 250 – 81C14:1 (mg/L) – – – 14 – –C16:0 (mg/L) 42 53 55 157 22 60C16:1 (mg/L) 4 5 5 130 2 38C17:1 (mg/L)c – – – 28 – 13C18:0 (mg/L) 8 11 11 34 5 28C18:1 (mg/L) 48 65 69 36 27 21C18:2 (mg/L) 14 20 21 – 5 –C18:3 (mg/L) 10 12 11 – 3 –Total fatty acid (mg/L) 126 166 172 687 64 267Fatty acid conversion (g fatty acid produced/g acetate consumed 0.05 0.04 0.02 0.07 0.02 0.086Fatty acid yield (% of theoretical yield)b 17.2 13.8 6.9 24.1 6.9 29.7

a Data are the average of two replicates.b The theoretical yield of fatty acid from acetate was assumed to be 0.29 g fatty acids/g acetic acid.c 2-Hexyl-cyclopropaneoctanoic acid.

Fig. 4. Acetic acid utilization by wild-type E. coli and engineered E. coli with acsgene (overexpress of acetyl-CoA synthase) in aerobic culture (37 �C)a,b. aThe culturemedium contained M9 salts, pure acetate and 2 g/L yeast extract. bThe error barsrepresent standard errors (n = 2).

Z. Liu et al. / Bioresource Technology 132 (2013) 166–170 169

acetate to the strain. With expressing acs gene, the engineeredE. coli significantly improved the efficiency of acetate utilizationto accumulate biomass. In 14 h culture, 3.4 g/L acetate has beenconsumed to accumulate approximate 0.9 g/L bacterial biomass(Fig. 4). Table. 1 showed that the engineered E. coli with acs, fadRand tesA genes accumulated 0.07 g fatty acids/g acetate from theculture on 9.5 g/L of pure acetate. In contrast, the same strain uti-lized the AD acetate solution, and produced 0.09 g fatty acid/g ace-tate. 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, engi-neered E. coli demonstrated a superior performance on acetate uti-lization efficiency. The yield on AD acetate solution wasapproximately four times higher than corresponding fungal culture(Table 1). Interestingly, during fatty acid accumulation, the engi-neered bacterium had higher yield on AD acetate than on pure ace-tate (Table 1), which indicated that the mutant strain utilized othernutrients (such as organic acids) in AD effluent for product synthe-sis. In-depth investigations are also needed to further explore therelationship between the engineered strain and other unidentifiedcompounds in the AD acetate solution.

Fatty acid composition further demonstrated the differences onfatty acid distribution between these two strains. In fermentationof engineered E. coli, more carbon resources were observed to flow

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 ace-tic acid, and over 70% from AD acetate solution. Meanwhile, fungalfermentation accumulated more long chain fatty acids such asC16:0, C18:0, C18:1 and C18:2, over 77% of total fungal fatty acidswere C16:0 and C18:1 (Table 1). This significant difference indi-cates that although both wild M. isabellina and engineered E. coliare capable of utilizing acetate as solo carbon source and accumu-lating fatty acids simultaneously, the carbon flows of these twostrains are significantly different.

4. Conclusion

This study utilized sewage sludge to produce fatty acids for bio-fuels production. AD was modified to treat sewage sludge andaccumulate acetate. Chemical control was found to be a morefavorable strategy than pH control for acetate accumulation duringthe AD. An engineered E. coli was created and showed significanthigher yield and productivity in fatty acid production than thatof wild-type M. isabellina, while both strains demonstrated strongcapabilities to utilize acetate from the AD for fatty acid accumula-tion. This study demonstrates a potential solution to select propermicrobial hosts for utilization of sewage sludge for advanced bio-fuel production.

Acknowledgements

This paper is based on research funded by the Bill & MelindaGates Foundation. The findings and conclusions contained withinare those of the authors and do not necessarily reflect positionsor policies of the Bill & Melinda Gates Foundation. The authors alsoacknowledge the Mass Spectrometry Facility at Michigan StateUniversity for providing the fatty acid composition analysis, andI-CARES center at Washington University for providing lab sup-plies. The authors also thank Dr. Fuzhong Zhang at WashingtonUniversity in St. Louis for his help to develop the engineeredE. coli strain.

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