ZYMOMONAS

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Biotechnol. J. 2014, 9 DOI 10.1002/biot.201300367 www.biotechnology-journal.com

1 Introduction

Global concern about the sustainable supply of crude oiland the environmental deterioration caused by the over-consumption of petroleum-derived products, particularlytransportation fuels, makes the development of alterna-tives essential. Biofuels from renewable biomass resourcesrepresent one alternative, and have been intensively stud-ied since the 1970s following the oil crisis [1, 2].

At present, fuel ethanol is the dominating biofuel, andis produced mainly by Saccharomycescerevisiae strainsvia the Embden-Meyerhof-Parnas (EMP) pathway. In thispathway two moles of ATP are produced per mole of glu-cose consumed, leading to a significant accumulation ofbiomass at the expense of ethanol yield [3]. In contrast toS. cerevisiae, Zymomonas mobilis metabolizes glucose

Research Article

Flocculating Zymomonas mobilis is a promising host to beengineered for fuel ethanol production from lignocellulosicbiomass

Ning Zhao1, Yun Bai1, Chen-Guang Liu1, Xin-Qing Zhao1, Jian-Feng Xu2 and Feng-Wu Bai1,3

1 School of Life Sciences and Biotechnology, Dalian University of Technology, Dalian, China2 Arkansas Biosciences Institute, Arkansas State University, AR, USA3 School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China

Whereas Saccharomyces cerevisiae uses the Embden-Meyerhof-Parnas pathway to metabolize glu-cose, Zymomonas mobilis uses the Entner-Doudoroff (ED) pathway. Employing the ED pathway,50% less ATP is produced, which could lead to less biomass being accumulated during fermen-tation and an improved yield of ethanol. Moreover, Z. mobilis cells, which have a high specific sur-face area, consume glucose faster than S. cerevisiae, which could improve ethanol productivity. Weperformed ethanol fermentations using these two species under comparable conditions to vali-date these speculations. Increases of 3.5 and 3.3% in ethanol yield, and 58.1 and 77.8% in ethanolproductivity, were observed in ethanol fermentations using Z. mobilis ZM4 in media containing~100 and 200 g/L glucose, respectively. Furthermore, ethanol fermentation by the flocculatingZ. mobilis ZM401 was explored. Although no significant difference was observed in ethanol yieldand productivity, the flocculation of the bacterial species enabled biomass recovery by cost-effec-tive sedimentation, instead of centrifugation with intensive capital investment and energy con-sumption. In addition, tolerance to inhibitory byproducts released during biomass pretreatment,particularly acetic acid and vanillin, was improved. These experimental results indicate thatZ. mobilis, particularly its flocculating strain, is superior to S. cerevisiae as a host to be engineeredfor fuel ethanol production from lignocellulosic biomass.

Keywords: Biofuels Ethanol fermentation White/industrial biotechnology Yield Zymomonas mobilis

Correspondence: Prof. Feng-Wu Bai, School of Life Sciences and Biotech-nology, Dalian University of Technology, 2 Linggong Rd., Dalian 116023,ChinaE-mail: [email protected]

Additional correspondence: Dr. Xin-Qing Zhao, School of Life Sciences and Biotechnology, Dalian University of Technology, 2 Linggong Rd.,Dalian 116023, ChinaE-mail: [email protected]

Abbreviations: DCW, dry cell weight; ED, Entner-Doudoroff; EMP, Embden-Meyerhof-Parnas; FBRM, focused-beam reflectance measurement; HG,high gravity; HMF, hydroxymethylfurfural; LG, low gravity; ORP, oxidore-duction potential; VHG, very high gravity

Received 23 AUG 2013Accepted 31 OCT 2013Accepted article online 06 NOV 2013

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2 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

explored, since the morphological change not only facili-tates biomass recovery by cost-effective sedimentationinstead of centrifugation, but also enhances tolerance toenvironmental stresses such as toxic byproducts releasedduring the pretreatment of lignocellulosic biomass.

2 Materials and methods

2.1 Strains, media and seed culture

Z.mobilis ZM4 (ATCC 31821), which has been intensive-ly studied for ethanol fermentation, was used in the study.The industrial strain S.cerevisiae ADY (Angel Yeast CO.,LTD, China) used for fuel ethanol production was selectedas the control to study the improvements in ethanol yieldand productivity of Z.mobilis under comparable fermen-tation conditions. To address the challenge in biomassrecovery with Z.mobilis, which needs high-speed cen-trifugation involving heavy capital investment on cen-trifuges and intensive energy consumption on centrifugerunning, the flocculating strain Z.mobilis ZM401 (ATCC31822) was selected, and compared with Z.mobilis ZM4to highlight its advantages in biomass recovery by sedi-mentation. In addition, the tolerance of the Z. mobilisstrains to inhibitory byproducts released during the pre-treatment of lignocellulosic biomass was also studied.

Medium comprising 20 g/L glucose, 10 g/L yeastextract and 2g/L KH2PO4 was used for the seed culture ofZ. mobilis ZM4 and ZM401 [12]. For S. cerevisiae ADYpropagation, medium comprising 30g/L glucose, 5g/Lyeast extract and 3g/L peptone was used [13]. Media con-taining 110 or 210g/L glucose, respectively, were used forethanol fermentations so that after inoculation, initial glu-cose within the fermentor was controlled at ~100g/L forlow-gravity (LG) fermentation and ~200g/L for high-grav-ity (HG) fermentation. The media were supplementedwith 10 g/L yeast extract for ethanol fermentation byZ. mobilis ZM4 and ZM401, and 5 g/L each of yeastextract and peptone for ethanol fermentation by S.cere-visiae ADY. In addition, very-high-gravity (VHG) mediumcontaining 300g/L glucose was used for feeding the fed-batch fermentation by Z. mobilis ZM4. All media weresterilized at 121C for 30min before inoculation.

A loop-full of stock culture was inoculated into 250mLErlenmeyer flask containing 100mL medium for seed cul-ture. Although Z.mobilis produces ethanol through theED pathway under anaerobic conditions, this species alsopossesses an oxidative phosphorylation pathway forgrowth, and no significant difference was observed in itsmetabolite profiles at the exponential growth phase [5, 14]. Thus, seed cultures for the Z. mobilis ZM4 andZM401 strains and S. cerevisiae ADY were performedovernight at 30C and 150 rpm to the mid-exponentialgrowth phase under microaerobic conditions by sealingthe flasks with regular gauze plugs.

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via the Entner-Doudoroff (ED) pathway, in which only onemole of ATP is produced per mole of glucose consumed.Consequently, less biomass is accumulated duringethanol fermentation, which in theory could improveethanol yield [4]. In addition, the relatively small bacterialcells provide a high specific surface area for sugar uptake[5], which could improve ethanol productivity.

Although these advantages have been speculated,Z.mobilis has never been used for fuel ethanol productionindustrially due to its narrow substrate spectrum of glu-cose, fructose and sucrose only [4]. Glucose is too costly forfuel ethanol production, and starch-based feedstocks suchas corn meal and cassava chips are used, which are lique-fied by amylase into dextrins, followed by the hydrolysis ofthe dextrins by glucoamylase. While glucose is the majorsugar produced during the saccharification process, othersugars such as maltose and maltotriose are also presentedin the mash, which can be fermened by S.cerevisiae [6]but not Z.mobilis. Ethanol yield from fructose and sucroseis much lower in Z.mobilis, since a significant level of sor-bitol is accumulated during the fermentation [4]. There-fore, Z.mobilis is not suitable for fuel ethanol productionfrom sugar- and starch-based feedstocks.

Sugar-based feedstocks such as sugarcane juice ormolasses are geographically limited to tropical and sub-tropical areas such as Brazil, and starch-based feedstocksare major food sources. Neither sugar- nor starch-basedfeedstocks are regarded as sustainable for large-scale pro-duction of fuel ethanol. Lignocellulosic biomass resources,particularly agricultural residues such as corn stover andrice and wheat straw, are alternative feedstocks for sus-tainable production of fuel ethanol [7]. The major carbo-hydrates of lignocellulosic biomass are hemicellulosesand cellulose, which can be hydrolyzed into hexose andpentose sugars. Since glucose is the only hexose sugarreleased from the hydrolysis of the cellulose component,the disadvantage of the narrow substrate spectrum withZ.mobilis would not be problematic for ethanol produc-tion from lignocellulosic biomass. In addition, Z.mobiliscan be readily engineered to provide pentose metabolicpathways for co-fermentation of hexose and pentose sug-ars, which has been explored since the 1990s [8, 9]. Thecompletion of the genome sequence of Z.mobilis and elu-cidation of its metabolic network [10, 11] will help indeveloping robust strains from this species for fuelethanol production from lignocellulosic biomass.

Recently, DuPont established a pilot plant for cellulosicethanol production in Tennessee, USA, in which a recom-binant Z. mobilis strain is being tested. Although theprogress seems exciting, how significant the improve-ment on ethanol yield and productivity by Z.mobilis com-pared to S.cerevisiae would be has not yet been addressedunder comparable conditions. We performed ethanol fer-mentations using Z.mobilis and S.cerevisiae with mediacontaining same glucose concentrations. Moreover, theadvantages of flocculating Z. mobilis were further


pyruvic acid were quantified by HPLC (Waters 2707,Waters 1525 binary HPLC pump;detectors: Waters 2998photodiode array detector and refractive index detector;column: Bio-Rad HPX-87H ion exclusion column, 300 7.8 mm; eluant: 0.005mol/L H2SO4 at a flow rate of0.4mL/min). Another byproduct, acetoin, of ethanol fer-mentation by Z. mobilis was analyzed by GC (Agilent6890 series GC system) equipped with flame ionizationdetector and capillary column (0.25mm diameter), withisobutanol as the internal standard.

The oxidoreduction potential (ORP) was monitored insitu during ethanol fermentations using an ORP electrode(Pt4805-DPAS-SC-K8 12 mm250 mm, Mettler Toledo).For ethanol fermentation using flocculating Z. mobilisZM401, a focused-beam reflectance measurement (FBRM)probe was inserted into the fermentor to monitor the sizedistribution of the bacterial flocs [16].

3 Results and discussion

3.1 Ethanol fermentations by Z. mobilis ZM4 and S. cerevisiae ADY

The time courses of glucose consumption, ethanol andbiomass accumulation, and ORP profiles during ethanolfermentations by Z.mobilis ZM4 and S.cerevisiae ADYwere monitored and recorded (Fig.1). Major byproductsproduced during the fermentations were analyzed(Table1) to evaluate the ethanol fermentation processes.

Only 2.62g(DCW)/L biomass was accumulated duringethanol fermentation by Z.mobilis ZM4 under the LG fer-mentation condition, much lower than the 7.17g(DCW)L1 accumulated during ethanol fermentation by S.cere-visiae ADY. An increase of 3.5% in ethanol yield wasachieved. Moreover, Z. mobilis ZM4 consumed glucosemuch faster, and ethanol fermentation was completed in12.5h (compared to the 20.0h required by S.cerevisiaeADY); ethanol productivity was increased by 58.1%.

However, under the HG fermentation conditions, a lagphase of ~20h was observed with Z.mobilis ZM4, duringwhich glucose uptake was extremely slow, and almost noethanol was produced. After that, exponential growthwas initiated and glucose utilization was facilitated. Nosimilar phenomenon was observed with ethanol fermen-tation by S. cerevisiae ADY, indicating that Z. mobilisZM4 was not as tolerant to osmotic stress as S.cerevisiaeADY. As a result, although only 3.30g(DCW)/L biomasswas accumulated by Z. mobilis ZM4, compared to7.13g(DCW)/L using S.cerevisiae ADY, no significant dif-ference was observed in ethanol yield and productivity forthe two species.

From the viewpoint of bioprocess engineering, fed-batch fermentation that is initiated with LG medium andfollowed by feeding VHG medium can effectively alleviatesubstrate inhibition. This strategy was thus employed to

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2.2 Batch and fed-batch ethanol fermentations

The harvested seed culture was inoculated into a fermen-tor (KoBioTech KF-2.5L, Korea) containing 1L medium forbatch fermentation. The inoculation level was controlledat 20% for Z.mobilis ZM4 and ZM401, and 10% for S.cere-visiae ADY. The fed-batch fermentation by Z.mobilis ZM4was initiated by inoculating the fermentor containing0.5L LG medium supplemented with 20g/L yeast extract,and 0.5L VHG medium, divided into two lots, was fed intothe fermentator when glucose was depleted. Ethanol fer-mentations were performed at 30C and 150rpm withoutaeration, and the pH was maintained automatically at 4.5by adding 2N NaOH.

2.3 Tolerance to inhibitory byproducts

Furfural, hydroxymethylfurfural (HMF), acetic acid andvanillin were added to the LG medium to study theirimpact on ethanol fermentations by Z.mobilis ZM401 andZM4. The amount of supplementation was decided basedon their concentrations in the hydrolysate of corn stoverdonated by COFCO, a large fuel ethanol producer in Chinaand also a pioneer in developing bioethanol from lignocel-lulosic biomass. Three concentrations of each supplementwere selected: 0.57, 0.86 and 1.15 g/L for furfural; 0.62,0.93, and 1.24g/L for HMF; 6.30, 8.40 and 10.50g/L foracetic acid; and 0.50, 0.75 and 1.00g/L for vanillin. Theethanol fermentations were carried out in flasks at 30Cand 150rpm under microaerobic conditions. Furthermore,0.57g/L furfural, 0.62g/L HMF, 6.30g/L acetic acid and0.50 g/L vanillin were added simultaneously to the LGmedium to study their synergistic effect on ethanol fer-mentations by Z.mobilis ZM401 and ZM4 in the fermen-tor. When acetic acid was added, the pH of the media wasadjusted to 6.0 using 2N KOH, which was similar to over-liming neutralization by Ca(OH)2 that has been recom-mended for industrial applications [15]. Finally, for ethanolfermentation with Z.mobilis ZM401 and ZM4, we tested aCOFCO hydro lysate of corn stover that had been preparedusing the dilute-acid steam-explosion pretreatment andhydrolysis of the cellulosic component by the cellulasesCtec3 (developed by Novozymes), and contained 80.0g/Lglucose, 33.0g/L xylose, 0.49g/L furfural, 0.28g/L HMFand 0.66g/L acetic acid.

Triplicate experiments were performed for all experi-ments, and statistical errors are reported with the experi-mental results.

2.4 Analytical methods

Dry cell weight (DCW) was used for biomass measure-ment. Glucose and ethanol was analyzed as describedpreviously [16]. Byproducts of ethanol fermentations byZ. mobilis and S. cerevisiae, including glycerol, aceticacid, lactic acid, citric acid, succinic acid, sorbitol and




Figure 1. Ethanol fermentations by S. cerevisiae ADY () and Z. mobilis ZM4 (o) and ZM401 () in media containing ~100 g/L glucose (AD) and ~200 g/Lglucose (EH). Triplicate experiments were performed, and their average values are illustrated. The error bars represent standard deviations. Arrows indi-cate the feeding of the concentrated medium in the fed-batch fermentation. Error analysis indicates the significance of the experimental results.


address the osmotic stress exerted on Z.mobilis ZM4 bythe HG medium under batch fermentation conditions.The VHG medium was fed at 10 and 20h, when glucosewithin the fermentor was almost depleted. As can beseen, no lag phase for cell growth and ethanol fermenta-tion was observed for Z.mobilis ZM4 under the fed-batchconditions, and the fermentation time was shortened to30 h, from 50 h required for the batch fermentation.Although more biomass (5.50 g(DCW)/L) was accumulat-ed with the fed-batch fermentation process, Z. mobilis

ZM4 produced 98.6g/L ethanol from 204.0g/L glucoseconsumed, increasing the ethanol yield to 0.483, equiva-lent to 94.6% of the theoretical maximum of 0.511. Thisrepresents an increase of 3.3% in ethanol yield comparedto that achieved with ethanol fermentation by S.cerevisi-ae ADY. This result indicates that the fed-batch processis an effective strategy for overcoming the disadvantageof Z.mobilis ZM4 in tolerating osmotic stress, and demon-strates its advantage in improved ethanol yield and pro-ductivity for more efficient fuel ethanol production.



Table 1. Ethanol fermentation results of Z. mobilis ZM4 and S. cerevisiae ADYa)

Fermentation parameters LG fermentation with ~100 g/L glucose HG fermentation with ~200 g/L glucose

S. cerevisiae ADY Z. mobilis ZM4 S. cerevisiae ADY Z. mobilis ZM4

Batch Batch Batch Fed-batchInitial glucose (g/L) 1020.71 97.00.71 2020.09 2000.00 1041.41Initial ethanol (g/L) 1.450.09 1.710.00 1.250.12 1.540.41 1.640.22Initial biomass (g/L) 0.250.01 0.250.00 0.300.03 0.270.02 0.300.71Residual glucose (g/L) 0.140.00 0.140.03 4.000.06 4.000.03 0.170.01Ethanol (g/L) 46.90.06 46.60.66 94.01.71 93.52.12 1000.02Biomass (g/L) 7.430.21 2.870.08 7.430.21 3.570.71 5.800.14Major byproducts (g/L)

Glycerol 2.100.20 0.300.10 4.000.88 0.600.20 0.550.00Pyruvate 1.300.20 0.500.10 0.960.07 0.000.01 0.060.01Acetate - 0.030.00 0.220.07 0.160.05 0.090.02Lactate 2.700.30 0.500.08 5.400.10 1.200.20 0.690.07Citric acid 1.700.02 0.470.30 0.100.03 0.210.02 0.120.02Succinate 1.700.03 2.700.28 1.100.09 3.600.10 1.100.50Sorbitol 0.860.20 0.500.21 0.350.01Acetoin 0.780.50 0.860.08 0.550.05Ethyl acetate 0.140.08 0.120.06 Isoamyl alcohol 0.100.04 0.090.02 0.100.03 0.070.02 0.040.02Acetaldehyde 0.030.00 0.030.00 0.030.00 0.040.00 0.030.00

Glucose consumed (g/L) 102 96.9 198 196 204Ethanol produced (g/L) 45.4 44.9 92.3 92.0 98.6Biomass accumulated (g/L) 7.17 2.62 7.13 3.30 5.50Fermentation time (h) 20.0 12.5 50.0 50.0 30.0Ethanol productivity (g/L per h) 2.27 3.59 1.85 1.84 3.29Specific growth rate (h1) 0.05 0.08 0.02 0.02 0.03Specific glucose uptake rate (h1) 0.71 2.96 0.56 1.19 1.24Specific ethanol productivity (h1) 0.32 1.37 0.26 0.56 0.60Biomass yield 0.07 0.03 0.04 0.02 0.03Ethanol yield 0.45 0.46 0.47 0.47 0.48Carbon flux distribution (%)

Ethanol 58.0 60.4 60.6 61.2 63.0CO2 29.0 30.2 30.3 30.6 31.5Byproducts detected 8.5 7.3 6.1 4.2 2.3Others 4.5 2.1 3.0 4.0 3.2

a) For the fed-batch process, the fermentation was initiated with the LG medium containing ~100 g/L glucose, and concentrated medium containing ~300 g/L glucosewas fed, as illustrated in Fig. 1, to guarantee that the amount of glucose fermented was the same as that in the HG fermentation. Concentrations of glucose, ethanol,biomass and byproducts from triplicate experiments were measured and averages with standard deviations recorded. All other parameters were calculated based on

these averages. Molar carbon flux to ethanol and detected byproducts was calculated as: and , respectively, where

MEthanol, M(Byproduct)i and MGlucose represent molar concentrations of ethanol, byproduct i produced and glucose consumed, and ni represents the number of carbonatoms in the molecule of byproduct i. Molar carbon flux to CO2 associated with ethanol production was estimated as 50% of that to ethanol production. All remainingcarbon flux was directed to undetected byproducts, biomass and CO2 associated with all byproduct formation and biomass synthesis.

%26

100MM

Ethanol

Glucose

%6

100( )nM

Mi Byproduct

Glucose

i



ORP reflects the net balance of reducing and oxidizingpower within culture and fermentation systems, and thusdirectly reflects the status of cell growth and product for-mation, particularly under microaerobic and anaerobicfermentation conditions [17]. Since Z.mobilis generatesless ATP and reducing power from the ED pathway, theORP level during ethanol fermentation by this speciesshould be higher than that monitored with ethanol fer-mentation by S.cerevisiae, particularly under the HG fer-mentation condition. This was validated by the quantita-tive comparison of the ORP profiles monitored duringethanol fermentations by Z.mobilis ZM4 and S.cerevisi-ae ADY shown in Figs.1D and H.

3.2 Major byproducts formation

In addition to biomass accumulation, byproducts forma-tion is another factor that compromises ethanol yield.Whereas the major byproducts produced during ethanolfermentation by S. cerevisiae have been characterized,including glycerol, ethyl acetate, acetaldehyde, and high-er alcohols [18], reports on the byproducts formed duringethanol fermentation by Z. mobilis are very limited.Recently, metabolic profiling was developed for Z.mobilis,and acetate, lactate, citrate, succinate and acetoin werepredicted as potential major byproducts [14]. To validatethese speculations and provide more insights on theimprovement of ethanol yield observed with Z. mobilisZM4, the major byproducts produced during ethanol fer-mentation by the two strains were analyzed, and illus -trated in Table1.

In addition to the glycerol, pyruvate, lactate, citricacid, succinate, isoamyl alcohol and acetaldehyde detect-ed in ethanol fermentation by S. cerevisiae ADY, twounique byproducts, sorbitol and acetoin, were producedduring ethanol fermentation by Z. mobilis ZM4, but noethyl acetate was detected. Mass balance was furtherperformed for evaluating carbon molar flux distributionsduring ethanol fermentations by Z. mobilis ZM4 andS.cerevisiae ADY. Under the batch LG fermentation con-dition, carbon molar flux to ethanol and CO2 duringethanol production was 87.0% for the EMP pathway inS.cerevisiae and 90.6% for the ED pathway in Z.mobilis.Although the improvement in carbon molar flux to ethanoland CO2 was not significant with Z.mobilis ZM4 underthe batch HG fermentation condition, because of the gen-eration of more byproducts such as sorbitol to address theosmotic stress [19], this disadvantage was overcomeeffectively under the fed-batch HG fermentation condi-tion, and a carbon molar flux directed to ethanol and CO2was as high as 94.5%. These experimental results clearlyconfirmed the improved ethanol production yield of thebacterial species.

3.3 Ethanol fermentation by the flocculatingZ. mobilis ZM401

The flocculation of microbial cells not only makes themself-immobilized within fermentors creating a high celldensity and improving productivity (demonstrated inethanol fermentation by the flocculating yeast [20]), it alsofacilitates biomass recovery since cell flocs can settle


Figure 2. Flocculation of Z. mobilis. (A) Morphology of ZM401 observed withSEM. (B) Chord length distribution ofthe bacterial flocs detected by FBRMduring ethanol fermentation in mediumcontaining ~200 g/L glucose. Li: Chordlength at the midpoint of channel bin i(m), and ti: chord counts per second.(C, D) Sedimentation performance ofthe flocculating and regular strainsZM401 and ZM4.


down quickly from fermentation broth. This has been inpractice in breweries for a long time [21]. Therefore,ethanol production by Z. mobilis ZM401, a flocculatingstrain, was explored and compared with that observedwith the regular Z.mobilis ZM4. The results indicated nosignificant difference between the two bacterial strains,except that, under the HG fermentation conditions,Z.mobilis ZM401 completed ethanol fermentation ~5hearlier (data not shown). This might be due to theimproved ethanol tolerance caused by the morphologicalchange, which was observed using scanning electronmicroscopy (SEM) and characterized by the FBRM sys-tem (Figs.2A and B). The sedimentation performance ofthe bacterial flocs was further qualitatively characterized(Fig. 2C) and compared to that observed with the non-flocculating strain ZM4 (Fig. 2D). The findings clearlyindicate that the flocculation of Z. mobilis ZM401 canmake biomass recovery by sedimentation viable. Sedi-mentation is the most economically competitive strategyfrom the viewpoint of engineering, compared to centrifu-gation that requires heavy capital investment for the cen-trifuges and intensive energy consumption for centrifugeoperation.

3.4 Tolerance to inhibitory byproducts

Theoretically, tolerance to environmental stresses can beimproved when microbial cells flocculate, due to enhancedquorum sensing [22]. The flocculation of Z. mobilis isexpected to improve its tolerance to inhibitory byprod-ucts presented in the hydrolysate of lignocellulosic bio-mass, such as furfural and HMF from degradation of pen-tose and hexose sugars, acetic acid from acetylated hemi-

celluloses, and vanillin from lignin [23]. These majorinhibitory byproducts were added to the LG medium con-taining ~10% glucose to study their effect on ethanol fer-mentations by the flocculating Z.mobilis ZM401 and theregular Z.mobilis ZM 4. The sugar content of the LG medi-um was equivalent to the total sugars in the hydrolysate ofcorn stover donated by COFCO for this research.

Table2 summarizes the experimental results. While nodifference was observed with fermentation performanceunder the furfural and HMF supplementation conditions,the flocculation of Z.mobilis did improve its tolerance tothe inhibition exerted by acetic acid and vanillin, twomajor byproducts released from side reactions associatedwith the degradation of hemicelluloses and lignin [23].More glucose was consumed by the flocculatingZ.mobilis ZM401 (97.4 and 63.0g/L) than the non-floccu-lating Z.mobilis ZM4 (67.5 and 34.0g/L) when 10.5g/Lacetic acid and 1.00g/L vanilin were supplemented. Con-sequently, more ethanol was produced by Z. mobilisZM401 than that by Z.mobilis ZM4 (46.2 and 25.3g/L vs.23.9 and 11.3g/L). The improved ethanol production wasin accordance with the increased biomass accumulation,1.30 and 0.90g(DCW)/L by Z.mobilis ZM401 vs. 1.08 and0.60g(DCW)/L by Z.mobilis ZM4. Moreover, fermentationtime was shorted from 48 to 30h for Z.mobilis ZM401when 10.5g/L acetic acid was supplemented.

The synergistic effects of furfural, HMF, acetic acidand vanillin on ethanol fermentations by Z.mobilis ZM401and ZM4 are illustrated in Figs.3AC. Although no sig-nificant impact on cell growth was observed, a significantimprovement in tolerance to these byproducts wasobserved with Z.mobilis ZM401 due to the morphologicalchange associated with the flocculation. As can be seen,



Table 2. Ethanol fermentations by Z. mobilis ZM4 and ZM401 from the simulated medium supplemented with furfural, 5-HMF, acetic acid and vanillina)

Glucose consumed (g/L) Ethanol produced (g/L) Biomass accumulated Fermentation(g(DCW)/L) time (h)

ZM4 ZM401 ZM4 ZM401 ZM4 ZM401 ZM4 ZM401

Furfural (g/L)0.69 99.90.00 99.90.00 42.40.48 47.00.30 1.450.21 1.400.09 24 240.92 99.60.30 99.80.10 41.42.50 45.31.24 1.200.09 1.100.07 24 241.15 97.80.17 97.90.15 41.70.85 45.12.30 1.250.07 1.100.15 24 24

HMF (g/L)0.62 99.90.00 99.90.00 47.60.92 48.00.50 2.050.20 2.000.05 24 240.93 99.90.00 99.90.00 41.90.56 46.40.42 1.900.05 1.800.09 24 241.24 97.70.16 97.90.20 39.80.84 44.60.80 1.750.08 1.700.10 24 24

Acetic acid (g/L)6.30 99.90.00 99.90.00 46.00.72 48.90.38 1.900.04 1.800.05 24 248.40 97.60.19 99.90.00 44.00.99 48.30.24 1.850.15 1.600.09 24 2410.50 67.51.00 97.40.13 23.93.67 46.20.98 1.080.10 1.300.20 48 36

Vanillin (g/L)0.50 104.00.00 104.00.00 47.33.00 49.61.30 1.560.09 1.550.07 24 240.75 104.00.00 104.00.00 43.21.20 47.43.00 1.200.08 1.100.06 24 241.00 34.02.00 63.03.00 11.13.40 25.35.30 0.600.10 0.900.04 48 48

a) All data were collected from triplicate experiments and averages with standard errors were recorded.



them with pentose metabolic pathways [24]. Until now,more effort has been made in engineering S.cerevisiae,with genes encoding xylose reductase and xylitol dehy-drogenase from Pichia stipitis to enable xylose to bemetabolized to xylulose. The xylulose is then directed tothe native pentose phosphate pathway, facilitated by theoverexpression of xylulokinase gene, for ethanol produc-tion by the EMP pathway. However, an imbalance of co-factors leads to an accumulation of xylitol, which signifi-cantly compromises ethanol yield [2527]. In contrast,only one enzyme xylose isomerase is needed to be engi-neered into Z.mobilis for xylose to be metabolized to xylu-lose [8, 28], which can ultimately solve this bottleneckwith strain development. Taking into account high


50.1g/L ethanol was produced by Z.mobilis ZM401 at48h compared to 48.4g/L ethanol produced at 54h byZ.mobilis ZM4. This indicates a significant increase inthe ethanol productivity by Z.mobilis ZM401, from 0.89 to1.04g/L/h, an increase of 16.8%. Similar results were alsoobserved during ethanol fermentation from thehydrolysate of corn stover donated by COFCO (Figs.3Dand E).

Developing robust strains is a prerequisite for eco-nomic fuel ethanol production from lignocellulosic bio-mass. To make complete use of pentose and hexose sug-ars released from hemicelluloses and cellulose, two majorstrategies have been developed (depending on the type ofhost strains S.cerevisiae or Z.mobilis) for engineering

Figure 3. Ethanol fermentations by Z. mobilis ZM4 (o) andZM401 () in the simulated medium containing ~100 g/L glu-cose, supplemented with 0.5 g/L vanillin, 0.5 mL/L furfural,0.5 mL/L 5-HMF and 6.0 mL/L acetic acid (AC), and in thehydrolysate of corn stover donated by COFCO (D, E). Triplicateexperiments were performed, and average values are illustrated.The error bars represent standard deviations.


ethanol yield and productivity, Z. mobilis could outper-form S.cerevisiae for ethanol production from lignocellu-losic biomass. In addition, the flocculating strain of thisspecies presents more advantages in cost-effective bio-mass recovery and improved tolerance to inhibitorybyproducts released during biomass pretreatment. Withthe completion of the genome sequencing of Z.mobilisZM401 [29], more fundamental knowledge on its floccula-tion and improved tolerance to toxic byproducts will begained through comparative genome analysis. This couldthus provide a better host strain for genetic modificationsfor more efficient production of fuel ethanol from lignocel-lulosic biomass.

4 Concluding remarks

For the first time, ethanol fermentations by S.cerevisiaeand Z.mobilis have been studied under comparable con-ditions to validate the hypotheses that Z.mobilis wouldlead to an improved ethanol yield due to the unique EDpathway with less ATP produced in the bacterial species,and enhanced ethanol productivity due to higher glucoseuptake associated with the high specific surface area ofthe much smaller bacterial cells. In addition, ethanol fer-mentation performance of the flocculating Z. mobilisZM401 was further studied and compared with thatobserved with the regular non-flocculating Z. mobilisZM4. Based on experimental results, we conclude thatcompared to S.cerevisiae ADY, a more than 3% increasein ethanol yield can be achieved with ethanol fermenta-tion by Z. mobilis ZM4. Moreover, ethanol productivitycan also be improved by Z. mobilis, depending on fer-mentation processes applied to the system. Under thebatch LG fermentation condition, a more than 50%increase in ethanol productivity was observed withZ.mobilis ZM4 compared to that with S.cerevisiae ADY.When the fed-batch strategy is applied to the HG fermen-tation, much higher ethanol productivity is obtained,which could lead to saving in capital investment on theproduction facilities. Also, under HG fermentation condi-tions, the improved ethanol titer in the fermentation brothcould save energy consumption for downstream ethanoldistillation and reduce the discharge of distillage from thedistillation system.

In addition, not only can the bacterial flocs be conve-niently recovered by cost-effect sedimentation, instead ofhigh-speed centrifugation required by non-flocculatingbacteria involving intensive capital investment and ener-gy consumption, but also their tolerance to inhibitorybyproducts released during the pretreatment of lignocel-lulosic biomass is improved. These merits make the floc-culating Z.mobilis a suitable host to be engineered withpentose metabolic pathways for fuel ethanol productionfrom lignocellulosic biomass.

Funding for this research was provided in part by theNational Natural Science Foundation of China, grantnumber 21276038, and the National High-Tech R&D Pro-gram, grant number 2012AA021205. The corn stoverhydrolysate used in this research was kindly donated byDr. F. Li at COFCO, which is gratefully appreciated.

The authors declare no conflict of interest.

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