Bioresource Technology 135 (2013) 199–206

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Bioresource Technology

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Bioethanol production from mannitol by a newly isolated bacterium,Enterobacter sp. JMP3

Jing Wang a, Young Mi Kim b, Hong Soon Rhee b, Min Woo Lee c,⇑, Jong Moon Park d,⇑a School of Environmental Science, Pohang University of Science and Technology, San 31, Hyoja-dong, Pohang 790-784, South Koreab Bioenergy Research Center, Pohang University of Science and Technology, San 31, Hyoja-dong, Pohang 790-784, South Koreac Department of Chemical Engineering, Keimyung University, 2800 Dalgubeoldaero, Dalseo-Gu, Daegu 704-701, South Koread Department of Advanced Nuclear Engineering, Advanced Environmental Biotechnology Research Center, School of Environmental Science and Engineering,Pohang University of Science and Technology, San 31, Hyoja-dong, Pohang 790-784, South Korea

h i g h l i g h t s

" A new Enterobacter sp. JMP3 capable of degrading Laminaria japonica was isolated." This bacterium showed high yields of ethanol production from mannitol and glucose." Mannitol was proved to be better carbon source for ethanol production than glucose." M1PDH pathway of mannitol dehydrogenation was verified in Enterobacter sp. JMP3.

a r t i c l e i n f o

Article history:Available online 16 October 2012

Keywords:BiorefineryBioethanolMannitolEnterobacterLaminaria japonica

0960-8524/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.biortech.2012.10.012

⇑ Corresponding authors. Tel.: +82 54 279 2275; faxtel.: +82 53 580 5236; fax: +82 53 580 5165 (M.W. L

E-mail addresses: mwlee@kmu.ac.kr (M.W.(J.M. Park).

a b s t r a c t

In this study a new bacterium capable of growing on brown seaweed Laminaria japonica, Enterobacter sp.JMP3 was isolated from the gut of turban shell, Batillus cornutus. In anaerobic condition, it produced highyields of ethanol (1.15 mol-EtOH mol-mannitol�1) as well as organic acids from mannitol, the major car-bohydrate component of L. japonica. Based on carbon distribution and metabolic flux analysis, it wasrevealed that mannitol was more favorable than glucose for ethanol production due to their differentredox states. This indicates that L. japonica is one of the promising feedstock for bioethanol production.Additionally, the mannitol dehydrogenation pathway in Enterobacter sp. JMP3 was examined and veri-fied. Finally, an attempt was made to explore the possibility of controlling ethanol production by alteringthe redox potential via addition of external NADH in mannitol fermentation.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Biofuels can be defined as liquid or gaseous fuels mainly madefrom biomass like plant matter and residues. They are produced bybiorefinery process which optimizes the use of biomass for gener-ating biofuels as well as biomaterials (Balat and Balat, 2009;Ragauskas et al., 2006). Bioethanol is one of the most widely usedbiofuels because of its higher oxygen content, better octane boos-ter characteristic, non-toxicity and so on (Sánchez and Cardona,2008). Traditionally, most bioethanol has been produced usingagricultural feedstocks (Mussatto et al., 2010). Nowadays,however, food security has emerged as a critical issue, so thatbioethanol production from renewable and cheap materials is

ll rights reserved.

: +82 54 279 8299 (J.M. Park),ee).Lee), jmpark@postech.ac.kr

attracting great attention (Balat and Balat, 2009; Tilman et al.,2009).

Marine biomass is one of the most promising candidates to beable to replace agricultural feedstocks. They have high productiv-ity, high CO2 capture capacity and lignin-free composition; more-over, they would not compromise food supply and cause aserious environmental issue since they do not require any arableland (John et al., 2011). According to the ‘‘Food and AgriculturalOrganization stats’’ (FAO, 2011), in 2008, the highest productionof cultured seaweed across the world was the brown seaweed,especially Laminaria japonica (4.8 million tons year�1). In SouthKorea, L. japonica is the second most abundant seaweed with anannual production of 0.29 million tons. L. japonica is mostly com-posed of carbohydrates, which take up to 60–67% of the total dryweight.

Among the carbohydrates present in L. japonica, the two mostabundant components are mannitol and alginate of which contentsreach 20–30% and 14.6–22.3% of the total dry weight, respectively

200 J. Wang et al. / Bioresource Technology 135 (2013) 199–206

(Cho et al., 1995; Choi et al., 2008; Horn et al., 2000a). Since algi-nate which is widely distributed in brown algae, especially in thecell walls (Horn et al., 2000a; Zhou et al., 2008) is a polysaccharide,the bioethanol production from it would need extra saccharifica-tion process. In the meantime, mannitol can be easily dissolvedfrom L. japonica, so that there would be no need of saccharificationor pretreatment. Considering the simpler fermentation process andhigher proportion of mannitol as well as its higher solubility, man-nitol has been chosen as the target substrate in this study.

Indeed, bioethanol production from seaweed biomass has beenintensively investigated by many researchers. Nowadays, it is evenrevealed that bioethanol can be directly produced from the sea-weed using an engineered microbial platform (Wargacki et al.,2012). However, there have still been only limited works con-ducted related to mannitol fermentation. Mannitol is known tobe rarely fermentable especially under anaerobic condition (Hornet al., 2000a), and only few microorganisms such as Lactococcus lac-tis MG1363 (Neves et al., 2002) and Zymobacter palmae (Horn et al.,2000b) have been reported as a mannitol degrading strain.

In this study, a new strain Enterobacter sp. JMP3 has been iso-lated from the gut of turban shell, Batillus cornutus. And the char-acteristics of the strain for mannitol fermentation wereinvestigated by comparison with glucose through carbon distribu-tion and metabolic flux analysis. Furthermore, the key pathway ofmannitol metabolism of Enterobacter sp. JMP3 was verified by en-zyme tests. To improve ethanol production from mannitol, redoxpotential was altered in fermentation system by the addition ofexternal NADH.

2. Methods

2.1. Isolation and identification of L. japonica degrading bacteria

Turban shell, B. cornutus, was purchased from a local fish marketin Jukdo, Pohang, South Korea to isolate bacteria that can feed on L.japonica. The guts of this marine animal were rinsed with distilledwater and dissected to take its digestive fluid. The digestive fluidwas suspended in 225 mL serum bottles and incubated underanaerobic condition at 30 �C and 170 rpm. Two different mediumwere used for cultivation: nutrient medium containing yeast ex-tract (3.0 g L�1), NaCl (5.0 g L�1) and peptone (2.0 g L�1); mediumconsisting of 1% L. japonica. After enrichment for over seven timesin L. japonica-containing medium, each diluted culture broth wasrepeatedly streaked on nutrient agar plates and incubated for24 h at 30 �C to select the colonies based on the morphological dif-ferences. Finally three different colonies were isolated and eachgenomic DNA was extracted using the Genomic DNA PreparationKit for Bacteria (Sol Gent Co. Ltd., Daejeon, South Korea). The ex-tracted DNA was amplified by polymerase chain reaction (PCR)with 27F primer (50- AGAGTTTGATCCTGGCTCAG-30) and 1492Rprimer (50- GGTTACCTTGTTACGACTT-30) (Hong et al., 2009) usinga thermal cycler (Eppendorf Mastercycler Gradient, Hamburg, Ger-many). PCR amplifications were conducted under the followingcondition: denaturation step at 94 �C for 5 min, 32 cycles of ampli-fication step at 94 �C for 45 s, 55 �C for 45 s, 72 �C for 1 min, fol-lowed by final extension step at 72 �C for 10 min. The PCRproducts were purified using SolGent PCR Purification Kit (Sol GentCo. Ltd., Daejeon, South Korea) and then ligated into pGEM-T Easyvector (Promega, Madison, WI, USA). The ligation mixture wastransformed into Escherichia coli Mach T1 (Invitrogen, Carlsbad,CA, USA) and transformed cells were screened on LB agar platescontaining X-gal and ampicilin. After cultivation of each trans-formed cell in LB medium with ampicilin for 12 h at 37 �C, plas-mids were extracted using AccuPrep� Plasmid Mini Extraction Kit(Bioneer, Daejeon, South Korea). Each plasmid of three bacteria

was sequenced commercially (SolGent Co. Ltd., Daejeon, SouthKorea) and the sequences were compared to related sequences inGenBank using the NCBI BLAST. For phylogenetic analysis, MEGA4software (Tamura et al., 2007) was used and bootstrap test wasconducted with 1000 replicates.

2.2. Scanning electron microscopy (SEM)

Enterobacter sp. JMP3 was cultivated on L. japonica medium.After sampled and centrifuged, the harvested cells and seaweedwere washed three times with PBS buffer. Then, the cells werefixed with 25% glutaraldehyde in PBS buffer and kept in 4 �C over-night. These samples were washed once again with PBS and dehy-drated using a series of ethanol [25% (v/v), 5 min; 50% (v/v), 5 min;75% (v/v), 5 min; 100% (v/v), 5 min]. Finally, the samples werespread on the silicon chip and dried in the desiccators for scanningelectron microscopy (SEM) analysis. For SEM analysis, the sampleswere coated with gold in a Cressington high-resolution sputtercoater 108 Auto (Cressington Scientific Instruments Ltd., Watford,UK), and examined using JEOL JSM-6510 scanning electron micro-scope (JEOL Ltd., Tokyo, Japan).

2.3. Fermentation study of Enterobacter sp. JMP3 with glucose andmannitol

Glucose and mannitol (Sigma–Aldrich Co. LLC, St. Louis, MO,USA) were used as carbon sources and the medium compositionwas as follows: 14 g L�1 K2HPO4, 6 g L�1 KH2PO4, 2 g L�1 (NH4)2

SO4, 1 g L�1 Trisodium citrate dehydrates, 0.2 g L�1 MgSO4, 5 g L�1

Peptone and 5 g L�1 glucose or mannitol. For inoculation, Enterobac-ter sp. JMP3 was aerobically pre-cultivated at 35 �C in the medium.At the late exponential growth phase, the pre-cultivated microbeswere centrifuged to concentrate and washed with distilled water.One millilitre of the inoculums was added into 100 mL of the fer-mentation medium to make the initial cell dry weight around0.03 g L�1. The fermentation system was set up in 250 mL flaskfor aerobic test and 160 mL serum bottle for anaerobic test, bothwith 100 mL of working volume. The fermentation medium wasthe synthetic medium stated above. The serum bottles were purgedwith N2 gas for 10 min for maintaining anaerobic condition. Thecultivation was carried out at 35 �C and 160 rpm. Initial cell dryweight was around 0.03 g L�1 and samples were taken periodicallyfor analysis. All experiments were performed in duplicate.

2.4. Carbon and redox balance of anaerobic metabolism of glucose andmannitol in Enterobacter sp. JMP3

By comparing the anaerobic fermentation results with glucoseand mannitol, the effect of different carbon sources on anaerobicmetabolism of Enterobacter sp. JMP3 was evaluated and quantified.The carbon balances were calculated based on the stoichiometricequation of glucose and mannitol conversion (see appendix), andthe concentration data of metabolites (ethanol, acetate, lactate,succinate, formate, carbon dioxide and hydrogen) were obtainedfrom high performance liquid chromatography (HPLC) and gaschromatography (GC) analysis. The corresponding metabolic coef-ficients were calculated for glucose and mannitol individually. Thebalance equations were modified from another report of anaerobicmetabolism of Enterobacter aerogenes (Converti and Perego, 2002).

2.5. Determination of mannitol dehydrogenation pathway

2.5.1. Cell extractionEnterobacter sp. JMP3 were cultivated in the synthetic medium

with 5 g L�1 of mannitol till late exponential phase and then centri-fuged at 4000 rpm for 10 min. After washing the cells with

J. Wang et al. / Bioresource Technology 135 (2013) 199–206 201

potassium phosphate buffer (50 mM, pH 7.4), they were suspendedin the buffer solution at a final concentration of 80% (w/v) withprotease inhibitor (Complete™ protease inhibitor cocktail tablets,Roche Diagnostics Korea Co. Ltd., Seoul, South Korea). The dosageamount of protease inhibitor was one tablet per 25 mL solution.The ultra-sonic machine was used to break the cell wall: 5 minfor five times with 1 min of intervals in an ice bath to keep thechilled condition.

2.5.2. Mannitol dehydrogenase (MDH) testTo check the mannitol dehydrogenase in Enterobacter sp. JMP3,

the reaction mixture was prepared as the following: potassiumphosphate buffer (50 mM, pH 7.4), NAD+ (1 mM), 70 mg L�1 of cellextracts from above, and mannitol (75 mM). The reaction was per-formed at 30 �C and initiated with the addition of mannitol. Andthen the absorbance at 340 nm was measured using spectropho-tometer to ascertain generation of NADH (Wisselink et al., 2004).The final reaction mixtures were analyzed by HPLC.

2.5.3. Mannitol 1-phosphate dehydrogenase (M1PDH) testThe mannitol 1-phosphate dehydrogenase activity was deter-

mined by the reduction reaction of fructose 6-phosphate. The reactionmixture contained sodium phosphate buffer (50 mM, pH 6), NADH(0.5 mM), 250 mg L�1 of cell extracts and fructose 6-phosphate(1 mM). The reaction was performed at 30 �C and initiated withthe addition of fructose 6-phosphate. And then the absorbance at340 nm was measured using spectrophotometer to ascertain theoxidation of NADH (Wisselink et al., 2005). The final reactionmixtures were analyzed by HPLC.

2.6. Effect of redox balance on ethanol production in Enterobacter sp.JMP3 by external NADH

Cells were sub-cultivated (1:100 dilution) in 25 mL serum bot-tles containing 10 mL synthetic medium as mentioned above (Sec-tion 2.3) using mannitol as the carbon source. One millimolar ofNADH was additionally added into the medium and control groupwithout NADH addition was set up to compare the effects. Themedium was purged with nitrogen gas for 15 min to attain theanaerobic condition. And the cells were cultivated at 35 �C in a ro-tary shaker at 160 rpm. After 24 h, cell growth and metaboliteswere analyzed by spectrophotometer and HPLC, respectively.

2.7. Analytical methods

The consumption of glucose and mannitol as well as the pro-duction of organic acids and ethanol were analyzed by HPLC (Mod-el 1100, Agilent Inc., Santa Clara, CA, USA) using a refractive indexdetector at 50 �C and Aminex HPX-87H column (300 mm �7.8 mm) with 5 mM H2SO4 as the mobile phase at a flow rate of0.6 mL min�1. Carbon dioxide and hydrogen gas were analyzedby GC (Model 6890 N, Agilent Technologies, Palo Alto, CA) usinga pulsed-discharge ionization detector and a Supelco Carboxen-1010 PLOT capillary column (30 m � 0.32 mm). Helium gas wasused as carrier gas and the temperatures of oven, inlet and detectorwere 120, 150, and 240 �C, respectively.

3. Results and discussion

3.1. Isolation and identification of L. japonica degrading bacteria andsubstrate growth test by SEM

3.1.1. Isolation and identification of L. japonica degrading bacteriaL. japonica degrading bacteria were isolated from the gut of tur-

ban shell, B. cornutus which can grow on L. japonica. Three bacteria

were isolated and their phylogenetic trees were constructed bycomparing nucleotide sequences based on available 16S rRNAsequences.

The three bacteria were identified as Serratia sp., Entercoccussp. and Enterobacter sp. and named as Serratia sp. JMP1, Entercoccussp. JMP2 and Enterobacter sp. JMP3. Among them, Enterobacter sp.JMP3 was selected for further study since it exhibited best perfor-mance for using mannitol and dry powder of L. japonica. Enterobac-ter sp. JMP3 showed high identities of 16S rRNA sequence andmorphology with other Enterobacter species; however, its bio-chemical characteristics were quite different from other Enterobac-ter species of API database (see Fig. S1 and Table S1).

Mannitol fermentation by Enterobacter species has rarely re-ported (Nakashimada et al., 2002) although many studies wereconducted focusing on utilization of glucose (Converti and Perego,2002; Rachman et al., 1997), glycerol (Ito et al., 2005; Lee et al.,2012), cellulose and hemicellulose (Bi et al., 2009b; ThirumalaiVasan et al., 2011), etc.

3.1.2. SEM observation of Enterobacter sp. JMP3 on L. japonicaThe growth of Enterobacter sp. JMP3 on L. japonica under both

aerobic and anaerobic condition was observed by SEM analysis(see Fig. S2). According to the result, Enterobacter sp. JMP3 hasshown to grow on L. japonica as attached forms to the surface.While rips of the seaweed surface were observed from the begin-ning, possibly cause by the heating in autoclave operation, thereappeared tendency of increasing rips according to the time, bothin aerobic and anaerobic condition. It can be suggested that themicrobial activity may lead to surface erosion. The degradation ofpolysaccharides of seaweed cell wall may have caused the erosion.

3.2. Fermentation study of Enterobacter sp. JMP3 with glucose andmannitol

3.2.1. Microbial growthFermentation tests with Enterobacter sp. JMP3 have conducted

under both aerobic and anaerobic condition. The microbial growthtogether with substrate consumption according to the time isshown in Fig 1. For both substrates, the cell growth reached thestationary phase at the same time when substrate was totally de-graded. In addition, under aerobic condition, there has appearedmore microbial density. Furthermore, in terms of substrates, itcan be observed that glucose is more easily consumed by Entero-bacter sp. JMP3 than mannitol.

3.2.2. Fermentation productsFig 2 shows the yields of major products in each condition when

the summation yield of all products has achieved the highestduring the fermentation process (see Fig. S3 for the whole time-profiles). The yield is exhibited in unit of the carbon molar of productper carbon molar of substrate (glucose or mannitol). From thisfigure, the following conclusions can be obtained: (1) ethanol, ace-tic acid and formic acid are the most abundant three products ingeneral; (2) for both carbon sources, higher yield of products wereachieved in anaerobic condition, except for lactic acid, since morecarbon source in substrate was used for microbial growth in aero-bic condition (Fig. 1); (3) mannitol can produce the highest yield ofethanol in anaerobic fermentation; (4) In terms of formic acid,succinic acid and ethanol production, mannitol was more favorablesubstrate than glucose.

3.2.3. Carbon distribution of glucose and mannitol in anaerobicfermentation by Enterobacter sp. JMP3

To further evaluate the ethanol production using mannitol, car-bon distributions were investigated on the basis of the anaerobicmetabolism of mannitol and glucose in Enterobacter species.

Table 1Stoichiometric coefficients of anaerobic fermentations of glucose and mannitol byEnterobacter sp. JMP3: (a) refer to the time when total yield of products reachesmaximum; (b) refer to the ultimate fermentation time. The coefficients accords to theconsumption of 1 mol glucose or mannitol.

Glucose (a) Glucose (b) Mannitol (a) Mannitol (b)

a 0.64 0.80 0.65 0.77b 0.05 0.06 0.07 0.04c 0.05 0.06 0.06 0.09d 0.26 0.08 0.21 0.11a1 0.70 1.47 0.68 1.17a2 0.71 0.82 1.07 1.12a3 0.57 0.78 0.23 0.422a � a1 0.58 0.13 0.62 0.37

Time (hr)0 10 20 30 40 50 60

Cel

l Dry

Wei

ght (

g L-1

)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Glucose_AGlucose_OMannitol_AMannitol_O

Time (hr)0 10 20 30 40 50 60

Subs

trate

Con

sum

ptio

n (g

L-1

)

0

1

2

3

4

5

6

Glucose_A Glucose_OMannitol_AMannitol_O

Fig. 1. Microbial growth (a) and substrate consumption (b) of Enterobacter sp. JMP3under different conditions: Mannitol_A, mannitol anaerobic fermentation; Manni-tol_O, mannitol aerobic fermentation; Glucose_A, glucose anaerobic fermentation;Glucose_O, glucose aerobic fermentation (same in the following figures).

Succinate Lactate Acetate Ethanol Formate Total products

Yiel

d (C

mol

C m

ol -1

Sub

stra

te)

0.0

0.2

0.4

0.6

0.8

1.0

Mannitol_A Mannitol_O Glucose_A Glucose_O

Fig. 2. The effect of carbon sources on ethanol production.

202 J. Wang et al. / Bioresource Technology 135 (2013) 199–206

Stoichiometric coefficients for glucose and mannitol were listed inTable 1 based on the carbon mass balance equations (Appendix).With consumption of one mole of glucose or mannitol, the carbondistribution as a result of various metabolites and cell mass weredisplayed in Table 1. Two sampling point were selected during

the fermentation process and calculated: the concentration whenshowed the maximum yield of all products (a) and after 50 h asthe final concentration (b). Similar work was described by Convertiand Perego (2002) to evaluate the effects of initial concentration ofglucose on carbon and energy balances in the anaerobic metabo-lism of E. aerogenes.

As shown in Table 1, the carbon distribution was changed bycarbon sources and fermentation phase. The effects of fermenta-tion phase on metabolic flux can be explained: (1) there appeareddecrease in biomass (d) at the end of fermentation process in bothmetabolisms of glucose and mannitol, which suggests a trend ofusing carbon source to accumulate biomass at early stage of fer-mentation while a shift to fermentation products later on; (2) com-pared with the decrease of biomass, the carbon distribution hasshifted to major products, resulting in increase of some of the or-ganic acids and ethanol: succinic acid (c), acetic acid (a3) and eth-anol (a2) in mannitol fermentation; and succinic acid (c), aceticacid (a3), ethanol (a2) and lactic acid (b) for glucose; (3) formic acidproduction (2a � a1) decreased in a relatively large degree in bothmetabolisms, while on the other hand, hydrogen production (a1)has increased accordingly. This might be involved in hydrogen pro-duction from formic acid (Appendix).

In terms of the effect of different carbon sources on carbon dis-tribution, there are several points to be noted: (1) while the per-centages of glucose and mannitol contributed in acetic acids andethanol production in all (a) merely differ from each other, an obvi-ous shift from acetic acid production (a3) to ethanol production(a2) can be examined when change the carbon source from glucoseto mannitol. Specifically, at the final stage of fermentation, aceticacid production (a3) decreased from 0.78 to 0.42 while ethanolproduction (a2) increased from 0.82 to 1.12. Considering that themetabolic pathways of these two products were linked, thereseems to be some factor driving carbon flux into ethanol produc-tion in the balance; (2) besides ethanol and acetic acid production,when changing glucose to mannitol, the increase in formic acid andsuccinic acid (c) and the decrease in lactic acid (b) and hydrogen(a1) production were also observed.

As stated above, mannitol might be more favorable carbonsource for ethanol production by Enterobacter sp. JMP3. Regardingto the anaerobic metabolism of mannitol and glucose in Enterobac-ter sp. JPM3, the driving force of redox potential, intracellular ratioof NADH/NAD+, offers a reasonable explanation. As referred to theAppendix, it should be noted that there would be one additionalmol of NADH generated by the oxidation of one mol of mannitolcompared with glucose (Quain and Boulton, 1987). This leads to in-crease of the intracellular NADH/NAD+ ratio. Thus, in order to keepthe redox balance inner cell environment, this redox potentialwould drive carbon flux into NADH-consuming pathways. Compar-ing to the metabolic pathway related to ethanol and acetic acid for-mation (see Fig. S4), it is noticed that with 1 mol of ethanol isgenerated, there would be 2 mol of NADH consumed; while acetic

Time (hr)0 10 20 30 40 50

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H (m

M)

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0.1

0.2

0.3

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MDH test: controlMDH test: reactionM1PDH test: controlM1PDH test: reaction

Fig. 3. NADH analysis for verifying the mannitol dehydrogenation pathway.

J. Wang et al. / Bioresource Technology 135 (2013) 199–206 203

acid pathway is not involved in redox balance. Thus it could besuggested that the redox potential acts as a driving force to pro-duce more ethanol in the branch at Acetyl-CoA. As depicted inFig. S4, mannitol generates additional NADH before enters glycoly-sis pathway (in either MDH or M1PDH pathway). As a result, thedifferences in the reduction–oxidation states of the two carbonsources have a significant effect on cellular NADH/NAD+ ratio andsubsequently on the final product composition.

Nicotinamide adenine dinucleotide (NAD), as a cofactor, in-volves in over 300 oxidation–reduction reactions. The NADH/NAD+ cofactor pair plays an essential role in microbial metabolism.When a carbon source (e.g. glucose and mannitol in this study) isconsumed by microorganisms, it would be oxidized using NAD+,and accordingly, NADH is generated as well as metabolic interme-diates. As it is crucially important to maintain the redox balance tocontinue cell growth, NADH must be oxidized to NAD+. While thistask would be fulfilled by oxygen as the final electron acceptor inaerobic process, under anaerobic growth, the regeneration ofNAD+ is accomplished using NADH to reduce metabolic intermedi-ates. So the variation of NADH/NAD+ ratio would have effects onthe metabolic distribution in fermentation process (Berrı́os et al.,2002; San et al., 2002). Many attempts have been made to improvethe production of target products by cofactor manipulation ofNADH/NAD+ through genetic engineering (Berrı́os et al., 2002;San et al., 2002). The results obtained in this study, showed a greatpotential of mannitol as a substrate for bioethanol production dueto its oxidation state.

3.2.4. Determination of mannitol dehydrogenation pathway inEnterobacter sp. JMP3

In upper stream of mannitol metabolism, two pathways areknown to exist when oxidized into fructose-6-phosphate (Wisse-link et al., 2002). As shown in Fig. S4, they are labeled MDH andM1PDH pathway. To verify the pathway followed in Enterobactersp. JMP3, each pathway was examined. The experiment has beendesigned to modify the oxidation–reduction reaction in each corre-sponding pathway where NADH/NAD+ conversion is accompanied.Therefore, if certain pathway is followed, the generation or con-sumption of NADH would be observed.

To verify the MDH pathway, reaction mixtures with mannitol,NAD+ and cell extracts from Enterobacter sp. JMP3 were incubatedat 30 �C for 1hr (Since mannitol-specific PTS, which is a kind of su-gar phosphotransferase system, would be disintegrated when thecell membrane breaks, adding mannitol is not supposed to initiateM1PDH pathway). Control samples were prepared with same pro-cedure but without cell extracts. If this pathway exists in Entero-bacter sp. JMP3, the redox reaction would carry on and as aresult, there would be formation of NADH. As plotted in Fig. 3,however, no generation of NADH was observed, thus, it suggestedthe MDH pathway do not exist in Enterobacter sp. JMP3.

Similarly, M1PDH pathway was examined. The reverse reactionin this pathway was verified due to the availability of reagents. The

Table 2Yield of ethanol and organic acids in fermentation tests in strains of Enterobacter (Unit: C

Strain Substrate Substrate conc.(g/l)

Succinate Lactate Acetat

E. sp. JMP3 Glucose 5 0.086 0.059 0.262E. sp. JMP3 Mannitol 5 0.094 0.039 0.082E. aerogenes Glucose 9 0.037 0.072 0.002E. aerogenes HU 101 Glucose 20 ND 0.145 0.057E. aerogenes Glucose 15 0.044 0.540 0.183E. aerogenes HU 101 Glucose 10 ND 0.181 0.049E. aerogenes HU 101 Mannitol 10 ND 0.196 0.022E. asburiae JDR-1 Glucose 2.5 ND 0.066 0.175E. asburiae JDR-1 Glucose 25 0.007 0.047 0.002

reaction mixtures were prepared with fructose 6-phosphate, NADHand cell extracts (without the extracts in control samples). Suppos-ing mannitol oxidized through this pathway, in the test, NADHconsumption would be detected with the formation of NAD+. Asexpected, NADH has decreased with time in reaction sampleswhile no change occurred in control samples (Fig 3). HPLC analysisfor end products of both samples was conducted, and fructose 6-phosphate, as the reactant, was detected in both. The relativeamount of product in the reaction samples was significantly lesserthan in the control samples. This indicated that the reactant wasconsumed. Also, compared with the control sample, a considerableadditional peak appeared in the reaction sample, which could indi-cate the formation of mannitol 1-phosphate.

According to the results above, it can be concluded that themannitol metabolism in Enterobacter sp. JMP3 follows theM1PDH pathway in Fig. S4. In our knowledge, this is the first timethis pathway has been reported in Enterobacter sp.

3.3. Effect of redox balance on ethanol production in Enterobacter sp.JMP3 by external NADH

Enterobacter sp. JMP3 showed significant increase in ethanolproduction from mannitol compared with glucose (Fig. 2). Further-more, the yield of ethanol as well as succinic acid and formic acidwere remarkably high compared with other Enterobacter sp. (Table2). In particular, ethanol yield was highest in both substrate(0.272 C mol/C mol glucose and 0.383 C mol/C mol mannitol). Thisindicates that Enterobacter sp. JMP3 has a more potential for etha-nol production from seaweed compared to other Enterobacter sp.reported up to now.

As mentioned before, the redox state of mannitol affectedNADH/NAD+ ratio and finally led to increase in the metabolicflux into ethanol pathway. To increase the target product, many

-mol/ C-mol products/substrate).

e Ethanol Formate Acetoin Butanediol Total Ref.

0.272 0.021 ND ND 0.700 This study0.383 0.053 ND ND 0.652 This study0.187 ND 0.022 0.357 0.675 Converti and Perego (2002)0.163 0.027 0.007 0.247 0.645 Rachman et al. (1997)0.240 0.000 ND 0.013 1.021 Lu et al. (2009)0.157 ND 0.009 0.242 0.639 Nakashimada et al. (2002)0.322 ND 0.006 0.130 0.677 Nakashimada et al. (2002)0.236 0.019 ND ND 0.496 Bi et al. (2009a)0.109 0.048 ND 0.223 0.437 Bi et al. (2009b)

EtOH Acetate Formate Lactate Succinate

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c. (g

L-1

)

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0.2

0.4

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1.2

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Control NADH

Fig. 4. The effect of external NADH on mannitol fermentation.

204 J. Wang et al. / Bioresource Technology 135 (2013) 199–206

attempts have been made such as controlling the oxidoreductionpotential in cell culture by altering pH (Nakashimada et al.,2002) or inactivation of competing pathways which consumereducing power (Neves et al., 2002); and external NADH additionis one of the simple approach among them (Zhang et al., 2009).

To investigate the effect of external NADH addition on NADH/NAD+ ratio and ethanol production, 1 mM of NADH was added tothe anaerobic medium. However, external NADH led to the meta-bolic flux into lactic and succinic acid rather than ethanol(Fig. 4). This result is extraordinarily odd considering that bothpathways are NADH-consuming. A possible explanation is thatEnterobacter sp. JMP3 might differently regulate its metabolismin the case of external NADH supply. When the intracellularNADH/NAD+ ratio is abruptly increased, the cell should find away to rapidly rebalance it. Indeed, lactic acid pathway seems tobe more efficient than ethanol pathway for this purpose becauselactic acid can be directly produced from pyruvate while ethanolpathway requires more steps to be converted (Fig. S4). For furtherincrease in ethanol production, it seems that genetic manipulationto inactivate the competing pathways, such as lactic acid pathway,need to be combined with this approach.

4. Conclusions

A new strain Enterobacter sp. JMP3 which is capable of produc-ing ethanol and organic acids using glucose and mannitol underboth aerobic and anaerobic conditions has been successfully iso-lated from the gut of turban shell, B. cornutus. When mannitolwas used as a sole carbon source under anaerobic condition, thismicroorganism showed the highest ethanol yield of 0.383 C-mol/C-mol suggesting that mannitol could be a more favorable carbonsource than glucose. Through carbon distribution and metabolicflux analysis, it was also found that intracellular NADH/NAD+ ratiocould significantly affect the metabolic pathway regulation for thegiven microorganism.

Acknowledgements

This research was supported by Basic Science Research Pro-gram through the National Research Foundation of South Korea(NRF) funded by the Ministry of Education, Science and Technol-ogy (Grant number 2011-0001108), the Advanced Biomass R&DCenter (ABC) of South Korea Grant funded by the Ministry of Edu-cation, Science and Technology (ABC-2011-0028387), Marine Bio-technology Program Funded by Ministry of Land, Transport andMaritime Affairs of South Korean Government, South Korea andthe Manpower Development Program for Marine Energy funded

by Ministry of Land, Transportation and Maritime Affairs (MLTM)of South Korean government and by WCU (World Class Univer-sity) program through the National Research Foundation of SouthKorea funded by the Ministry of Education, Science and Technol-ogy (R31-30005).

Appendix A. Anaerobic metabolism of glucose in Enterobactersp. JMP3

A.1. Pyruvate – formate lyase activity

aGlucoseþ 2aNADþ þ 2aADPþ 2aPi

þ

! 2aPyruvateþ 2aNADH2 þ 2aATPþ 2aH2O ð1Þ

2aPyruvateþ 2aCoA-SH! 2aAcetyl-S-CoAþ 2aFormate ð2Þ

Net (Eqs. (1) and (2)):

aGlucoseþ 2aNADþ þ 2aADPþ 2aPi þ 2aCoA-SH

! 2aFormateþ 2aNADHþ2 þ 2aATPþ 2aH2O

þ 2aAcetyl-S-CoA ð3Þ

c1: Hydrogen production

a1Formate! a1CO2 þ a1H2 ð4Þ

c2: Ethanol production

a2Acetyl-S-CoAþ2a2NADHþ2 !a2Ethanolþ2a2NADþþa2CoA-SH

ð5Þ

c3: Acetate production

a3Acetyl-S-CoAþ a3Pi ! a3Acetylphosphateþ a3CoA-SH ð6Þ

a3Acetylphosphateþ a3ADP! a3Acetateþ a3ATP ð7Þ

Net (Eqs. (6) and (7)):

a3Acetyl-S-CoAþ a3ADPþ a3Pi

! a3Acetateþ a3CoA-SHþ a3ATP ð8Þ

Net (Eqs. (3)-(5) and (8)):

aGlucoseþ ð2a2 � 2aÞNADHþ2 þ ð2aþ a3ÞADPþ 2að2a

þ a3ÞPi

! ð2a� a1ÞFormateþ a2Ethanolþ a3Acetateþ a1H2

þ 2aH2Oþa1CO2 þ ð2a2 � 2aÞNADþ þ ð2aþ a3ÞATP ð9Þ

A.2. Lactate production

bGlucoseþ 2bNADþ þ 2bADPþ 2bPi

! 2bPyruvateþ 2bNADHþ2 þ 2bATPþ 2bH2O ð10Þ

2bPyruvateþ 2bNADHþ2 ! 2bLactateþ 2bNADþ ð11Þ

Net (Eqs. (10) and (11))

bGlucoseþ 2bADPþ 2bPi ! 2bLactateþ 2bATPþ 2bH2O ð12Þ

A.3. Succinate production

cGlucoseþ 2cNADþ þ 2cPi ! 2cPhosphoenolypyruvate

þ 2cNADHþ2 þ 2cH2O ð13Þ

J. Wang et al. / Bioresource Technology 135 (2013) 199–206 205

2cPhosphoenolypyruvateþ 2cCO2 þ 4cNADHþ2! 2cSuccinateþ 2cPi þ 4cNADþ ð14Þ

Net (Eqs. (13) and (14))

cGlucoseþ 2cNADHþ2 þ 2cCO2

! 2cSuccinateþ 2cNADþ þ 2cH2O ð15Þ

A.4. Cell growth

dGlucoseþ 1:5dNH3 þ 0:873dNADHþ2! 6dCH1:75O0:5N0:25 þ 0:873dNADþ þ 3dH2O ð16Þ

where

aþ bþ cþ d ¼ 1 ð17Þ

Appendix B. metabolism of mannitol in Enterobacter sp. JMP3

B.1. Pyruvate – formate lyase activity

aManntiolþ 3aNADþ þ 2aADPþ 2aPi

! 2aPyruvateþ 3aNADHþ2 þ 2aATPþ 2aH2O ð10Þ

2aPyruvateþ 2aCoA-SH! 2aAcetyl-S-CoAþ 2aFormate ð20Þ

Net (Eqs. (10) and (20)):

aMannitolþ 3aNADþ þ 2aADPþ 2aPi þ 2aCoA-SH

! 2aFormateþ 3aNADHþ2 þ 2aATPþ 2aH2O

þ 2aAcetyl-S-CoA ð30Þ

a01: Hydrogen production

a1Formate! a1CO2 þ a1H2 ð40Þ

a02: Ethanol production

a2Acetyl-S-CoAþ 2a2NADHþ2 ! a2Ethanolþ 2a2NADþ

þ a2CoA-SH ð50Þ

a03: Acetate production

a3Acetyl-S-CoAþ a3Pi ! a3Acetylphosphateþ a3CoA-SH ð60Þ

a3Acetylphosphateþ a3ADP! a3Acetateþ a3ATP ð70Þ

Net (Eqs. (60) and (70)):

a3Acetyl-S-CoAþ a3ADPþ a3Pi

! a3Acetateþ a3CoA-SHþ a3ATP ð80Þ

Net (Eqs. (30)-(50) and (80)):

aMannitolþ ð2a2 � 3aÞNADHþ2 þ ð2aþ a3ÞADPþ 2að2aþ a3ÞPi ! ð2a� a1ÞFormateþ a2Ethanolþ a3Acetateþ a1H2

þ 2aH2Oþ a1CO2 þ ð2a2 � 3aÞNADþ þ ð2aþ a3ÞATP ð90Þ

B.2. Lactate production

bMannitolþ 3bNADþ þ 2bADPþ 2bPi

! 2bPyruvateþ 3bNADHþ2 þ 2bATPþ 2bH2O ð100Þ

2bPyruvateþ 2bNADHþ2 ! 2bLactateþ 2bNADþ ð110Þ

Net (Eqs. (100) and (110))

bMannitolþ bNADþ þ 2bADPþ 2bPi

! 2bLactateþ bNADHþ2 þ 2bATPþ 2bH2O ð120Þ

B.3. Succinate production

cMannitolþ 3cNADþ þ 2cPi ! 2cPhosphoenolypyruvate

þ 3cNADHþ2 þ 2cH2O ð130Þ

2cPhosphoenolypyruvateþ 2cCO2 þ 4cNADHþ2! 2cSuccinateþ 2cPi þ 4cNADþ ð140Þ

Net (Eqs. (130) and (140))

cMannitolþ cNADHþ2 þ 2cCO2 ! 2cSuccinateþ cNADþ þ 2cH2O

ð150Þ

B.4. Cell growth

dMannitolþ 1:5dNH3 þ 0:127dNADþ

! 6dCH1:75O0:5N0:25 þ 0:127dNADHþ2 þ 3dH2O ð160Þ

where

aþ bþ cþ d ¼ 1 ð170Þ

References

Balat, M., Balat, H., 2009. Recent trends in global production and utilization of bio-ethanol fuel. Appl. Energy 86 (11), 2273–2282.

Berrı́os-Rivera, S.J., Bennett, G.N., San, K.-Y., 2002. Metabolic engineering ofEscherichia coli: increase of NADH availability by overexpressing an NAD+-dependent formate dehydrogenase. Metab. Eng. 4 (3), 217–229.

Bi, C., Rice, J.D., Preston, J.F., 2009a. Complete fermentation of xylose andmethylglucuronoxylose derived from methylglucuronoxylan by Enterobacterasburiae strain JDR-1. Appl. Environ. Microbiol. 75 (2), 395–404.

Bi, C., Zhang, X., Ingram, L.O., Preston, J.F., 2009b. Genetic engineering ofEnterobacter asburiae strain JDR-1 for efficient production of ethanol fromhemicellulose hydrolysates. Appl. Environ. Microbiol. 75 (18), 5743–5749.

Cho, D.-M., Kim, D.-S., Lee, D.-S., Kim, H.-R., Pyeun, J.-H., 1995. Trace componentsand functional saccharides in marine algae 2. Dietary fiber contents anddistribution of the alga polysaccharides. J. Korean Fish Soc. 28 (3), 270–278.

Choi, J.-S., Shin, S.H., Ha, Y.-M., Kim, Y.C., Kim, T.B., Park, S.M., Choi, I.S., Song, H.J.,Choi, Y.J., 2008. Mineral contents and physiological activities of dried sea tangle(Laminaria japonica) collected from gijang and wando in Korea. J. Life Sci. 18 (4),474–481.

Converti, A., Perego, P., 2002. Use of carbon and energy balances in the study of theanaerobic metabolism of Enterobacter aerogenes at variable starting glucoseconcentrations. Appl. Microbiol. Biotechnol. 59 (2–3), 303–309.

FAO, 2011 FAO, 2011. Available from <http://www.fao.org/figis/servlet/TabSelector> (accessed June 2012).

Hong, J.Y., Sato, E.F., Nishikawa, T., Hiramoto, K., Inoue, M., 2009. Effect ofobstructive jaundice and nitric oxide on the profiles of intestinal bacterialflora in wild and iNOS�/� mice. J. Clin. Biochem. Nutr. 44 (2), 160–167.

Horn, S.J., Aasen, I.M., Emptyvstgaard, K., 2000a. Ethanol production from seaweedextract. J. Ind. Microbiol. Biotechnol. 25 (5), 249–254.

Horn, S.J., Aasen, I.M., Østgaard, K., 2000b. Production of ethanol from mannitol byZymobacter palmae. J. Ind. Microbiol. Biotechnol. 24 (1), 51–57.

Ito, T., Nakashimada, Y., Senba, K., Matsui, T., Nishio, N., 2005. Hydrogen and ethanolproduction from glycerol-containing wastes discharged after biodieselmanufacturing process. J. Biosci. Bioeng. 100 (3), 260–265.

John, R.P., Anisha, G.S., Nampoothiri, K.M., Pandey, A., 2011. Micro and macroalgalbiomass: a renewable source for bioethanol. Bioresour. Technol. 102 (1), 186–193.

Lee, S.J., Kim, S.B., Kang, S.W., Han, S.O., Park, C., Kim, S.W., 2012. Effect of crudeglycerol-derived inhibitors on ethanol production by Enterobacter aerogenes.Bioproc. Biosyst. Eng. 35 (1–2), 85–92.

Lu, Y., Zhao, H., Zhang, C., Lai, Q., Wu, X., Xing, X.H., 2009. Expression of NAD+-dependent formate dehydrogenase in Enterobacter aerogenes and itsinvolvement in anaerobic metabolism and H2 production. Biotechnol. Lett. 31(10), 1525–1530.

Mussatto, S.I., Dragone, G., Guimarães, P.M.R., Silva, J.P.A., Carneiro, L.M., Roberto,I.C., Vicente, A., Domingues, L., Teixeira, J.A., 2010. Technological trends, globalmarket, and challenges of bio-ethanol production. Biotechnol. Adv. 28 (6), 817–830.

206 J. Wang et al. / Bioresource Technology 135 (2013) 199–206

Nakashimada, Y., Rachman, M.A., Kakizono, T., Nishio, N., 2002. Hydrogenproduction of Enterobacter aerogenes altered by extracellular and intracellularredox states. Int. J. Hydrogen Energy 27 (11–12), 1399–1405.

Neves, A.R., Ramos, A., Shearman, C., Gasson, M.J., Santos, H., 2002. Catabolism ofmannitol in Lactococcus lactis MG1363 and a mutant defective in lactatedehydrogenase. Microbiology 148 (11), 3467–3476.

Quain, D.E., Boulton, C.A., 1987. Growth and metabolism of mannitol by strains ofSaccharomyces cerevisiae. J. Gen. Microbiol. 133 (7), 1675–1684.

Rachman, M.A., Furutani, Y., Nakashimada, Y., Kakizono, T., Nishio, N., 1997.Enhanced hydrogen production in altered mixed acid fermentation of glucoseby Enterobacter aerogenes. J. Ferment. Bioeng. 83 (4), 358–363.

Ragauskas, A.J., Williams, C.K., Davison, B.H., Britovsek, G., Cairney, J., Eckert, C.A.,Frederick, W.J., Hallett, J.P., Leak, D.J., Liotta, C.L., Mielenz, J.R., Murphy, R.,Templer, R., Tschaplinski, T., 2006. The path forward for biofuels andbiomaterials. Science 311 (5760), 484–489.

Sánchez, Ó.J., Cardona, C.A., 2008. Trends in biotechnological production of fuelethanol from different feedstocks. Bioresour. Technol. 99 (13), 5270–5295.

San, K.-Y., Bennett, G.N., Berrı́os-Rivera, S.J., Vadali, R.V., Yang, Y.-T., Horton, E.,Rudolph, F.B., Sariyar, B., Blackwood, K., 2002. Metabolic engineering throughcofactor manipulation and its effects on metabolic flux redistribution inEscherichia coli. Metab. Eng. 4 (2), 182–192.

Tamura, K., Dudley, J., Nei, M., Kumar, S., 2007. MEGA4: molecular evolutionarygenetics analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24 (8), 1596–1599.

Thirumalai Vasan, P., Sobana Piriya, P., Immanual.Gilwax.Prabhu, D., John Vennison,S., 2011. Cellulosic ethanol production by Zymomonas mobilis harboring an

endoglucanase gene from Enterobacter cloacae. Bioresour. Technol. 102 (3),2585–2589.

Tilman, D., Socolow, R., Foley, J.A., Hill, J., Larson, E., Lynd, L., Pacala, S., Reilly, J.,Searchinger, T., Somerville, C., Williams, R., 2009. Beneficial biofuels – the food,energy, and environment trilemma. Science 325 (5938), 270–271.

Wargacki, A.J., Leonard, E., Win, M.N., Regitsky, D.D., Santos, C.N.S., Kim, P.B., Cooper,S.R., Raisner, R.M., Herman, A., Sivitz, A.B., Lakshmanaswamy, A., Kashiyama, Y.,Baker, D., Yoshikuni, Y., 2012. An engineered microbial platform for directbiofuel production from brown macroalgae. Science 335 (6066), 308–313.

Wisselink, H.W., Mars, A.E., Van Der Meer, P., Eggink, G., Hugenholtz, J., 2004.Metabolic engineering of mannitol production in Lactococcus lactis: influence ofoverexpression of mannitol 1-phosphate dehydrogenase in different geneticbackgrounds. Appl. Environ. Microbiol. 70 (7), 4286–4292.

Wisselink, H.W., Moers, A.P.H.A., Mars, A.E., Hoefnagel, M.H.N., De Vos, W.M.,Hugenholtz, J., 2005. Overproduction of heterologous mannitol 1-phosphatase:a key factor for engineering mannitol production by Lactococcus lactis. Appl.Environ. Microbiol. 71 (3), 1507–1514.

Wisselink, H.W., Weusthuis, R.A., Eggink, G., Hugenholtz, J., Grobben, G.J., 2002.Mannitol production by lactic acid bacteria: a review. Int. Dairy J. 12 (2–3), 151–161.

Zhang, C., Ma, K., Xing, X.-H., 2009. Regulation of hydrogen production byEnterobacter aerogenes by external NADH and NAD+. Int. J. Hydrogen Energy34 (3), 1226–1232.

Zhou, M.H., Han, F.F., Li, J., Zhao, X.W., 2008. Isolation and identification of a novelalginate-degrading bacterium Ochrobactrum sp. Songklanakarin. J. Sci. Technol.30 (2), 135–140.