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Continuous hydrogen production by the hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1

Journal of Biotechnology116(2005)

271–282

Continuous hydrogen production by the hyperthermophilic archaeon,Thermococcus kodakaraensis KOD1 Tamotsu Kanai a,Hiroyuki Imanaka a,Akihito Nakajima b,Kenetsu Uwamori b,

Yoshiyuki Omori b,Toshiaki Fukui a,Haruyuki Atomi a,Tadayuki Imanaka a,?

a Department of Synthetic Chemistry and Biological Chemistry,Graduate School of Engineering,Kyoto University,

Katsura,Nishikyo-ku,Kyoto615-8510,Japan

b Taiyo Nippon Sanso Corporation,Shinagawa-ku,Tokyo142-8558,Japan

Received19March2004;received in revised form12November2004;accepted18November2004

Abstract

The hydrogen(H2)production potential of the hyperthermophilic archaeon,Thermococcus kodakaraensis KOD1was evaluated at85?C.In batch cultivation using a complex medium supplemented with elemental sulfur(S0),evolution of H2S and CO2was observed in the gas phase.When S0was omitted and pyruvate or starch was added in the medium,the cells produced H2at high levels instead of H2S.As the level of H2appeared to correlate with the speci?c growth rate,analysis in continuous cultures was performed to develop a continuous H2production system.In a steady-state condition at a dilution rate of0.2h?1,a continuous H2production rate(per gram dry weight,gdw)of24.9and14.0mmol gdw?1h?1was observed in media supplemented with pyruvate and starch,respectively.In both cultivations,a high accumulation of acetate and alanine was found as metabolites. When the dilution rates were elevated in the medium with pyruvate,steady-state growth was observed up to0.8h?1,and a maximum H2production rate of59.6mmol gdw?1h?1was obtained.Based on the experimental results along with data of the entire genome sequence,the metabolic pathway of the strain relating to starch and pyruvate degradation is discussed.

Keywords:Hydrogen production;Hyperthermophile;Archaea;Thermococcus;Hydrogenase

1.Introduction

In recent years,hydrogen gas(H2)is attracting widespread attention as a clean,non-polluting fuel.

?Corresponding author.Tel.:+81753832777;

fax:+81753832778.

E-mail address:imanaka@sbchem.kyoto-u.ac.jp(T.Imanaka).However,since H2is not a primary energy source,it must be produced from other energy resources.The main process at present to manufacture H2is steam re-forming of natural gas and petroleum,a process that totally depends on fossil fuel and generates CO2as a byproduct(Rostrup-Nielsen,2001).

One of the methods to circumvent this problem is to utilize the H2production potential of microorgan-

272T.Kanai et al./Journal of Biotechnology116(2005)271–282

isms.A large number of microbes living in anaer-obic conditions are known to produce H2as a fer-mentative means of disposing excess reducing equiv-alents(Kosaric and Lyng,1988;Nandi and Sengupta, 1998;Das and Veziroglu,2001).In terms of H2pro-ductivity,research on fermentative H2production has been focused mainly on two bacterial genera.One is Clostridium,whose members are obligate anaero-bic heterotrophs producing H2by fermenting carbohy-drates such as glucose,starch,and xylose(Karube et al., 1976;Taguchi et al.,1992,1994;Kataoka et al.,1997). The other genus,Enterobacter,is recently gaining more attention because of its high H2productivity.Enter-obacter aerogenes was the?rst species in this genus reported for its fermentative H2production(Tanisho et al.,1983)and several other groups searching for H2-producing microbes have also independently iso-lated various strains of E.aerogenes(Yokoi et al.,1995; Rachman et al.,1997).A newly isolated species,En-terobacter cloacae IIT-BT08,was also shown to have a high H2-producing potential(Kumar and Das,2000, 2001).More recently,reports on fermentative H2pro-duction using thermophilic bacteria have been pub-lished.Such organisms include Thermotoga neapoli-tana(van Ooteghem et al.,2002),Thermotoga el?i (van Niel et al.,2002),and Caldicellulosiruptor sac-charolyticus(van Niel et al.,2002).

In this report,we demonstrate a high H2-producing potential in the hyperthermophilic archaeon,Ther-mococcus kodakaraensis KOD1.The organism was isolated from a geothermal spring in a coastal area of Japan and grows within a temperature range of 60–100?C(Morikawa et al.,1994;Atomi et al.,2004). The archaeal strain belongs to the order Thermococ-cales,which includes two major genera,Pyrococcus and Thermococcus(Itoh,2003).Members of the or-der Thermococcales are anaerobic heterotrophs that utilize peptide-related substrates with elemental sulfur (S0)as an electron acceptor(Blumentals et al.,1990; Adams,1994;Bonch-Osmolovskaya and Mirosh-nichenko,1994;Adams et al.,2001).A small number of Thermococcales species can also grow on carbo-hydrates without the requirement of S0.For example, Pyrococcus furiosus can utilize maltose,starch,glyco-gen,or cellobiose(Kengen et al.,1996),while Ther-mococcus litoralis can use starch,maltose,cellobiose, or sucrose(Rinker and Kelly,1996).In both cases, H2is produced as a reduced end product(Schicho et al.,1993;Sch¨a fer and Sch¨o nheit,1993;Kengen and Stams,1994),but the H2production potentials of these hyperthermophilic archaea was not the center of attention and a comparison with other H2-producing microbes was not discussed in these reports(Nandi and Sengupta,1998).In this study,we have examined the H2-producing potential of T.kodakaraensis,and showed that H2productivity of the strain was compara-ble to or even higher than those of Enterobacter species.

2.Materials and methods

2.1.Strain and media

T.kodakaraensis KOD1(JCM12380)(Morikawa et al.,1994;Atomi et al.,2004)was routinely grown in MA-YT medium with the following composition;

4.8g L?1Marine Art SF A salt,26.4g L?1Marine Art SF B salt as arti?cial sea salts(Senju Pharma-ceutical,Osaka,Japan),5g L?1yeast extract(Nacalai tesque,Kyoto,Japan),and5g L?1of tryptone(Nacalai tesque).The pH of the medium was around6.9.In the case of cultivation with S0,sulfur powder(Wako pure chemical industries,Osaka,Japan)was added at a concentration of2g L?1after autoclaving the MA-YT medium.In the case of cultivation with pyruvate or starch,5g L?1sodium pyruvate(Nacalai tesque)or 5g L?1soluble starch(Nacalai tesque)was added to the MA-YT medium,respectively,before autoclaving.

2.2.Cultivation of T.kodakaraensis KOD1

Batch cultures of T.kodakaraensis KOD1were performed anaerobically with a gas-lift fermenter de-signed for cultivation of hyperthermophiles(TSF100, Taiyo Nippon Sanso Corporation,Tokyo,Japan).In a10L cultivation vessel,7L of medium was intro-duced and cultivation was performed at85?C with con-tinuous agitation using an equipped rotor at100rpm. The evolved gas metabolites were?ushed out by N2, which was introduced continuously into the vessel at a rate of100mL min?1using a digital mass?ow con-troller CMQ9200(Yamatake,Yokohama,Japan).Cell growth was monitored periodically by measuring the absorbance of the culture at660nm(A660).The pH was continuously monitored with a pH electrode GST-5425C(Toa DKK,Tokyo,Japan).When cultivation was performed using medium containing starch,0.2N

T.Kanai et al./Journal of Biotechnology116(2005)271–282273

NaOH solution was added at appropriate intervals to prevent the culture pH from dropping below6.0.

Continuous culture experiments were performed us-ing the same cultivation apparatus as described above. Fresh medium was supplied into the vessel using a peri-staltic pump(Master?ex,Illinois,USA)to maintain a dilution rate of0.2h?1.The volume of the culture was monitored with a water level sensor(B.E.Marubishi, Tokyo,Japan),which was connected to a pump for cul-ture discharging to maintain a culture volume of3L. When cultivating on starch medium,culture pH was maintained at6.8by automatic titration with the addi-tion of0.2N NaOH solution.In washout experiment, a smaller vessel of3L was used and the working vol-ume was set at1L.Cultivation was performed at85?C with continuous agitation at50rpm.N2was introduced continuously into the vessel at a rate of100mL min?1, and dilution rates were changed from0.4to0.9h?1.

2.3.Analysis of gas composition

The amounts of H2,CO2,H2S,O2,and N2in the exhaust gas were determined periodically during culti-vation by three gas chromatographs equipped with ther-mal conductivity detectors(GC14A,Shimadzu,Ky-oto,Japan).H2was measured by sequential separation using columns packed with Adsorb-P-1(Nishio Kogyo, Tokyo,Japan)and MS-5A(GL Science,Tokyo,Japan), at a temperature of50?C using N2as carrier gas.Quan-ti?cation of O2,CO2and N2was performed by sequen-tial separation using columns packed with Thermon-1000(Shinwa Chemical Industries,Kyoto,Japan),MS-5A,and Porapak-Q(Dow Chemical,Midland,Michi-gan,USA)at a temperature of80?C using He as carrier gas.H2S was measured with a single column packed with Thermon-1000(Shinwa Chemical Industries),at

a temperature of70?C using He as carrier gas.

2.4.Analysis of substrates,fermentation products, and biomass

Concentrations of acetate and pyruvate in the medium were determined enzymatically using the F-kit for acetate and pyruvate(Roche Diagnostics,Basel, Switzerland).Starch concentration was determined by the phenol–sulfuric acid method using l-glucose (Nacalai tesque)as a standard(McCready et al.,1950). Concentrations of each amino acid and NH3in the medium were determined with an amino acid ana-lyzer L-8800(Hitachi,Tokyo,Japan)after acid hy-drolysis(Moore and Stein,1963).Concentrations of organic acids were determined by high-performance liquid chromatography using a Sim-pack SPR-H col-umn(Shimadzu)and an electrical conductivity detector (Shimadzu).Samples were prepared by diluting cul-ture medium with deionized water.Cell density was monitored by measuring absorbance of culture medium at660nm(A660).Biomass was estimated from the cell density data by calculation using previously de-termined calibration information.

3.Results

3.1.Analyses of gas metabolites during batch cultivation in different growth media

Cultivation of T.kodakaraensis KOD1was rou-tinely performed using complex medium based on ar-ti?cial seawater,tryptone,and yeast extract(MA-YT medium).To compare gas metabolites generated by the species,batch cultivation of T.kodakaraensis KOD1 was performed using MA-YT medium supplemented either with(i)S0,(ii)pyruvate(MA-YTP medium), or(iii)soluble starch(MA-YTS medium).The highest growth rate was obtained in the MA-YT+S0medium, reaching a maximum cell density after4h of cultiva-tion(Fig.1A).The highest speci?c growth rate(?max) of1.39h?1was recorded between1and2h.As for gas metabolites,high levels of H2S and CO2were pro-duced throughout the cultivation period with maximal rates at3h of cultivation(17.3and10.8mmol L?1h?1 for H2S and CO2,respectively).The cells also gen-erated a detectable level of H2(0.27mmol L?1h?1at 3h),but its production rate was less than2%compared with that of H2S.In comparison,slower growth was ob-served for cells grown in the MA-YTP and MA-YTS media,reaching their maximal cell concentrations after 13and24h of cultivation,respectively(Fig.1B and C). A?max of0.495h?1was recorded in MA-YTP medium between5and6h,while that of0.408h?1was recorded in the MA-YTS medium between19and20h.Under both growth conditions,T.kodakaraensis cells gener-ated high levels of H2and CO2whereas H2S production could not be detected.As for cells grown in MA-YTP medium,the H2production rate was almost compara-ble to that of CO2throughout the cultivation periods

274T.Kanai et al./Journal of Biotechnology 116(2005)

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Fig.1.Gas composition and growth of batch cultivation of T.kodakaraensis cells under different medium conditions.(A)MA-YT medium supplemented with 0.2%S 0;(B)MA-YT medium supplemented with 0.5%(w/v)sodium pyruvate;(C)MA-YT medium supplemented with 0.5%(w/v)soluble starch.Upper panels:Time course of cell growth as monitored by absorbance at 660nm.Lower panels:Time course of gas composition as monitored by methods described in Section 2.H 2(circles),N 2(diamonds),CO 2(triangles),and H 2S (crosses)were quanti?ed.

(Fig.1B),reaching its maximum (3.88mmol L ?1h ?1)at 11h of cultivation.On the other hand,the H 2pro-duction rate of cells grown in MA-YTS medium was about two times higher than that of CO 2(Fig.1C),and the highest H 2production rate was detected at 22h of cultivation (3.16mmol L ?1h ?1).Under all growth conditions,the sum of gas metabolites (CO 2,H 2S,and H 2)along with N 2was nearly 100%throughout the cultivation periods,suggesting that no other gas was generated to signi?cant levels.

In MA-YTS medium,we observed that the pH of the medium signi?cantly decreased with prolongation of culture,indicating the possibility of organic acid for-mation.Therefore,the acetate content in the medium was determined.After 30h of cultivation in MA-YTS medium,acetate was detected at a concentration of 9mM.Although a decrease in pH was not observed in the MA-YTP medium,an even higher accumulation of acetate (20mM)was observed at 13h of cultiva-tion.The stable pH may be due to the consumption of pyruvate correlating with the increase in acetate con-centration.

3.2.Analysis of H 2production during continuous culture

Fig.1indicates that H 2and other gas metabo-lites are generated in a growth-associated man-ner.Therefore,to develop a system for long-term H 2production by T.kodakaraensis KOD1,contin-uous cultivation was performed.At a dilution rate (D )of 0.2h ?1,a constant generation of H 2(and CO 2)was observed in both MA-YTP and MA-YTS media.As for H 2production rate per unit cul-ture,a higher production rate of 9.46mmol L ?1h ?1was observed in MA-YTP medium compared to 6.70mmol L ?1h ?1in the MA-YTS medium.The H 2production rate per unit biomass was calculated to be 24.9and 14.0mmol gdw ?1h ?1for cells grown in MA-YTP and MA-YTS media,respectively (Table 1).As was also observed in batch cultures,the CO 2production rate with pyruvate-grown cells was al-most equivalent to that of H 2,whereas with starch-grown cells,CO 2production was nearly half of H 2production.

T.Kanai et al./Journal of Biotechnology116(2005)271–282275 Table1

Cell densities and gas production rates under steady-state growth conditions(D=0.2h?1)

Medium Cell density(gdw L?1)H2(mmol gdw?1h?1)CO2(mmol gdw?1h?1) MA-YTP0.3824.925.9

MA-YTS0.4814.07.65

Continuous cultivation was performed with a working volume of3L at a dilution rate of0.2h?1.gdw:gram dry weight.

3.3.Analyses of metabolites in the liquid phase

The observed difference in gas composition for both continuous cultures re?ects different modes of metabolism depending on the carbon source.To fur-ther analyze the intracellular metabolism in detail,con-sumption of substrates and accumulation of fermenta-tion products in the medium was determined(Table2). The pyruvate consumption rate was26.0mmol h?1in MA-YTP medium.The consumption rate of starch was 6.03mmol h?1(expressed as glucose units)in the MA-YTS medium.In both cases,more than half of the ini-tial substrate concentration was observed in the ef?uent (data not shown),indicating that these were not growth-limiting substrates.

Next,as both media contain tryptone as a source of amino acids,concentrations of each amino acid in the initial medium and in the ef?uent were compared in both cases(Fig.2).A striking difference was observed in the alanine concentration.In the MA-YTP medium, an increase of14.9mM,corresponding to a production rate of8.94mmol h?1,could be detected.Although to a lower extent,an increase of4.55mM,corresponding to a production rate of2.73mmol h?1,was observed in the MA-YTS medium.It should be noted that a slight increase in proline concentration was also detected in the ef?uent of both media.

With the exceptions of alanine and proline,most other amino acids were consumed during fermen-tation(Fig.2).Although the total amino acid con-sumption was three times higher in MA-YTP medium than in MA-YTS medium(Table2),a common ten-dency existed where hydrophobic amino acids were the most consumed.The amino acids that signi?-cantly decreased in MA-YTP medium were leucine (65%),phenylalanine(60%),isoleucine(55%),aspar-tic acid+asparagine(40%),valine(37%),and me-thionine(35%),while those in MA-YTS medium were leucine(32%),phenylalanine(28%),isoleucine (20%),and methionine(20%).A preference for hy-drophobic amino acid consumption has also been re-ported for P.furiosus(Krahe et al.,1996).On the other hand,a difference in consumption levels of glu-tamic acid+glutamine was observed between the two medium conditions.A signi?cant decrease in glu-tamic acid+glutamine concentration was observed in the MA-YTP medium(26%),while the concentra-tion did not change during cultivation in MA-YTS

Table2

Consumption of substrates and formation of fermentation products under steady-state growth conditions(D=0.2h?1)

(A)Consumption of substrates(mmol h?1)

Medium Pyruvate Starch a Amino acids b NH3 MA-YTP26.0–7.20 3.59 MA-YTS– 6.03 2.400.07

(B)Formation of fermentation products(mmol h?1)

Medium Acetate Alanine Biomass c Ala/Ac d

MA-YTP18.408.94 1.810.49

MA-YTS6.78 2.73 2.020.40 Continuous cultivation was performed with a working volume of3L at a dilution rate of0.2h?1.

a Amount of starch is expressed as glucose equivalents.

b Alanine is not included in the sum.

c Base

d on th

e assumption that the organic fraction o

f cells is C5H7NO2and that this comprises90%of dcw(McCarty,1975).

d Ratio of alanin

e with acetate.

276T.Kanai et al./Journal of Biotechnology 116(2005)

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Fig.2.Concentration of each amino acid in the culture medium during continuous cultivation.(A)MA-YT medium supplemented with 0.5%(w/v)sodium pyruvate;(B)MA-YT medium supplemented with 0.5%(w/v)soluble starch.Fresh media supplied to (medium)and broth retrieved from (ef?uent)the cultivation vessels were analyzed.Dilution rate (D )was set as 0.2h ?1for both medium conditions.

medium.This resembled the changes in NH 3levels;a decrease rate of 3.59mmol h ?1was observed in MA-YTP medium,whereas virtually no consumption was observed in the MA-YTS medium (Table 2).Although it is generally dif?cult to obtain good stoichiometry when cultivation is performed with complex medium,the stoichiometric data of our experiment is summa-rized in Table 3.The data shows that,during con-Table 3

Stoichiometric data of fermentation products under a steady-state growth condition (D =0.2h ?1)Substrate H 2CO 2Acetate Alanine Pyruvate 1.09 1.140.710.34Starch

3.33

1.83

1.12

0.45

The products are expressed as mol per mol of pyruvate or glucose equivalent.

tinuous culture in starch medium,a high H 2-yield (=mol of H 2produced/mol of substrate consumed)of 3.33was obtained,which corresponds to 83%of the theoretical value for complete starch oxidation to acetate.

3.4.Effect of the dilution rate on H 2production Continuous cultivation was performed with increas-ing dilution rates from 0.4h ?1,and its effects on cell weight and H 2production rate were examined (Fig.3).Using the MA-YTP medium,cell densities decreased with the increase in dilution rate until 0.5h ?1,and then maintained a relatively constant value between 0.5and 0.8h ?1.A further increase in dilution rate to 0.9h ?1led to a washout of the cells.As for H 2production rate per unit biomass,levels increased with increasing

T.Kanai et al./Journal of Biotechnology116(2005)271–282

277

Fig.3.Effect of the dilution rate on the continuous cultivation of T. kodakaraensis.Upper panel:Effect of the dilution rate on cell weight. Lower panel:Effect of the dilution rate on the H2production rate per cell weight.Cells were cultivated in MA-YT medium supplemented with0.5%(w/v)sodium pyruvate.

dilution rate until0.6h?1and then leveled off.At a di-lution rate of0.8h?1,the maximum H2production rate was observed,at a high level of59.6mmol gdw?1h?1. This is simply supposed to be due to an increase in metabolic?ux,because,while production rates of both acetate and alanine increased with an increase in dilution rates,the proportion of alanine synthesis against acetate synthesis did not decrease(data not shown).4.Discussion

The present study reports the development of a con-tinuous H2production system using the hyperther-mophilic archaeon,T.kodakaraensis KOD1.At low dilution rates with the substrates pyruvate and starch, H2production rates per unit biomass were comparable to those reported for Enterobacter species(Table4). Moreover,higher H2production rates were observed with an elevation in dilution rate,reaching a max-imum of59.6mmol gdw?1h?1at a dilution rate of 0.8h?1,indicating a high H2-producing potential of T.kodakaraensis KOD1.

Continuous culture experiments of Thermococcales species have been examined for P.furiosus and T. litoralis,as a means to understand the metabolism and bioenergetics in these species(Raven et al.,1992; Rinker and Kelly,2000).These studies put emphasis on stoichiometric analysis,and the potential of these hyperthermophilic archaea for H2production was not focused upon(Nandi and Sengupta,1998).One study with P.furiosus has applied a nutrient-rich medium, similar to our case,in a continuous cultivation using maltose as a carbohydrate source(Schicho et al.,1993). Although clear values have not been stated in the text, an approximate estimation from a?gure shows that average H2production rates of80mmol gdw?1h?1 have been achieved at dilution rates between0.4and 0.65h?1.The results together with those in this study indicate a strikingly high potential of carbohydrate-degrading Thermococcales species as fermentative H2 producers.

Table4

Comparison of H2production rates per unit biomass

Organism Main substrate Cultivation method H2production rate

(mmol gdw?1h?1)

Reference

Photosynthetic bacteria and algae Organic acids etc.–<6Hillmer and Gest(1977) Clostridium butyricum Glucose B a7.3c Karube et al.(1976) E.aerogenes E82005Glucose B a17c Tanisho et al.(1987) E.cloacae IIT-BT08Sucrose B a29.6c Kumar and Das(2000) P.furiosus DSM3638Maltose C(0.4–0.65)b80d Schicho et al.(1993) T.kodakaraensis KOD1Pyruvate C(0.2)b24.9This study

T.kodakaraensis KOD1Starch C(0.2)b14.0This study

T.kodakaraensis KOD1Pyruvate C(0.8)b59.6This study

a Batch cultivation.

b Continuous cultivation;dilution rates(h?1)are indicated in the parentheses.

c Maximum H2production rate is indicated.

d Averag

e values estimated from a plot in reference(Schicho et al.,1993)at dilution rates between0.4and0.65.

278T.Kanai et al./Journal of Biotechnology116(2005)271–282

As for H2production rate per unit culture,values of 6.70–9.46mmol L?1h?1were obtained at a dilution rate of0.2h?1(Table1).At the current stage,these values are lower than those reported with Enterobac-ter species.An H2evolution rate of58mmol L?1h?1 was reported using a self-?occulated cell system with

E.aerogenes AY-2(Rachman et al.,1998),and that of

75.6mmol L?1h?1was also reported using immobi-lized cell systems with E.cloacae IIT-BT08(Kumar and Das,2001).To achieve a higher H2production rate per unit culture,the cell density during cultivation becomes the important factor.At the moment,the use of microbial support carriers and self-?occulated cell systems has not been examined for T.kodakaraensis. Therefore,the use of these techniques will contribute to increasing cell density during cultivation,resulting in the increase of H2production rate per unit culture.

Recently,several reports on microbial H2produc-tion using thermophilic bacteria have been reported. For example,batch cultivation of T.neapolitana was performed at70?C using glucose or soluble starch as a primary carbon source and a H2production rate of5.97×10?1mmol L?1h?1was reported(van Ooteghem et al.,2002,2004).Another study shows batch cultivation of two thermophilic bacteria that were grown at70?C in medium containing sucrose(for C. saccharolyticus)and at65?C in medium containing glucose(for T.el?i)(van Niel et al.,2002).In the case of T.neapolitana,H2and CO2were produced in a sim-ilar ratio as that of T.kodakaraensis grown on starch (H2:CO2,2:1)(van Ooteghem et al.,2002).This fact together with the production of alanine as an end prod-uct by T.neapolitana(Ravot et al.,1996)shows a clear similarity in carbohydrate metabolism between these two species,although they belong to different domains (archaea and bacteria).

The use of hyperthermophiles in continuous cul-ture at high temperature offers several advantages.For example,continuous cultivation usually requires strict care during operation due to the constant risk of con-tamination.Cultivation of hyperthermophiles is rela-tively free from such risks as the high temperature prevents the contamination of ordinary microbes.An-other advantage is that the solubility of various macro-molecules such as starch increases dramatically at high temperatures.Therefore,starch liquefaction processes, which are usually important to increase soluble sub-strate concentrations,become unnecessary.Simplify-ing the cultivation process should contribute in lower-ing the total cost of fermentative H2production.

To develop a strain with higher H2production poten-tial,it is important to understand the metabolic path-way(s)underlying H2production.In this aspect,the future use of T.kodakaraensis KOD1in fermentative H2production will be bene?cial,because a wealth of information is available on the biochemical and physiological aspects of the species(Tachibana et al., 1996,1997;Rahman et al.,1998;Siddiqui et al.,1998; Imanaka et al.,2002;Imanaka and Atomi,2002;Rashid et al.,2002a,b;Kanai et al.,2003).Moreover,the entire genome sequence of the species has recently been de-termined(Fukui et al.,2005).Taking into account the genome information as well as genetic and biochemi-cal data from carbohydrate-degrading Thermococcales species(Kengen et al.,1996;Verhees et al.,2003),the carbohydrate metabolism of the species has been pro-posed(see below).

Another favorable point of this archaeon is the avail-ability of chromosomal gene disruption or replacement technology(Sato et al.,2003),which is not common in other hyperthermophiles.This will enable us to ratio-nally undertake molecular breeding of the strain.For example,although T.kodakaraensis KOD1cannot uti-lize maltose as a carbon source because of the lack of maltose transporter(Fukui et al.,2005),introduc-tion of the genes encoding the respective transporter of other hyperthermophiles(such as P.furiosus and T. litoralis(DiRuggiero et al.,2000))will develop a new strain that can produce H2from maltose.Another target for molecular breeding of T.kodakaraensis KOD1is the present necessity of both yeast extract and tryptone for the ef?cient growth(and consequently H2produc-tion).The genome sequence data shows the absence of biosynthetic genes for the amino acids Ile,Val,Leu and Arg,as well as of genes for thiamine cofactor biosyn-thesis.Introducing heterologous genes of the respective pathways from other hyperthermophilic archaea could alleviate the requirements of these complex compounds in T.kodakaraensis KOD1.

The glycolytic pathway proposed in T.kodakaraen-sis KOD1is summarized in Fig.4.Starch is initially degraded by the functions of?-amylase(and related en-zymes)secreted from the cells(Tachibana et al.,1996; Dong et al.,1997a,b;Rashid et al.,2002a),and the re-sulting malto-oligosaccharides are subsequently trans-ported into the cells,where they are further degraded

T.Kanai et al./Journal of Biotechnology116(2005)271–282

279

Fig.4.Proposed metabolic pathway of starch and pyruvate degradation in T.kodakaraensis.Dotted arrows indicate reactions covered by multiple enzymes.Abbreviations used are:DHAP,dihydroxyacetone phosphate;GAP,glyceraldehyde-3-phosphate;2OG,2-oxoglutarate;Pi,inorganic phosphate;Fd(ox),oxidized ferredoxin;Fd(red),reduced ferredoxin;GDH,glutamate dehydrogenase;AlaAT,alanine aminotransferase;POR, pyruvate:ferredoxin oxidoreductase;ACS,acetyl-CoA synthetase;MBH,membrane-bound hydrogenase.For some substrates,the numbers of carbon atoms are described inside the parentheses.

into glucose units(Koning et al.,2002).In Thermo-coccales,metabolism of glucose units into pyruvate is catalyzed through a modi?ed Embden–Meyerhof path-way(Selig et al.,1997;Verhees et al.,2003),and all orthologue genes are present on the genome of T.ko-dakaraensis KOD1.As for pyruvate metabolism,an oxidative pathway exists where pyruvate is converted to acetate(Sch¨a fer and Sch¨o nheit,1991,1992;Rinker and Kelly,2000).Through the oxidative degradation of glucose units into acetate,the conversion of glyceralde-hyde3-phosphate to3-phosphoglycerate(Mukund and Adams,1995;van der Oost et al.,1998)and the oxida-tive decarboxylation of pyruvate into acetyl-CoA and CO2(Kletzin and Adams,1996)generate the reduced form of ferredoxin that provides the reducing force for membrane-bound hydrogenase(MBH)to produce H2 (Sapra et al.,2000;Silva et al.,2000).The latest report shows that H2production in P.furiosus MBH is further coupled to proton translocation across the plasma mem-brane,thereby generating a proton-motive force,with which ATP synthesis is driven by membrane-bound ATP synthetase(Sapra et al.,2003).This respiratory coupling of H2production to ATP synthesis may be the reason of growth-dependent H2production observed for T.kodakaraensis.

Besides the oxidative metabolism of pyruvate, another branch,that generates l-alanine as a re-duced end product of carbohydrate fermentation has been reported in Thermococcales(Kengen and Stams, 1994;Kobayashi et al.,1995;Rinker and Kelly,2000).

280T.Kanai et al./Journal of Biotechnology116(2005)271–282

Alanine is formed by alanine aminotransferase directly from pyruvate via transamination with glutamate.In P. furiosus and T.litoralis,alanine production was found to increase with increased H2partial pressure(Kengen and Stams,1994;Rinker and Kelly,2000),suggest-ing that the generation of H2and alanine are compet-itive means of disposing intracellular reducing equiv-alents.As a signi?cant accumulation of alanine was also observed in this study,preventing alanine forma-tion should contribute to an increase in H2production of T.kodakaraensis KOD1.

It is noteworthy that cells grown on MA-YTS medium produced less H2(per unit biomass)than those grown on MA-YTP medium.However,an appropriate explanation for this phenomenon from a metabolic view point is dif?cult,as our cultures were performed with complex media containing signi?cant amounts of both yeast extract(0.5%,w/v) and tryptone(0.5%,w/v),whose components are not strictly de?ned.When calculation of an energetic yield(Y ATP)was performed based on the proposed carbohydrate metabolism and the amount of end products produced,a large value of26.5(g-biomass mol?1ATP)was obtained for cultivation on MA-YTS medium.This may indicate the existence of other pathway(s)that generates ATP(thus resulting in an increase of biomass)from unidenti?ed component(s) in our medium without the generation of H2. Acknowledgements

This research was partially supported by Kansai Re-search Foundation for technology promotion,Grant-in-Aid for Young Scientists,2004(to T.K.). References

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