Biological production by moderately thermophilic anaerobic

HP Goorissen and AJM Stams Laboratory of Microbiology, Wageningen University and Research centre Wageningen The Netherlands

Abstract This study focuses on the biological production of hydrogen at moderate temperatures (65-75 C) by anaerobic bacteria. A survey was made to select the best (moderate) for hydrogen production from cellulolytic biomass. From this survey we selected Caldicellulosiruptor saccharolyticus (a gram-positive bacterium) and elfii (a gram-negative bacterium) as potential candidates for biological hydrogen production on mixtures of C5-C6 sugars. and were used as model substrates to describe growth and hydrogen production from hydrolyzed biomass. Mixed substrate utilization in batch cultures revealed differences in the sequence of substrate consumption and in catabolite repression of the two microorganisms. The regulatory mechanisms of catabolite repression in these microorganisms are not known yet.

Keywords: Biological hydrogen production, thermophiles, Caldicellulosiruptor saccharolyticus, Thermotoga elfii, catabolite repression

Introduction Hydrogen can be seen as a true ‘green’ alternative for fossil fuels in case of biological production from biomass, since there is no net CO2 production. Mesophilic hydrogen has been studied extensively; however, thermophilic microorganisms are regarded to be superior with respect to hydrogen production rates and yields (Claassen et al 1999). We aimed at biological hydrogen production via a two-step process in which a thermophilic heterotrophic fermentation from biomass is followed by a phototrophic fermentation on fatty acids, produced in the heterofermentative reactor (for more information about this process see: www.biohydrogen.nl). To be able to select the best thermophiles for the required process we performed a literature survey. The pentose sugar xylose is one of the major constituents of hemicellulose and therefore abundant in nature. The last decades much effort has been put into the utilization of xylose for the production of alternative energy sources, like ethanol (Hahn-Hägerdal et al, 1994, Kim et al, 1999). However, the conversion of xylose in an alcoholic fermentation is incomplete and problematic (Claassen et al, 1999). Moreover, the utilization of pentoses in hemicellulose-derived sugar mixtures is delayed and may be incomplete. Residual sugars lower the product yield and cause problems in the downstream processing of fermentation products. Clearly, more information on xylose degradation in mixtures of saccharides is required. Miscanthus, a lignocellulosic biomass was selected as a substrate for the bioprocess. Chemical and mechanical pretreatment of this biomass, followed by enzymatic hydrolysis, resulted in a mixture of saccharides consisting mainly (60% of 62.5% total polysaccharides) of glucose and xylose in a ratio of 2:1 (de Vrije et al, 2002). Carbon catabolite repression is a regulatory mechanism that causes sequential utilization of carbohydrates. Via catabolite repression the presence of a carbon source in the medium can repress the utilization of alternative carbon sources, thereby causing differences in sugar utilization. The mechanism of catabolite repression is well studied in mesophilic species, and from these studies we learned that catabolite repression is regulated by different mechanisms in gram-negative and in gram- positive organisms (Cook et al, 1994, Saier et al, 1996). We compared xylose utilization by C. saccharolyticus a gram-positive moderate thermophilic cellulolytic strain that can produce hydrogen, , lactate and traces of ethanol from C6 and C5 sugars (Rainey et al, 1995) with T. elfii a gram- negative thermophilic non-cellulolytic strain (van Niel et al, 2003, Kadar et al, 2003) in order to optimize a thermophilic hydrogen production process from biomass.

1 Materials and Methods Organisms and media C. saccharolyticus (DSM 8903) and T. elfii (DSM 9442) were purchased from the Deutsche Sammlung von Mikroorganimsen und Zellkulturen (Braunschweig, Germany). For growth of C. saccharolyticus, a bicarbonate buffered medium was used. It consisted of (per l): KH2PO4 0.41 g, Na2HPO4 0.53 g, CaCl2 0.11 g, MgCl2 0.1 g, NH4HCO3 0.44 g, NaHCO3 3.75 g, Na2S·9H2O 0.5 g, yeast extract 1 g, resazurin 1 mg, vitamin solution 1 ml and trace element solution 1 ml. The vitamin solution consisted of (mg/l) biotin 20, folic acid 20, pyridoxine-HCl 100, thiamine-HCl x 2 H2O 50, riboflavin 50, nicotinic acid 50, cobalamin, 50, D-Ca-pantothenate 50, p-aminobenzoic acid 50, lipoic acid 50. The trace elements solution consisted of (per l) FeCl2·4H2O 1.5 g, ZnCl2 70 mg, MnCl2·4H2O 0.1 g, H3BO3 6 mg, CoCl2·6H2O 0.19 g, CuCl2·2H2O 2 mg, NiCl2·6H2O 24 mg, Na2MoO4·H2O 36 mg, Na2WO4 15 mg, Na2SeO3·5H2O 15 mg. The gas phase consisted of 80% N2 and 20% CO2 and the pH was 7.2. Substrates were added from sterile stock solutions of 1 M. For experiments in a controlled batch reactor a modified medium (http://www.dsmz.de/media/med640.htm) was used as described by van Niel et al (2003). T. elfii was grown on a modified DSM 664-medium (http://www.dsmz.de/media/med644.htm) without tryptone but with yeast extract (3 g/l) and with a

trace element solution that is used for (http://www.dsmz.de/media/med141.htm).

¢¡¤£¦¥ §©¨ £¦¥ ¨ ¦ ¦¡¤ ¤¦ ¦

Hydrogen was measured on a 406 Packard gas chromatograph equipped with a thermal conductivity detector (TCD, 100 mA). The gases were separated at 100 ºC on a molecular sieve column (13x, 180 cm by ¼ inch, 60-80 mesh) with argon as the carrier gas. Optical density was measured spectrophotometrically at 620 nm on a Hitachi U-1100 spectrophotometer. Pentoses and hexoses were analyzed on a Dionex BioLC system equipped with an electrochemical conductivity detector. The saccharides were separated at 30 ºC on a Carbopack PA2 column with 0.2% NaOH as eluens. Fatty acids and ethanol were measured by HPLC, as described previously (Stams et al, 1993).

Results and discussion

Selection of the best candidate for thermophilic hydrogen production from (poly-)saccharides The main thermophilic saccharolytic hydrogen producers are classified in the kingdom of the Eubacteria and belong to the genera Clostridium, Caldicellulosiruptor, Fervidobacterium Thermoanaerobacter, Thermotoga, and Thermococcus (see Table 1). The vast majority of known thermophilic cellulolytic anaerobic hydrogen-producing bacteria belongs to the genus Clostridium, consisting of spore-forming bacteria that grow at temperatures up to 60°C. The product pattern of most Clostridium species is quite divers and includes ethanol, lactate, acetate butyrate, propionate, and H2 . To our knowledge maximum yields on glucose above 3 mol H2/mol glucose were never reported for mesophilic species. The genus Caldicellulosiruptor consists of four closely related species, with Caldicellulosiruptor saccharolyticus (DSM 8903) as the type strain. Caldicellulosiruptor species are non-spore-forming and have narrower product profiles than closely related Clostridium species. The temperature range for Clostridium species is limited to about 60°C whereas Caldicellulosiruptor species can grow up to 80°C. C. saccharolyticus gives a hydrogen yield on glucose close to the theoretical value of 4 mol H2/mol glucose (unpublished results) The order of Thermotogales include hydrogen-producing species from the genera Thermotoga, Thermosipho and Fervidobacterium, which are all characterized morphologically by a typical sheath-like outer structure referred to as toga. Thermosipho species are very sensitive to and require elemental sulfur as an electron sink. The genus Thermotoga represents the deepest branch in the kingdom of Eubacteria, with most isolates from marine hydrothermal environments. An exception is Thermotoga lettingae, isolated from a thermophilic sulfidogenic bioreactor (Balk et al, 2002). Thermotoga species can grow up to slightly higher temperatures (90°C) than Caldicellulosiruptor species. Their nutrient requirements are more complex; depending on the species they require yeast extract, peptone, and NaCl, since most Thermotoga species are moderately halophilic. Fervidobacterium species are known to produce unique spheroids and are quite sensitive to antibiotics. In contrast with Thermotoga species the members of the genus are not halophilic. Thermoanaerobacterium species are diverse regarding NaCl requirement and substrate profiles. However, they are all capable of slightly acidic growth and can produce elemental sulfur from

2 thiosulfate. On 16S rDNA level the similarity with C saccharolyticus is around 82%.The majority of Thermoanaerobacter species is spore-forming and requires thiosulfate or sulfur as terminal electron acceptor. Some of them are able to use xylose as a substrate at relatively high growth temperatures (85°C) Members of the Thermococcales represent a unique group of hyperthermophilic microorganisms belonging to the Euryarchaeota branch within the Archaea domain. In general they can grow at extremely high temperatures and except the Thermococcus genus, they are not taken into account here. Species belonging to the genus Thermococcus are halophilic and can grow on a wide range of proteinaceous and saccharolytic substrates. From this survey we concluded that species from the genus Caldicellulosiruptor might be the most suitable for a biological hydrogen production process from cellulolytic biomass at a temperature of 70°C. They have a broad substrate profile, a relatively small product profile with high H2 concentrations, and low nutrient requirements. To compare physiological features and to optimize the process we included a low NaCl requiring Thermotoga strain in our studies.

Growth of C. saccharolyticus in a minimal medium containing glucose and xylose C. saccharolyticus has a preference for xylose if grown in mixtures with glucose (fig. 1a and 1b), although this xylose repression is concentration dependent. We used different xylose concentrations (5 or 33 mM) combined with 36-43 mM glucose concentrations. If the initial xylose concentration was high (33 mM), xylose acted as a repressor. At a low initial xylose concentration, glucose repressed xylose utilization. To investigate whether the unusual diauxic pattern of C. saccharolyticus at high pentose concentrations is typical for the xylose/glucose mixture, we studied /glucose and /glucose utilization as well. Similar results were obtained (results not shown), although some small differences in lactate production were observed. During growth on arabinose the onset of lactate production started earlier and the lactate production during growth on ribose was negligible. Hydrogen production was similar for growth on all pentose/glucose mixtures.

Growth of T. elfii in a minimal medium containing glucose and xylose Experiments with T. elfii (fig. 2a and 2b) revealed that xylose and glucose were used simultaneously if the xylose concentration was high (~40 mM). At low xylose concentration (~ 5 mM) glucose repressed xylose utilization similar to the pattern observed for C. saccharolyticus, although no lactate production was found. Simultaneous substrate utilization has been described before for other thermophilic species such as Thermoanaerobacter thermohydrosulfuricus (Cook et al, 1993). For the application of the overall process, complete degradation is required. From our batch culture studies it is clear that residual concentrations sugars remain. However, this is likely due to product inhibition. Experiments with C. saccharolyticus were repeated in controlled batch cultures in order to exclude the influence of hydrogen accumulation and acidification of the medium on the substrate utilization pattern and product formation. Results are shown in fig. 3a and b. Similar results were obtained as for the batch culture experiments, although lactate production was more pronounced. From our experiments it can be concluded that for a thermophilic hydrogen production process on cellulolytic biomass, the concentration of the pentose sugar in the mixture determines the utilization pattern, and that this pattern is depending on the strain that is used. In experiments in continues cultures we could show that C. saccharolyticus is a robust microorganism, able to adapt easily to changing conditions (results not published). However, to obtain full degradation of pentose/glucose mixtures with varying substrate concentrations in a hydrogen production process from biomass, the utilization of mixed cultures will be essential.

3 Table 1 Potential candidates for a biological thermophilic hydrogen producing process from cellulolytic biomass Strain Topt (T range) Substrates utilised1 td Fermentation products Gram Origin Special requirements/ comments2 Ref. (°C) stain Acetothermus paucivorans 58 glucose, nr acetate, H2, CO2 - requires vitamin B12 Dietrich 1988 Anaerocellum 72-75 cellulose, starch, cellobiose, 110 min lactate, acetate, H2, CO2, ethanol + hot spring, boggy meadow YE required; sens. to antibiotics Svetlichnyi 1990 thermophilum (40-83) mono- disacch. Caldicellulosiruptor kristjanssonii 78 cellulose, starch, cellobiose, 120 acetate, H2, CO2 (lactate, ethanol) - biomat slightly alkaline hot spring, Iceland Bredholt 1999 (45-82) pectin min lactoaceticus 68 starch, cellobiose, xylan, pectin, 120 min lactate, acetate, H2 (CO2, ethanol) - alkaline hot spring, hveragerdi, Iceland limited substrate range Mladenovska 1995 (50-78) disacch. owensensis 75 cellulose, starch, cellobiose, 7.3 h lactate, acetate, H2, CO2, ethanol - sediment sample Owens Lake, Calif. Huang et al 1998 (50-80) pectin, disacch. saccharolyticus 70 cellulose, starch cellobiose, n.r. lactate, acetate, H2, CO2, ethanol - wood in a geothermal spring, Taupo, NZ Rainey et al 1994 (45-80) Clostridium celluloflavum 60 cellulose, carbohydrates nr acetate, H2, CO2, ethanol - nr produces yellow pigment He 1991 (45-70) cellulosi 55-60 cellulose nr acetate, H2, CO2, ethanol - manure Yanling 1991 (40-65) josui 45 ball-milled cellulose, Avicel, nr CO2, H2, acetate, ethanol + /- thai compost limited substrate range Sukhumavasi et al 1988 (25-60) cellobiose, xylan, starch- stercorarium 65 cellulose, starch, xylan 16 h CO2, H2, acetate, ethanol, lactate - compost Madden 1983 (60-70) succinogenum 50-53 cellulose, carbohydrates nr succinate, acetate, ethanol, CO2, H2 - silage on milk cow field Tan 1993 (37-65) thermocopriae 60 cellulose, starch, cellobiose, nr butyrate, acetate, lactate, ethanol - compost Jin et al 1988 (47-74) H2S thermocellum 60 cellulose,starch- xylan- nr acetate, H2, CO2, ethanol, lactate + McBee 1954 thermopapyrolyticum cellulose, carbohydrates nr butanol, butyrate, acetate, lactate, nr riverside mud Mendez 1991 ethanol, H2S , CO2, H2 uzonii 65 starch, cellobiose, cellulose- nr CO2, H2, acetate, ethanol, lactate + hot spring Kamchatka peninsula Krivenko et al 1990 (50-75) Dictyoglomus 72 cellulose, starch, cellobiose, 240 min CO2, H2, acetate, ethanol, lactate - hot springs, Uzon volcano crater S0 inhibition Svetlichnyi and turgidus (48-86) pectin, sugars Svetlichnaya 1988 Fervidobacterium nodosum 70 starch, sugars, (cellulose, nr CO2, H2, acetate, ethanol, lactate - hot spring, nz Patel et al 1985 (41-79) cellobiose, nt) islandicum 65 cellulose, starch, sugars, nr CO2, H2, acetate, ethanol, lactate - Icelandic hot spring Huber et al 1990 (50-80) (cellulose nt) pennivorans 70 (50-80) starch, xylose CO2, H2, acetate, ethanol, lactate - hot spring Azores islands Friedrich 1996 Spirocheta 66-68 cellulose, CMC, starch, 70 min CO2, H2, acetate, isobutyrate hot spring littorial Kuril island moderate halophile NaClopt. = 1.5% Aksenova et al 1990 thermophila (40-73) cellobiose, simple sugars Thermotoga elfii 66 sugars (starch, 2.8 h CO2, H2, acetate - oil well, Africa YE, bio-trypticase, and S0 required; Ravot et al 1995 (50-72) cellulose,cellobiose n.t.) hypogea 70-75 xylan, sugars (cellulose, nr CO2, H2, acetate, alanine - oil well, Africa YE required Fardeau et al 1997 (56-90) cellobiose, starch nt)

4 Strain Topt (T range) Substrates utilised1 td Fermentation products Gram Origin Special requirements/ comments2 Ref. (°C) stain lettingae 65 starch, cellobiose, pectin, sugars CO2, H2, acetate, alanine - sulfidogenic reactor Balk et al 2002 maritima 80 starch, sugars (cellulose, 75 min CO2, H2, acetate, lactate - Ggothermal heated see floor, Italy tungsten increases activity; max 108 H. Huber et al., 1986 (50-90) cellobiose nt) cells/ml; H2, S0 inhibition naphtophila 80 (48-86) starch, pectin, sugars 59 min CO2, H2, acetate, lactate nr Kubiki oil reservoir NaCl 0.1-6.0% (opt. 1.0 %) Takahata et al 2001 neapolitana 80 starch, sugars,pectin 45 min CO2, H2, acetate, lactate, alanine - shallow, submarine, hot spring sensitive to antibiotics Jannasch et al 1988 (55-90) (cellulose,cellobiose nt) petrophila 80 (47-88) starch, cellulose, pectin, sugars 54 CO2, H2, acetate, lactate nr Kubiki oil reservoir NaCl 0.1-5.5% (opt. 1.0 %) Takahata et al 2001 subterranea 70 peptone, YE, casein, glucose+/-, 285 min n.r. - Paris oil well H2, S0 inhibition; YE required Jeanthon et al 1995 (50-75) +/- Thermoanaerobacter brockii 55-60 cellulose, starch, cellobiose, nr CO2, H2, acetate, lactate + oil field Lee et al 1993 (40-75) mono-, disacch. cellulolyticus 75 cellulose, starch, cellobiose, nr CO2, H2, acetate, lactate, - hot spring optimum pH= 8.0, tungsten Taya et al 1988 (50-85) mono-, disacch. requirement ethanolicus 70 starch, cellobiose, mono-, nr ethanol , CO2, H2, acetate, lactate +/- hot spring, Yellowstone Wiegel and Ljungdahl 1981 (37-78) disacch. subterraneus 65 polysacch, cellobiose, starch 2.5h CO2, H2, acetate, lactate, alanine + oil field halophile, YE required Fardeau et al 2000 (35-80) tengcongensis 75 starch, cellobiose, mono-, nr CO2, H2, acetate freshwater hotspring H2 inhibition Xue et al 2001 (50-80) disacch. thermhydrosulfuricus 55 mono-, disacch. nr CO2, H2, acetate beet sugar factory Klaushofer and Parkkinen 1965, Lee et al 1993 Thermoanaerobacterium polysaccharolyticum 65-68 xylose, cellulose-, starch-, nr ethanol, CO2, H2, acetate, formate, + waste canning factory Cann et al. 2000 (45-72) polysacch, lactate (formerly Clostridium) xylose, cellulose-, starch-, nr ethanol, CO2, H2, acetate, formate, nr thermosaccharolyticum polysacch lactate thermosulfurigens >60 (<75) starch, polysacch, pectin nr nr Lee et al 1993 zeae starch, polysacch, cellulose- nr ethanol, CO2, H2, acetate, formate, + waste pile canning factory Cann et al 2001 lactate Thermococcus litoralis 88 disachharides, starch CO2, H2, nr shallow thermal submarine spring halophile, exopolysaccharide Neuner 1990 (55-98) formation stetteri 76 (55-94) proteins, starch, nr CO2, H2, nr marine volcanic crater requires S0 for growth on starch Miroshnichenko et al 1989 waiotapuensis 85 proteins, AA, maltose, starch, 54 min CO2, H2, nr freshwater hot spring halophile Gonzalez et al 1999 (60-90) pyruvate, cellobiose- Thermosipho geolei 70 cellulose, starch, cellobiose, nr CO2, H2, acetate, alanine nr- continental oil reservoir halophile, H2 inhibition Haridon et al 2001 45-75 mono-, disacch. melanesiensis 70 mono-, disacch. nr CO2, H2, acetate, alanine - gills of a deep see vent hydrothermal halophile, rich medium, So required Antoine et al 1997 45-80 mussel for H2 production 1Only relevant (poly-)saccharides are mentioned, 2 only characteristics relevant for the desired hydrogen production process are taken into account; nr = not reported, nt = not tested

5 45 25 A 40

35 20 )

M 30 ) m ( % 15 (

n

25 n o i e t g a r o t r

n 20 d e y

c 10 H n

o 15 C

10 5

5

0 0 0 10 20 30 40 50 Time (h)

45 25 B 40

20 35 )

M 30 ) m ( % 15 (

n n

o 25 i e t g a r o t r

n 20 d e y

c 10 H n

o 15 C

10 5

5

0 0 0 10 20 30 40 50 Time (h)

Fig 1a and 1b. Growth of C. saccharolyticus at high (a) and low (b) xylose concentrations in mixtures of glucose and xylose. Symbols: * hydrogen; ÷, glucose; ø, xylose; æ, acetate; p, lactate

6 45 25 A 40

20 35 )

M 30 ) m ( % 15 (

n

25 n o i e t g a r o t r

n 20 d e y

c 10 H n

o 15 C

10 5

5

0 0 0 10 20 30 40 50 Time (h)

45 25 B 40

20 35 )

M 30 ) m ( % 15 (

n

25 n o i e t g a r o t r

n 20 d e y

c 10 H n

o 15 C

10 5

5

0 0 0 10 20 30 40 50 Time (h)

Fig 2a and 2b. Growth of T.elfii at high (a) and low (b) xylose concentrations in mixtures of glucose and xylose. Symbols: * hydrogen; ÷, glucose; ø, xylose; æ, acetate; p, lactate

7 40 A 35

30 ) M m

( 25

n o i t

a 20 r t n e c

n 15 o C 10

5

0 0 10 20 30 40 50 Time (h)

40 B 35

30 ) M 25 m (

n o t

a 20 r t n e

n 15 o C

10

5

0 0 10 20 30 40 50 Time (h)

Fig 3a and 3b. Growth of C. saccharolyticus in controlled batch cultures with mixtures of glucose and xylose. Symbols ÷, glucose; ø, xylose; æ, acetate; p, lactate

8 Acknowledgements Part of this research was support with a grant of the Dutch Programme EET (Economy, Ecology Technology) a joint initiative of the Ministry of Economic Affairs, Education, Culture, and Sciences and of Housing, Spatial planning, and the Environment. The programme is run by the EET Programme Office, a partnership of Senter Novem

References

Aksenova EY, Svetlichnyi A, Zavazin GA (1990) Spirocheta thermophila sp. nov., a thermophilic marine spirochete isolated from a littoral hydrotherm of Shiashkotan island. Mikrobiologiya 59, 1050-1057 Antoine E, Cilia V, Meunier JR, Guezennec J, Lesongeur F, Barbier G (1997) Thermosipho melanesiensis sp. nov., a new thermophilic anaerobic bacterium belonging to the order Thermotogales, isolated from deep-sea hydrothermal vents in the southwestern Pacific Ocean. Int. J. Syst. Bacteriol. 47, 1118-1123 Balk M, Weijma J, Stams A JM (2002). Thermotoga lettingae sp. nov., a novel thermophilic methanol degrading bacterium isolated from a thermophilic anaerobic bioreactor. Int. J. Syst. Evol. Microbiol 52,1361-1368 Bredholt S, Sonne-Hansen J, Nielsen P, Mathrani IM, Ahring B (1999) Caldicellulosiruptor kristjanssonii sp.nov., a cellulolytic, extremelly thermophilic, anaerobic bacterium. Int. J. Syst. Bacteriol. 49, 991-996 Cann ISK, Stroot PG, Mackie KR, White BA, Mackie RI (2001) Characterization of two novel saccharolytic, anaerobic thermophiles, Thermoanaerobacterium polysaccharolyticum sp.nov. and Thermoanaerobacter zeae sp.nov., and emendation of the genus Thermoanaerobacterium. Int. J. Syst. Evol. Microbiol. 51:293-202 Claassen PAM, van Lier JB, Lopez Contreras AM, van Niel EWJ, Sijtsma L, Stams AJM, de Vries SS, Weusthuis RA (1999) Utilisation of biomass for the supply of energy carriers. Appl. Environ. Microbiol. 52, 741-755 Cook GM, Janssen PH, Morgan HW (1993) Simultaneous uptake and utilisation of glucose and xylose by Clostridium thermohydrosulfuricum. FEMS Microbiol. Lett. 109, 55-62. Cook GM, Janssen PH, Russell, JB, and Morgan HW (1994) Dual mechanisms of xylose uptake in the thermophilic bacterium Thermoanaerobacter thermohydrosulfuricus. FEMS Microbiol. Lett. 116, 257-262 Dietrich G, Weiss N, Winter J (1988) Acetothermus paucivorans, gen. nov. sp. nov., a strictly anaerobic, thermophilic bacterium from sewage sludge, fermenting hexoses to acetate, CO2, and H2 System. Appl. Microbiol.10, 174-179 Fardeau M-L, Ollivier B, Patel BKC, Magot M, Thomas P, Rimbault A, Rocchiccioli F, Garcia J-L (1997) Thermotoga hypogea sp. nov., a xylanolytic, thermophilic bacterium from an oil- producing well. Int. J. Syst. Bacteriol. 47,1013-1019 Fardeau M-L, Magot M, Patel BKC, Thomas P, Garcia J-L, Ollivier B (2000) Thermoanaerobacter subterraneus sp. nov., a novel isolated from oil field water. Int. J. Syst. Evol. Microbiol 50, 2141-2149 Fond O, Engasser J-M, Matta-El-Amouri G, Petitdemange E (1986) The acetone butanol fermentation on glucose and xylose. I. Regulation and kinetics in fed-batch cultures. Biotechnol. Bioeng. 28, 167-175 Friedrich A, Antranikian G (1996) Keratin degradation by Fervidobacterium pennavorans, A novel anaerobic species of the order Thermotogales. Appl. Environ. Microbiol. 62, 2875-2882 Gonzalez JM, Scheckells D, Viebahn M, Krupatkina D, Borges KM, Robb FT (1999) Thermococcus waiotapuensis sp. nov., an extremely thermophilic archaeon isolated from a freshwater hot spring. Arch. Microbiol. 172, 95-101 Hahn-Hägerdal B, Jeppson H, Skoog K, Prior, BA (1994) Biochemistry and physiology of xylose fermentation by yeasts. Enzyme Microb. Technol. 16, 933-943 L'Haridon S, Miroshnichenko ML, Hippe H, Fardeau ML, Bonch-Osmolovskaya E, Stackebrandt E Jeanthon C (2001) Thermosipho geolei sp. nov., a thermophilic bacterium isolated from a continental petroleum reservoir in Western Siberia. Int. J. Syst. Evol. Microbiol 51, 1327-1334 He YL, Ding YF, Long YQ (1991) Two cellulolytic Clostridium species: Clostridium cellulosi sp. nov., and Clostridium cellulofermentans Int. J. Syst. Bacteriol.41,306-309 Huang C-Y, Patel BK, Mah RA, Baresi L (1998) Caldicellulosiruptor owensensis sp. nov., an anaerobic, extremely themophilic xylanlolytic bacterium. Int. J. Syst. Bacteriol. 48, 91-97

9 Huber H, Langworthy T A, König H, Thomm M, Woese C R, Sleytr UB, Stetter KO (1986) Thermotoga maritima sp. nov. represents a new genus of unique extremely thermophilic eubacteria growing up to 90 °C. Arch. Microbiol. 144, 324-333 Huber RC, Woese R, Langworthy TA, Kristjansson JK, Stetter KO (1990) Fervidobacterium islandicum sp. nov., a new extremely thermophilic eubacterium belonging to the "Thermotogales". Arch. Microbiol. 154:105-111 Inada T, Kimata K, Aiba H (1996) Mechanisms responsible for glucose- diauxie in Escherichia coli:challenge to the cAMP model. Genes Cell 1, 293-301 Jannasch H, Huber R, Belkin S, Stetter KO (1988). Thermotoga neapolitana sp. nov. of the extremely thermophilic eubacterial genus Thermotoga. Arch. Microbiol. 150,103-104. Jeanthon C, Reysenbach A-L, L'Haridon S, Gambacorta A, Pace NR, Glenat P, Prieur D (1995) Thermotoga subterranea sp. nov., a new thermophilic bacterium isolated from a continental oil reservoir. Arch. Microbiol. 164:91-97 Jin F, Yamasato K, Toda K (1988) Clostridium thermocopriae sp. nov., a cellulolytic thermophile from animal feces, compost, soil, and a hot spring in Japan Int. J. Syst. Bacteriol. 38:279-281 Kadar Z, de Vrije T, Budde MAW, Szengyel Z, Reczy K, Claassen PAM (2003) Hydrogen production from paper sludge hydrolysate. Appl. Biochem. Biotechnol. 105-108, 557-566. Kim JS, Oh KK, Kim SW, Jeong YS, Hong SI (1999) Ethanol production from lignocellulosic biomass by simultaneous saccharification and fermentation employing the reuse of yeast and enzyme. J. Microbiol. Biotechnol. 9, 297-302 Klaushofer H, Parkkinen E (1965) Zur Frage der Bedeutung aerober und anaerober thermophiler Sporenbildner als Infektionsursache in Rübenzuckerfabriken. I. Clostridium thermohydrosulfuricum, eine neue Art eines saccharose-abbauenden, thermophilen, schwefelwasserstoff-bildenden Clostridium, Z Zuckerind, 90, 445-449 Krivenko VV, Vadachloriya RM, Chermykh NA, Mityushina LL, Krasil'nikova EN (1990) Clostridium uzonii sp. nov., an anaerobic thermophilic glycolytic bacterium isolated from hot springs in the Kamchatka peninsula. Mikrobiologiya 59:1058-1066 Lacis LS and Lawford HG (1991) Thermoanaerobacter ethanolicus growth and product yield from elevated levels of xylose or glucose in continuous cultures. Appl. Environ. Microbiol. 57, 579- 585 Lee YE, Jain MK, Lee C, Lowe SE, Zeikus JG (1993) Taxonomic distinction of saccharolytic thermophilic anaerobes: Description of Thermoanaerobacterium xylanolyticum new genus new species, and Thermoanaerobacterium saccharolyticum, new genus new species: Reclassification of Thermoanaerobium brockii, Clostridium thermosulfurogenes and Clostridium thermohydrosulfuricum E100-69 as Thermoanaerobacter brockii new combination, Thermoanaerobacterium thermosulfurigenes new combination, and Thermoanaerobacter thermohydrosulfuricus new combination, respectively; and transfer of Clostridium thermohydrosulfuricum 39E to Thermoanaerobacter ethanolicus. Int. J. Syst. Bacteriol. 43:41- 51 Madden RH (1983) Isolation and characterization of Clostridium stercorarium sp. nov., celullolytic thermophile. Int. J. Syst. Bacteriol. 33:837-840 McBee RH (1954) The characteristics of Clostridium thermocellum. J. Bacteriol. 67,505-506 Mendez BS, Pettinari MJ, Ivanier SE, Ramos CA, Sineriz F (1991) Clostridium thermopapyrolyticum, new species, a cellulollytic thermophile. Int. J. Syst. Evol. Microbiol 41, 281-283 Miroshnichenko ML, Bonch-Osmolovskaya EA, Neuner A, Kostrikina NA, Chernych NA, Alekseev VA (1989) Thermococcus stetteri sp. nov., a new extremely thermophilic marine sulfur- metabolizing archaebacterium. Syst. Appl. Microbiol.12, 257-262 Mladenovska Z, Mathrani IM, Ahring BK (1995) Isolation and characterization of Caldicelullosiruptor lactoaceticus sp. nov., an extremely thermophilic, cellulolytic, anaerobic bacterium. Arch. Microbiol.163:223 Neuner A, Jannasch HW, Belkin S, Stetter KO (1990) Thermococcus litoralis sp.nov. a species of extremely thermophilic archaebacteria. Arch. Microbiol. 153:205-207 Niel van, EWJ, Claassen PAM, Stams AJM (2003) Substrate and product inhibition of hydrogen production by the extreme thermophile, Caldicellulosiruptor saccharolyticus. Biotechnol. Bioeng. 81, 255-262. Patel BKC, Morgan HW, Daniel RM (1985) Fervidobacterium nodosum gen. nov., and sp. nov., a new chemoatitrophic, caldoactive anaerobic bacterium. Arch. Microbiol. 141:63-69 Rainey FA, Donnison AM, Janssen PH, Saul D, Rodrigo A, Bergquist PL, Daniel RM, Stackebrandt E, Morgan HW (1994) Description of Caldicellulosiruptor saccharolyticus gen. nov., sp. nov: An

10 obligately anaerobic, extremely thermophilic, cellulolytic bacterium. FEMS Microbiology Letters 120:263-266 Ravot G, Fardeau M-L, Magot M, Patel BKC, Prensier G, Egan A, Garcia J-L, Ollivier B (1995) Thermotoga elfii sp. nov., a novel thermophilic bacterium from an african oil-producing well. Int. J. Syst. Bacteriol. 45:308-314 Saier Jr., Chauvaux MH, Cook S, Deutscher, GM, Paulsen J, Reizer IT, and Ye JJ. (1996) Catabolite repression and inducer control in Gram-positive bacteria. Microbiology 142, 217-230. Saier Jr., MH (1998) Multiple mechanisms controlling carbon metabolism in bacteria. Biotechnol. Bioeng. 58, 170-174 Stams AJM, van Dijk JB, Dijkema C, Plugge CM (1993) Growth of syntrophic propionate-oxidizing bacteria with fumarate in the absence of methanogenic bacteria. Appl. Environ. Microbiol. 59, 1114-1119 Sukhumavasi J, Ohmiya K, Shimizu S, Ueno K (1988) Clostridium josui sp. nov., a cellulolytic, moderate thermophilc species from thai compost. Int. J. Syst. Bacteriol. 38:179-182 Svetlichnyi VA, Svetlichnaya TP (1988) Dictyoglomus turgidus sp. nov., a new extreme thermophilic eubacterium isolated from hot springs in the Uzon volcano crater. Mikrobiologiya 57:435-441 Svetlichnyi VA, Svetlichnaya TP Chernykh NA and Zavarzin GA (1990) Anaerocellum thermophilum gen. nov., sp. nov.: an extremely thermophilic celluloytic eubacterium isolated from hot springs in the Valley of Geysers. Mikrobiologiya 59:871-879 Takahata Y, Nishijima M, Hoaki T, Maryama T (2001). Thermotoga petrophila sp. nov. and Thermotoga naphtophila sp. nov., two hyperthermophilic bacteria from the Kubuki oil reservoir in Niiga, Japan. Int. J. Syst. Evol. Microbiol 51,1901-1909. Taya M, Hinoki H, Yagi T, Kobayashi T (1988) Isolation and characterization of an extremely thermophilic, cellulolytic, anaerobic bacterium. Appl. Microbiol. Biotechnol. 29:474-479 Thattai M and Shraima BI (2003) Metabolic switching in the sugar phosphotransferase system of Escherichia coli. Biophys. J. 85, 744-754 Vrije de T, Haas de GG, Tan GB, Keijsers, ERP and Claassen, PAM (2002) Pretreatment of Miscanthus for hydrogen production by Thermotoga elfii. Int. J. Hydrogen Energy 27, 1381- 1390 Wiegel J, Ljungdahl LG (1981) Thermoanaerobacter ethanolicus gen. nov., sp. nov., a new extreme thermophilic anaerobic bacterium. Arch. Microbiol. 128:343-348 Windberger E, Huber R, Tricone A, Fricke H, Stetter KO (1989) Thermotoga thermarum sp. nov. and Thermotoga neapolitana occuring in African continental solfataric springs. Arch. Microbiol. 151:506-512 Xue Y, Xu Y, Liu Y, Ma Y, Zhou P (2001). Thermoanaerobacter tengcongensis sp. nov., a novel saccharolytic, thermophilic bacterium isolated from a hot spring in Tengcong, China. Int. J. Syst. Evol. Microbiol 51,1335-1341

11