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Pii: s0360-3199(02)00115-5International Journal of Hydrogen Energy 27 (2002) 1391–1398 Distinctive properties of high hydrogen producing extreme thermophiles, Caldicellulosiruptor saccharolyticus and E.W.J. van Niela;b; ∗, M.A.W. Buddeb, G.G. de Haasb, F.J. van der Walb, aLaboratory for Microbiology, Wageningen University, H. van Suchtelenweg 4, 6703 CT Wageningen, The Netherlands bAgrotechnological Research Institute (ATO B.V.), Department of Bioconversion, P.O. Box 17, 6700 AA Wageningen, The Netherlands Growth and hydrogen production by two extreme thermophiles during sugar fermentation was investigated. In cultures of Caldicellulosiruptor saccharolyticus grown on sucrose and Thermotoga elÿi grown on glucose stoichiometries of 3:3 mol of hydrogen and 2 mol of acetate per mol C6-sugar unit were obtained. The hydrogen level was about 83% of the theoretical maximum. C. saccharolyticus and T. elÿi reached maximum cell densities of 1:1 × 109 and 0:8 × 109 cells=ml, respectively, while their maximum hydrogen production rates were 11.7 and 5:1 mmol=g dry weight=h, respectively. For growth of C.
saccharolyticus on sucrose, a biomass yield of 45:1 g DW=mol sucrose and a YATP of 11.3–14.1 were calculated. Replacement of yeast extract by casamino acids, plus proline and vitamins in the medium of C. saccharolyticus resulted in similar yields of hydrogen production on sucrose, but diminished the rate by about 30%. Both yeast extract and tryptone were required by T. elÿi, and appeared to function as sources of carbon, nitrogen and energy. In the absence of tryptone, T. elÿi converted 26% of the glucose to another by-product, resulting in a lower yield of hydrogen. Growth of T. elÿi ceased prior to glucose depletion, but the culture continued to ferment glucose to hydrogen and acetate until all glucose was consumed.
? 2002 International Association for Hydrogen Energy. Published by Elsevier Science Ltd. All rights reserved.
Keywords: Extreme thermophile; Hydrogen production; Sugar fermentation; Yields ment is only obtained when either the fossil fuel derived carbon dioxide is sequestered or when hydrogen is produced In view of the abatement of the greenhouse gas carbon from renewable resources. Various hydrogen production dioxide, there is a globally increasing interest in the replace- techniques exploiting these resources are still under study.
ment of carbonized fuels by hydrogen. The combustion of Biological hydrogen production has already been investi- hydrogen results in the formation of water with virtually gated with several types of microorganisms [1–5]. So far, no other emissions. However, production of hydrogen especially phototrophic microorganisms and mesophilic and from fossil fuels is concurrent with carbon dioxide produc- moderate thermophilicheterotrophicmicroorganisms have tion. Therefore, the real beneÿt for carbon dioxide abate- been considered , but not thermophiles growing above 60◦C. In previous studies with (hyper)thermophiles, hydro- ∗ Corresponding author. Agrotechnological Research Institute gen was considered as an undesired by-product e.g. [7–9].
(ATO BV), Department of Bioconversion, P.O. Box 17, 6700 AA Interestingly, yields of hydrogen close to the theoretical Wageningen, The Netherlands. Tel.: +31-317-475315; fax: +31- stoichiometry, according to the reaction: E-mail address: [email protected] (E.W.J. van Niel).
Glucose + 2H2O → 2 acetate + 2CO2 + 4H2 0360-3199/02/$ 22.00 ? 2002 International Association for Hydrogen Energy. Published by Elsevier Science Ltd. All rights reserved.
E.W.J. van Niel et al. / International Journal of Hydrogen Energy 27 (2002) 1391–1398 were obtained [8,10], which are superior to yields found respectively. The bioreactors were inoculated with 100 ml before with other fermentative microorganisms e.g. .
cultures of either organism (containing ca. 8×108 cells=ml), This report presents the biological production of hydro- which had been pregrown on sucrose or glucose. The cul- gen from glucose, the main sugar in lignocellulosic and tures of C. saccharolyticus and T. elÿi were continuously starchy biomass, and sucrose, the main component from en- stirred at a rate of 350 and 100 rpm, respectively, and ergy crops like Sweet Sorghum and sugar cane. The stoi- sparged with N2 at a ow of 6–7 and 18 l=h, respectively.
chiometries and hydrogen product yields were determined For replacement of yeast extract in the medium of C.
for sugar fermentations of two extreme thermophilicbacte- saccharolyticus, casamino acids (5 g=l) with or without ad- ria, i.e. Caldicellulosiruptor saccharolyticus  and Ther- ditional amino acids (proline, glutamine and cysteine; each 0:5 g=l) were tested in 250-ml crimp seal asks containing a bicarbonate-bu ered medium . Tests to investigate the e ect of yeast extract and tryptone on the growth of T. elÿi were performed in 100-ml crimp seal asks. All C. saccharolyticus (DSM 8903) and T. elÿi (DSM 9442) were purchased from the Deutsche Sammlung von Mikroor- Hydrogen was measured with a 406 Packard gas chro- ganismen und Zellkulturen (Braunschweig, Germany).
matograph equipped with a thermal conductivity detector (TCD, 100 mA). The gases were separated at 100◦C on a molecular sieve column (13×; 180 cm by 1 in, 60–80 mesh) with argon as carrier gas. Amino acids were deter- mined by HPLC (Pharmacia) on a Nova-Pak C18 column (250 × 4:6 mm2 ID) and separated and eluted with a gradi- for C. saccharolyticus was modiÿed to our ent of acetonitril (26–70%) in 35 mM Na-acetate (pH 5.7) requirements and consisted of (per l): K2HPO4 1:5 g, and 4% DMF. The ow rate was 1:0 ml=min and the col- KH2PO4 0:75 g, NH4Cl 0:9 g, NaCl 0:9 g, MgCl2 · umn temperature was 40◦C. Prior to separation in the col- 6H2O 0:4 g, DTT 0:2 g, yeast extract 1:5 g, resazurin 1 mg, umn, the samples were derivatised with dabsylchloride, a vitamin solution 1 ml, trace elements solution 1 ml. Su- chromophoric compound. The dabsylated products were de- crose (10 g=l) was used as the C- and energy source.
tected at 436 nm. The quantity of produced hydrogen during The vitamin solution consisted of (mg=l): biotin 20, folic the exponential growth phase was determined by integra- acid 20, pyridoxine-HCl 100, ribo avine 50, thiamin-HCl tion of the hydrogen production rate over time. The protein 50, nicotinamide 50, cobalamin 50, p-aminobenzoicacid in the culture uid was determined according to Bradford 50, lipoic acid 50, pantothenic acid 50. The trace ele- . The optical density of C. saccharolyticus cultures ments solution consisted of (per l): FeCl2 · 4H2O 1:5 g, was measured spectrophotometrically at 620 nm using a Hi- ZnCl2 70 mg, MnCl2 · 4H2O 0:1 g, H3BO3 6 mg, CoCl2 · tachi U-1100 spectrophotometer. Cells were counted un- 6H2O 0:19 g, CuCl2 · 2H2O 2 mg, NiCl2 · 6H2O 24 mg, der phase contrast with a Burker–Turk counting chamber.
Na2MoO4 · H2O 36 mg, Na2WO4 15 mg, Na2SeO3 · A relation between OD and cell number was calculated: 5H2O 15 mg. T. elÿi was grown on DSM664-medium [cell number] = (8:06[OD]620 − 0:49) · 108 cells=ml (R2 = 0:995). An amount of 1012 cells was equivalent to 734 mg out tryptone (5 g=l), containing glucose (10 g=l) and a dry weight (DW). The OD of T. elÿi cultures was measured trace elements solution that is used for methanogens at 580 nm using a Pharmacia spectrophotometer. A relation between OD and cell number was calculated: (3:87[OD]580+ was adjusted to pH 7.4 prior to autoclaving. Glucose, sodium 0:052) · 108 cells=ml (R2 = 0:99). The relation between OD carbonate and trace elements were autoclaved separately.
and cell DW of T. elÿi was found to be: (0:459 · [OD]580 − 0:076) g DW=l. Sucrose was measured as a reducing sugar with the anthron=sulfuricacid method .
Glucose and organic acids were analyzed by HPLC (Ther- moQuest, USA) on a column for organic acids (Polyspher Batch cultures of C. saccharolyticus and T. elÿi were OAHY, Merck, Germany) and detected by di erential re- grown in a jacketed 3-l bioreactor (Applikon, The Nether- fractometry. The mobile phase was 0:01 N H2SO4, with a lands) at a working volume of 1 l. The pH was monitored ow rate of 0:6 ml=min. The working temperature was 60◦C.
by an Applikon Biocontroller 1030 and maintained at pH 7 The glucose concentration in the supernatant of T. elÿi cul- and 7.4, respectively, with 1 N NaOH or KOH, while the tures was measured enzymatically using the Biotrol Glucose temperature was kept thermostatically at 70◦C and 65◦C, E.W.J. van Niel et al. / International Journal of Hydrogen Energy 27 (2002) 1391–1398 The biomass yield (YSX, g DW=mol sucrose), and the yield of acetate (YAX, mol acetate=g DW) were calculated via Protein (mg/L)
respectively, with X = biomass (mg protein=l), S = sucrose concentration (mM), A = acetate concentration (mM) and t = time (h). To ÿnd a value for the growth rate (h−1), was ÿtted through the data points of biomass. The growth rate and yield factors were found by applying the least-squares regression method (with Excel, Microsoft).
The elemental composition of the biomass of both organ- isms was assumed to be CH1:8O0:5N0:2.
Several carbohydrates were shown to support growth Fig. 1. Growth of C. saccharolyticus in a 1-l batch culture on 10 g and hydrogen production by both C. saccharolyticus and sucrose=l. (A) (•) Optical density; (♦) protein concentration in the culture uid. (B) ( ) sucrose; (◦) hydrogen; (4) acetate; T. elÿi. The substrates tested ranged from polysaccharides (cellulose, carboxy methyl cellulose (CMC) and starch), to disaccharides (cellobiose and sucrose) and monosaccha- rides (glucose, fructose and xylose). CMC did not support of acetate and 5:9 mol of hydrogen. Taking into account that growth of either microorganism and T. elÿi failed to grow 14.1% of the sucrose consumed was assimilated, 1 mol of on sucrose. The experiments were all done using the me- sucrose was fermented to 4 mol of acetate and 6:6 mol of hy- dia described in Section 2. An attempt to increase the drogen. The molar carbon and hydrogen balance of the con- bu er strength of the medium for T. elÿi was unsuccessful sumed sucrose in the stationary phase could be completed: because growth was completely inhibited by 50 mM phos- Sucrose + H2O → Biomass+Acetate+CO2 phate. This bu er concentration had no e ect on growth and hydrogen production of C. saccharolyticus cultures, but stabilized the pH. Since the cultures in the reactor were sparged with N2 the partial hydrogen pressure (pH2) was (272) (91) (38) (168) (—) (15) (155) (H): Like H2O, the amount of CO2 was not measured, but was 3.1. Growth of C. saccharolyticus on sucrose assumed to be produced with acetate at a ratio of 1:1.
Continuous sparging with N2 resulted in the formation Growth and hydrogen production by a sucrose ferment- of foam at the start of the exponential phase, indicating the ing culture of C. saccharolyticus were followed (Fig. 1).
occurrence of cell lysis. Indeed, from the onset of the expo- The results of the fermentation are given in Table 1. The nential growth phase, protein could be detected in the culture maximum cell density and hydrogen production rate found uid and accumulated to about 35 mg=l (Fig. 1A). From the at the end of the exponential growth phase were 404 mg increase in OD (Fig. 1A), an apparent speciÿc growth rate protein=l and 11:7 mmol H2=g DW=h, respectively. Total of 0:073 h−1 was calculated. Subsequently, values for YSX hydrogen production was linearly correlated with cell num- and YAX of 45:1 g DW=mol sucrose, 0:07 mol acetate=g DW ber (R2 = 0:98), revealing that about 100 mmol of H2 were being formed per 1012 cells (Table 1). Acetate was the main At the onset of the stationary phase, sucrose was not com- pletely consumed. At that stage, the rates of cell growth and Firstly, the growth and hydrogen production of a culture hydrogen production declined and lactate production started of T. elÿi in medium that also contained tryptone were fol- (Fig. 1). The stoichiometry of the sucrose fermentation dur- lowed (Fig. 2). The results of the fermentation (Table 1) are ing the exponential phase per mole of sucrose was: 3:2 mol similar to those obtained with C. saccharolyticus on sucrose, E.W.J. van Niel et al. / International Journal of Hydrogen Energy 27 (2002) 1391–1398 Fermentation parameters of C. saccharolyticus on sucrose and T. elÿi on glucose with and without tryptone in the medium (mmole/L)
Fig. 2. Growth of T. elÿi in a 1-l batch culture on 10 g glucose=l.
Fig. 3. Growth of T. elÿi in a 1-l batch culture on 10 g glucose=l, but (•) Optical density; ( ) glucose; (◦) hydrogen; (4) acetate.
without tryptone. (•) Optical density; ( ) glucose; (◦) hydrogen; except for the relatively low hydrogen productivity during Here as well the CO2 was not measured, but was assumed the exponential growth phase (equivalent to 5:1 mmol H2=g to be produced along with acetate at a ratio of 1:1.
DW=h). Acetate was the main by-product of the fermenta- Secondly, growth and hydrogen production were fol- lowed in a culture of T. elÿi in medium without tryptone The culture entered the stationary phase before all the (Fig. 3). A higher productivity (8:9 mmol H2=g DW=h) was glucose was consumed (Fig. 2). Therefore, accurate values obtained than in the presence of tryptone, but the hydrogen for the yield factors could not be determined. Instead, esti- yield decreased by about 50%. In addition, the stoichiom- mations for YSX and YAX, being 35 g DW=mol glucose and etry changed to 1 mol of glucose: 1:2 mol of acetate and 0:06 mol acetate=g DW, respectively, were derived from the 2:8 mol of H2. The molar carbon and hydrogen balance of available fermentation data. Both hydrogen and acetate con- the consumed glucose at the end of the fermentation could tinued to be produced until all glucose was consumed. From the decline in optical density it was assumed that the cells lysed, but since there were high concentrations of tryptone Glucose + H2O → Biomass + Acetate + CO2 + H2; present, this could not be proven by protein determination.
The stoichiometry of the glucose fermentation during the exponential phase was per mole of glucose: 2 mol of acetate With an assumed CO2 production equal to acetate, only The molar carbon and hydrogen balance of the consumed 74% of the glucose-carbon was found in the products given glucose at the end of the fermentation could be completed, in (7) and only 81% of the hydrogen could be retraced in assuming that assimilation of components from tryptone those products, suggesting that another compound was pro- duced. The analysis for organic acids and alcohols was vir- tually negative. Further attempts to determine nitrogenous 2O → Biomass + Acetate + CO2 + H2 products failed due to a high background caused by compo- nents of yeast extract. Consequently, values for YSX and YAX of 39:3 g DW=mol glucose, 0:03 mol acetate=g DW were E.W.J. van Niel et al. / International Journal of Hydrogen Energy 27 (2002) 1391–1398 tryptone were more than doubled. At low concentrations of E ect of replacements for yeast extract on relative hydrogen pro- yeast extract and without tryptone, the production remained duction yields and rates of C. saccharolyticus cultures low, suggesting that tryptone also acted as a carbon- and energy source similar to yeast extract. In the presence of glucose, 2 g yeast extract=l seemed to be insu cient to sus- tain productivity, unless 2 g tryptone=l was added. Higher concentrations of both nitrogen sources did not improve fer- mentation performance signiÿcantly (Table 4).
Yeast extract (YE), casamino acids (CA). Hydrogen production About 83% of the theoretically obtainable hydrogen (i.e.
yield on medium with yeast extract was set to 100%.
4 mol of H2 per 1 mol of glucose) is produced by C. sac- charolyticus and T. elÿi cultures during sugar fermentation.
This is comparable to results found with T. maritima on 3.3. E ect of medium components on growth of C.
glucose  and P. furiosus on maltose . The results are superior to what is normally found for hydrogen produc- tion during sugar fermentation. Frequently, 25–50% of the C. saccharolyticus did not grow on yeast extract as the theoretical hydrogen yield are obtained from similar sugar sole C- and energy source. However, in the presence of a fermentations with mesophilicand moderate thermophilic sugar, production of biomass and hydrogen improved when microorganisms [6,16]. The results with (hyper)thermophiles higher concentrations of yeast extract were added. The e ect agree with the fact that hydrogen production becomes more was most pronounced on biomass production, which dou- exergonicwith increasing temperatures , showing one bled when yeast extract was increased from 0.05 to 2 g=l.
of the advantages of applying these organisms for hydrogen The e ect on the hydrogen yield was less; it went up from production. This also applies for the reaction of acetate to 18.1 to 26:2 mmol=l culture. Without any organic nitrogen hydrogen and carbon dioxide, though it is still constraint source, C. saccharolyticus grew slowly on sucrose and pro- thermodynamically. A complete conversion of glucose to duced only half the amount of hydrogen as compared to 12 hydrogen and 6 carbon dioxide is possible at elevated the control with yeast extract and no lactate was produced temperatures (e.g. 70◦C) provided that the pH For optimal growth of C. saccharolyticus, casamino acids With C. saccharolyticus and T. elÿi maximum cell densi- plus vitamins could not replace yeast extract in the medium.
ties in the order of 109 cells=ml could be reached. Maximum Proline or cysteine appeared to be also required for obtaining cell concentrations one order of magnitude lower have nor- hydrogen yields similar to those found on a complex medium mally been found for (hyper)thermophiles [7,8,10]. An ex- (Table 2). In the presence of casamino acids, cysteine or planation for these relatively low cell concentrations is still proline alone increased the hydrogen production rate, but lacking. It might be that nutrients other than the substrate when combined, the production rate was augmented to a were limiting, or that one or more fermentation products became inhibiting. However, this was seemingly disproved when fresh cultures of P. furiosus were successfully grown 3.4. E ect of medium components on growth of T. elÿi in spent medium . It was argued that some type of cell density inhibition was the cause of low maximum cell num- An attempt was made to replace yeast extract by a combi- bers. In our case, growth of T. elÿi on glucose ceased pre- nation of casamino acids and a vitamins solution, but even maturely, while the fermentation continued until all glucose when this medium was supplemented with the amino acids was consumed. A similar phenomenon has been found for that are absent in casamino acids, growth remained virtu- the growth of Clostridium cellulolyticum on cellobiose , ally nil (Table 3). The hydrogen production observed in the which could be traced to an ine ciently regulated carbon absence of yeast extract could be fully accounted for by ow. Although this could explain the low cell numbers of the carry-over from the precultures. T. elÿi appeared to be T. elÿi, more study is necessary to elucidate the mechanism completely dependent on a medium enriched with yeast ex- behind its ine cient growth. In the case of C. saccharolyti- tract. Furthermore, T. elÿi showed considerable growth and cus, low cell numbers could be partly explained by cell lysis.
hydrogen production on yeast extract alone. This was fur- According to the protein concentrations at the start of the sta- ther tested using media with various concentrations of yeast tionary phase, in total about 7.9% of the biomass had lysed extract, tryptone, and glucose (Table 4). Without glucose, during the exponential growth phase. It has been established doubling of cell density and hydrogen production was only that cell lysis is caused by salts, such as sodium acetate, obtained when the concentration of both yeast extract and that slowly accumulate in the fermentation broth .
E.W.J. van Niel et al. / International Journal of Hydrogen Energy 27 (2002) 1391–1398 E ect of replacements for yeast extract on hydrogen yields and OD in cultures of T. elÿi with and without glucose Casamino acids +vitamin solution+ amino acid supplementa aThe amino acid supplement was cysteine, alanine, asparagine, proline, glutamine, serine and tryptophan, added at 0:2 g=l each.
Hydrogen production yield and ÿnal OD at the end of the exponential growth phase.
E ect of the concentrations of yeast extract and=or tryptone on the growth and hydrogen production by T. elÿi at the end of the fermentation The molar growth yield of C. saccharolyticus on sucrose It might be possible that such low culture densities can reach (YSX) was about 45 g DW=mol sucrose. Assuming a value high speciÿc rates, because of lower dissolved hydrogen for YATP of 10 g DW=mol ATP , the biomass yield indi- concentrations. High cell densities are a ected earlier by cates an ATP yield of about 4 mol ATP=mol sucrose. Hence, dissolved hydrogen, because they produce more hydrogen the YATP is ca. 11:3 g DW=mol ATP, which agrees well with per unit volume  and may cause retention of hydrogen the reciprocal of YAX (14:1 g DW=mol acetate), implying that the net energy gain on sucrose is equal to 1 ATP per 1 Yeast extract was required for the optimal growth of C.
acetate produced. However, applying the same calculation saccharolyticus, but did not act as a primary C- and energy to the yield factors obtained with T. elÿi, an ATP yield of 3.5 source. Interestingly, in the absence of the organic nitrogen, –4 mol=mol glucose is found, which is twice the theoretical no lactate was produced, which could partially explain the maximum. This implies not only that T. elÿi uses another non-proportional increase of hydrogen production in these C- and energy source in addition to glucose, but also that the cultures. Since lactate acts as a sink for electrons, the in- carbon balances cannot be completed. Determination of the crease in hydrogen production will su er from this dissipa- other carbon sources used and the identity of other products tion of reducing power. It is unclear whether the switch to formed was not possible due to the high background caused lactate, often observed at high hydrogen concentrations in by the myriad compounds in yeast extract and tryptone. It cultures of C. saccharolyticus , resulted from the avail- is known that T. elÿi can produce high quantities of alanine ability of organicnitrogen or from the supplementation with , and this may very well be the product formed in the fermentation on glucose in the absence of tryptone.
Cultures of C. saccharolyticus reached identical hydro- In this study maximum speciÿc hydrogen production rates gen production yields when yeast extract was replaced by of 5–12 mmol H2=g DW=h were found. This is about 2–5 casamino acids provided that in addition either proline or times lower than for T. maritima (26:9 mmol H2=g DW=h), cysteine was present. The addition of both proline and which was calculated from the data of Schroder et al . The cysteine was less successful, probably because of an antag- latter culture had a cell density in the order of 108 cells=ml.
onistic e ect. However, in all alternatives to yeast extract, E.W.J. van Niel et al. / International Journal of Hydrogen Energy 27 (2002) 1391–1398 the hydrogen production rates were lower, but could possi-  Brown SH, Kelly RM. Cultivation techniques for bly be further increased by micronutrients such as nucleic hyperthermophilic Archaebacteria: continuous culture of Pyrococcus furiosus at temperatures near 100◦C. Appl The lack of growth of T. elÿi on sucrose is in contrast to the ÿndings of Ravot et al. , who observed growth on  Schroder C, Selig M, Schonheit P. Glucose fermentation sucrose in the presence of thiosulfate as electron acceptor.
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