International Journal of Hydrogen Energy 27 (2002) 1391 – 1398
www.elsevier.com/locate/ijhydene
Distinctive properties of high hydrogen producing extreme
thermophiles, Caldicellulosiruptor saccharolyticus and
Thermotoga eli
E.W.J. van Niela; b; ∗ , M.A.W. Buddeb , G.G. de Haasb , F.J. van der Walb ,
P.A.M Claassenb , A.J.M. Stamsa
a Laboratory
b Agrotechnological
for Microbiology, Wageningen University, H. van Suchtelenweg 4, 6703 CT Wageningen, The Netherlands
Research Institute (ATO B.V.), Department of Bioconversion, P.O. Box 17, 6700 AA Wageningen, The Netherlands
Abstract
Growth and hydrogen production by two extreme thermophiles during sugar fermentation was investigated. In cultures of
Caldicellulosiruptor saccharolyticus grown on sucrose and Thermotoga eli 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. eli 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. eli, and appeared to function as sources of carbon, nitrogen and energy. In the absence of tryptone, T. eli converted
26% of the glucose to another by-product, resulting in a lower yield of hydrogen. Growth of T. eli 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
1. Introduction
In view of the abatement of the greenhouse gas carbon
dioxide, there is a globally increasing interest in the replacement of carbonized fuels by hydrogen. The combustion of
hydrogen results in the formation of water with virtually
no other emissions. However, production of hydrogen
from fossil fuels is concurrent with carbon dioxide production. Therefore, the real bene@t for carbon dioxide abate∗ Corresponding author. Agrotechnological Research Institute
(ATO BV), Department of Bioconversion, P.O. Box 17, 6700 AA
Wageningen, The Netherlands. Tel.: +31-317-475315; fax: +31317-475347.
E-mail address: e.w.j.niel@ato.dlo.nl (E.W.J. van Niel).
ment is only obtained when either the fossil fuel derived
carbon dioxide is sequestered or when hydrogen is produced
from renewable resources. Various hydrogen production
techniques exploiting these resources are still under study.
Biological hydrogen production has already been investigated with several types of microorganisms [1–5]. So far,
especially phototrophic microorganisms and mesophilic and
moderate thermophilic heterotrophic microorganisms have
been considered [6], but not thermophiles growing above
◦
60 C. In previous studies with (hyper)thermophiles, hydrogen was considered as an undesired by-product e.g. [7–9].
Interestingly, yields of hydrogen close to the theoretical
stoichiometry, according to the reaction:
Glucose + 2H2 O → 2 acetate + 2CO2 + 4H2
0360-3199/02/$ 22.00 ? 2002 International Association for Hydrogen Energy. Published by Elsevier Science Ltd. All rights reserved.
PII: S 0 3 6 0 - 3 1 9 9 ( 0 2 ) 0 0 1 1 5 - 5
(1)
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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
before with other fermentative microorganisms e.g. [6].
This report presents the biological production of hydrogen from glucose, the main sugar in lignocellulosic and
starchy biomass, and sucrose, the main component from energy crops like Sweet Sorghum and sugar cane. The stoichiometries and hydrogen product yields were determined
for sugar fermentations of two extreme thermophilic bacteria, i.e. Caldicellulosiruptor saccharolyticus [11] and Thermotoga eli [12].
2. Materials and methods
respectively. The bioreactors were inoculated with 100 ml
cultures of either organism (containing ca. 8×108 cells=ml),
which had been pregrown on sucrose or glucose. The cultures of C. saccharolyticus and T. eli were continuously
stirred at a rate of 350 and 100 rpm, respectively, and
sparged with N2 at a Low of 6 –7 and 18 l=h, respectively.
For replacement of yeast extract in the medium of C.
saccharolyticus, casamino acids (5 g=l) with or without additional amino acids (proline, glutamine and cysteine; each
0:5 g=l) were tested in 250-ml crimp seal Lasks containing
a bicarbonate-buMered medium [13]. Tests to investigate
the eMect of yeast extract and tryptone on the growth of
T. eli were performed in 100-ml crimp seal Lasks. All
experiments were done in duplicate.
2.1. Organism
C. saccharolyticus (DSM 8903) and T. eli (DSM 9442)
were purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany).
2.2. Media
The DSM640-medium (http://www.dsmz.de/media/
med640.htm) for C. saccharolyticus was modi@ed to our
requirements and consisted of (per l): K2 HPO4 1:5 g,
KH2 PO4 0:75 g, NH4 Cl 0:9 g, NaCl 0:9 g, MgCl2 ·
6H2 O 0:4 g, DTT 0:2 g, yeast extract 1:5 g, resazurin 1 mg,
vitamin solution 1 ml, trace elements solution 1 ml. Sucrose (10 g=l) was used as the C- and energy source.
The vitamin solution consisted of (mg=l): biotin 20, folic
acid 20, pyridoxine-HCl 100, riboLavine 50, thiamin-HCl
50, nicotinamide 50, cobalamin 50, p-aminobenzoic acid
50, lipoic acid 50, pantothenic acid 50. The trace elements solution consisted of (per l): FeCl2 · 4H2 O 1:5 g,
ZnCl2 70 mg, MnCl2 · 4H2 O 0:1 g, H3 BO3 6 mg, CoCl2 ·
6H2 O 0:19 g, CuCl2 · 2H2 O 2 mg, NiCl2 · 6H2 O 24 mg,
Na2 MoO4 · H2 O 36 mg, Na2 WO4 15 mg, Na2 SeO3 ·
5H2 O 15 mg. T. eli was grown on DSM664-medium
(http://www.dsmz.de/media/med664.htm) with or without tryptone (5 g=l), containing glucose (10 g=l) and a
trace elements solution that is used for methanogens
(http://www.dsmz.de/media/med141.htm). The medium
was adjusted to pH 7.4 prior to autoclaving. Glucose, sodium
carbonate and trace elements were autoclaved separately.
2.3. Cultivation
Batch cultures of C. saccharolyticus and T. eli were
grown in a jacketed 3-l bioreactor (Applikon, The Netherlands) at a working volume of 1 l. The pH was monitored
by an Applikon Biocontroller 1030 and maintained at pH 7
and 7.4, respectively, with 1 N NaOH or KOH, while the
◦
◦
temperature was kept thermostatically at 70 C and 65 C,
2.4. Analyses
Hydrogen was measured with a 406 Packard gas chromatograph 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 14 in, 60 –80
mesh) with argon as carrier gas. Amino acids were determined by HPLC (Pharmacia) on a Nova-Pak C18 column
(250 × 4:6 mm2 ID) and separated and eluted with a gradient of acetonitril (26 –70%) in 35 mM Na-acetate (pH 5.7)
and 4% DMF. The Low rate was 1:0 ml=min and the col◦
umn temperature was 40 C. Prior to separation in the column, the samples were derivatised with dabsylchloride, a
chromophoric compound. The dabsylated products were detected at 436 nm. The quantity of produced hydrogen during
the exponential growth phase was determined by integration of the hydrogen production rate over time. The protein
in the culture Luid was determined according to Bradford
[14]. The optical density of C. saccharolyticus cultures
was measured spectrophotometrically at 620 nm using a Hitachi U-1100 spectrophotometer. Cells were counted under phase contrast with a BOurker–TOurk counting chamber.
A relation between OD and cell number was calculated:
[cell number] = (8:06[OD]620 − 0:49) · 108 cells=ml (R2 =
0:995). An amount of 1012 cells was equivalent to 734 mg
dry weight (DW). The OD of T. eli cultures was measured
at 580 nm using a Pharmacia spectrophotometer. A relation
between OD and cell number was calculated: (3:87[OD]580 +
0:052) · 108 cells=ml (R2 = 0:99). The relation between OD
and cell DW of T. eli was found to be: (0:459 · [OD]580 −
0:076) g DW=l. Sucrose was measured as a reducing sugar
with the anthron=sulfuric acid method [15].
Glucose and organic acids were analyzed by HPLC (ThermoQuest, USA) on a column for organic acids (Polyspher
OAHY, Merck, Germany) and detected by diMerential refractometry. The mobile phase was 0:01 N H2 SO4 , with a
◦
Low rate of 0:6 ml=min. The working temperature was 60 C.
The glucose concentration in the supernatant of T. eli cultures was measured enzymatically using the Biotrol Glucose
Enzymatique Color Kit (Merck).
1393
1.6
40
The biomass yield (YSX , g DW=mol sucrose), and the yield
of acetate (YAX , mol acetate=g DW) were calculated via
1.2
30
0.8
20
0.4
10
(2)
and
(3)
respectively, with X = biomass (mg protein=l), S = sucrose
concentration (mM), A = acetate concentration (mM) and
t = time (h). To @nd a value for the growth rate (h−1 ),
dX=dt = X
(4)
was @tted 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 organisms was assumed to be CH1:8 O0:5 N0:2 .
0
0
0
20
Several carbohydrates were shown to support growth
and hydrogen production by both C. saccharolyticus and
T. eli. The substrates tested ranged from polysaccharides
(cellulose, carboxy methyl cellulose (CMC) and starch),
to disaccharides (cellobiose and sucrose) and monosaccharides (glucose, fructose and xylose). CMC did not support
growth of either microorganism and T. eli failed to grow
on sucrose. The experiments were all done using the media described in Section 2. An attempt to increase the
buMer strength of the medium for T. eli was unsuccessful
because growth was completely inhibited by 50 mM phosphate. This buMer concentration had no eMect 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
kept below 1000 Pa.
3.1. Growth of C. saccharolyticus on sucrose
Growth and hydrogen production by a sucrose fermenting culture of C. saccharolyticus were followed (Fig. 1).
The results of the fermentation are given in Table 1. The
maximum cell density and hydrogen production rate found
at the end of the exponential growth phase were 404 mg
protein=l and 11:7 mmol H2 =g DW=h, respectively. Total
hydrogen production was linearly correlated with cell number (R2 = 0:98), revealing that about 100 mmol of H2 were
being formed per 1012 cells (Table 1). Acetate was the main
by-product of the fermentation.
At the onset of the stationary phase, sucrose was not completely consumed. At that stage, the rates of cell growth and
hydrogen production declined and lactate production started
(Fig. 1). The stoichiometry of the sucrose fermentation during the exponential phase per mole of sucrose was: 3:2 mol
60
80
200
90
150
60
100
30
50
0
0
0
3. Results
40
Time (h)
(a)
Concentration (mM)
dA=dt = YAX dX=dt;
20
40
Time (h)
(b)
60
Hydrogen (mmole/L)
dX=dt = YSX dS=dt
OD
2.5. Determination of yield factors
Protein (mg/L)
E.W.J. van Niel et al. / International Journal of Hydrogen Energy 27 (2002) 1391 – 1398
80
Fig. 1. Growth of C. saccharolyticus in a 1-l batch culture on 10 g
sucrose=l. (A) ( ) Optical density; (♦) protein concentration in
the culture Luid. (B) ( ) sucrose; ( ) hydrogen; (4) acetate;
() lactate.
•
◦
of acetate and 5:9 mol of hydrogen. Taking into account that
14.1% of the sucrose consumed was assimilated, 1 mol of
sucrose was fermented to 4 mol of acetate and 6:6 mol of hydrogen. The molar carbon and hydrogen balance of the consumed sucrose in the stationary phase could be completed:
Sucrose + H2 O → Biomass + Acetate + CO2
+ Lactate + H2 ;
(148) (—)
(21)
(272) (91) (38)
(84)
(42)
(8)
(—)
(168) (—)
(15)
(155)
(5)
(C);
(H):
Like H2 O, the amount of CO2 was not measured, but was
assumed to be produced with acetate at a ratio of 1:1.
Continuous sparging with N2 resulted in the formation
of foam at the start of the exponential phase, indicating the
occurrence of cell lysis. Indeed, from the onset of the exponential growth phase, protein could be detected in the culture
Luid and accumulated to about 35 mg=l (Fig. 1A). From the
increase in OD (Fig. 1A), an apparent speci@c growth rate
of 0:073 h−1 was calculated. Subsequently, values for YSX
and YAX of 45:1 g DW=mol sucrose, 0:07 mol acetate=g DW
were found.
3.2. Growth of T. eli on glucose
Firstly, the growth and hydrogen production of a culture
of T. eli in medium that also contained tryptone were followed (Fig. 2). The results of the fermentation (Table 1) are
similar to those obtained with C. saccharolyticus on sucrose,
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E.W.J. van Niel et al. / International Journal of Hydrogen Energy 27 (2002) 1391 – 1398
Table 1
Fermentation parameters of C. saccharolyticus on sucrose and T. eli on glucose with and without tryptone in the medium
C. saccharolyticus
T. eli
(with tryptone)
T. eli
(without tryptone)
Final
cell density
(109
cells=ml)
Final
dry weight
(mg=l)
MAX
(h−1 )
Productivity
(mmol H2 =l=h)
Speci@c H2
production
(mmol=1012
cells)
1.1
0.8
808
886
0.073
ND
8.4
2.7
100
95
1.0
1082
0.11
4.5
46
2.5
1.5
100
1
50
0.5
0
40
80
2
50
1
25
0
0
120
0
75
OD
2
150
3
100
Concentration
(mmole/L)
200
OD
Concentration (mmole/L)
ND is not determined.
0
0
10
Time (h)
20
Time (h)
30
40
Fig. 2. Growth of T. eli in a 1-l batch culture on 10 g glucose=l.
( ) Optical density; ( ) glucose; ( ) hydrogen; (4) acetate.
Fig. 3. Growth of T. eli in a 1-l batch culture on 10 g glucose=l, but
without tryptone. ( ) Optical density; ( ) glucose; ( ) hydrogen;
(4) acetate.
except for the relatively low hydrogen productivity during
the exponential growth phase (equivalent to 5:1 mmol H2 =g
DW=h). Acetate was the main by-product of the fermentation.
The culture entered the stationary phase before all the
glucose was consumed (Fig. 2). Therefore, accurate values
for the yield factors could not be determined. Instead, estimations for YSX and YAX , being 35 g DW=mol glucose and
0:06 mol acetate=g DW, respectively, were derived from the
available fermentation data. Both hydrogen and acetate continued 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
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
and 3:3 mol of H2 .
The molar carbon and hydrogen balance of the consumed
glucose at the end of the fermentation could be completed,
assuming that assimilation of components from tryptone
and=or yeast extract was insigni@cant:
Here as well the CO2 was not measured, but was assumed
to be produced along with acetate at a ratio of 1:1.
Secondly, growth and hydrogen production were followed in a culture of T. eli in medium without tryptone
(Fig. 3). A higher productivity (8:9 mmol H2 =g DW=h) was
obtained than in the presence of tryptone, but the hydrogen
yield decreased by about 50%. In addition, the stoichiometry changed to 1 mol of glucose: 1:2 mol of acetate and
2:8 mol of H2 . The molar carbon and hydrogen balance of
the consumed glucose at the end of the fermentation could
not be completed:
•
◦
Glucose + H2 O → Biomass + Acetate + CO2 + H2
(300) (—) (36) (176) (88) (—) (C);
(600) (145) (65)
(352) (—)
(291)
(H):
(6)
•
◦
Glucose + H2 O → Biomass + Acetate + CO2 + H2 ;
(205) (—) (44) (73) (36) (—) (C);
(410) (75) (79)
(146) (—)
(170)
(7)
(H):
With an assumed CO2 production equal to acetate, only
74% of the glucose-carbon was found in the products given
in (7) and only 81% of the hydrogen could be retraced in
those products, suggesting that another compound was produced. The analysis for organic acids and alcohols was virtually negative. Further attempts to determine nitrogenous
products failed due to a high background caused by components of yeast extract. Consequently, values for YSX and YAX
of 39:3 g DW=mol glucose, 0:03 mol acetate=g DW were
found.
E.W.J. van Niel et al. / International Journal of Hydrogen Energy 27 (2002) 1391 – 1398
Table 2
EMect of replacements for yeast extract on relative hydrogen production yields and rates of C. saccharolyticus cultures
Medium
components
Hydrogen
production
yield (%)
Hydrogen
production
rate (%)
YE
100
100
CA
29
21
CA + glutamine
28
22
CA + proline
100
72
CA + cysteine
100
72
CA + proline + cysteine
61
52
Yeast extract (YE), casamino acids (CA). Hydrogen production
yield on medium with yeast extract was set to 100%.
3.3. E?ect of medium components on growth of C.
saccharolyticus
C. saccharolyticus did not grow on yeast extract as the
sole C- and energy source. However, in the presence of a
sugar, production of biomass and hydrogen improved when
higher concentrations of yeast extract were added. The eMect
was most pronounced on biomass production, which doubled when yeast extract was increased from 0.05 to 2 g=l.
The eMect on the hydrogen yield was less; it went up from
18.1 to 26:2 mmol=l culture. Without any organic nitrogen
source, C. saccharolyticus grew slowly on sucrose and produced only half the amount of hydrogen as compared to
the control with yeast extract and no lactate was produced
throughout the fermentation.
For optimal growth of C. saccharolyticus, casamino acids
plus vitamins could not replace yeast extract in the medium.
Proline or cysteine appeared to be also required for obtaining
hydrogen yields similar to those found on a complex medium
(Table 2). In the presence of casamino acids, cysteine or
proline alone increased the hydrogen production rate, but
when combined, the production rate was augmented to a
lesser extent (Table 2).
3.4. E?ect of medium components on growth of T. eli
An attempt was made to replace yeast extract by a combination of casamino acids and a vitamins solution, but even
when this medium was supplemented with the amino acids
that are absent in casamino acids, growth remained virtually nil (Table 3). The hydrogen production observed in the
absence of yeast extract could be fully accounted for by
the carry-over from the precultures. T. eli appeared to be
completely dependent on a medium enriched with yeast extract. Furthermore, T. eli showed considerable growth and
hydrogen production on yeast extract alone. This was further tested using media with various concentrations of yeast
extract, tryptone, and glucose (Table 4). Without glucose,
doubling of cell density and hydrogen production was only
obtained when the concentration of both yeast extract and
1395
tryptone were more than doubled. At low concentrations of
yeast extract and without tryptone, the production remained
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 insuTcient to sustain productivity, unless 2 g tryptone=l was added. Higher
concentrations of both nitrogen sources did not improve fermentation performance signi@cantly (Table 4).
4. Discussion
About 83% of the theoretically obtainable hydrogen (i.e.
4 mol of H2 per 1 mol of glucose) is produced by C. saccharolyticus and T. eli cultures during sugar fermentation.
This is comparable to results found with T. maritima on
glucose [8] and P. furiosus on maltose [10]. The results are
superior to what is normally found for hydrogen production during sugar fermentation. Frequently, 25 –50% of the
theoretical hydrogen yield are obtained from similar sugar
fermentations with mesophilic and moderate thermophilic
microorganisms [6,16]. The results with (hyper)thermophiles
agree with the fact that hydrogen production becomes more
exergonic with increasing temperatures [17], showing one
of the advantages of applying these organisms for hydrogen
production. This also applies for the reaction of acetate to
hydrogen and carbon dioxide, though it is still constraint
thermodynamically. A complete conversion of glucose to
12 hydrogen and 6 carbon dioxide is possible at elevated
◦
temperatures (e.g. 70 C) provided that the pH2 is kept
below 100 Pa [18].
With C. saccharolyticus and T. eli maximum cell densities in the order of 109 cells=ml could be reached. Maximum
cell concentrations one order of magnitude lower have normally been found for (hyper)thermophiles [7,8,10]. An explanation for these relatively low cell concentrations is still
lacking. It might be that nutrients other than the substrate
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
in spent medium [19]. It was argued that some type of cell
density inhibition was the cause of low maximum cell numbers. In our case, growth of T. eli on glucose ceased prematurely, while the fermentation continued until all glucose
was consumed. A similar phenomenon has been found for
the growth of Clostridium cellulolyticum on cellobiose [20],
which could be traced to an ineTciently regulated carbon
Low. Although this could explain the low cell numbers of
T. eli, more study is necessary to elucidate the mechanism
behind its ineTcient growth. In the case of C. saccharolyticus, low cell numbers could be partly explained by cell lysis.
According to the protein concentrations at the start of the stationary phase, in total about 7.9% of the biomass had lysed
during the exponential growth phase. It has been established
that cell lysis is caused by salts, such as sodium acetate,
that slowly accumulate in the fermentation broth [13].
1396
E.W.J. van Niel et al. / International Journal of Hydrogen Energy 27 (2002) 1391 – 1398
Table 3
EMect of replacements for yeast extract on hydrogen yields and OD in cultures of T. eli with and without glucose
Hydrogen production yield (%)
OD580
Medium components
Glucose
(4 g=l)
Without
glucose
Glucose
(4 g=l)
Without
glucose
Yeast extract
Casamino acids+vitamin solution
Casamino acids +vitamin solution+ amino acid supplementa
100
14
14
40
4
6
0.52
0.03
0.03
0.32
0.04
0
a The
amino acid supplement was cysteine, alanine, asparagine, proline, glutamine, serine and tryptophan, added at 0:2 g=l each.
Hydrogen production yield and @nal OD at the end of the exponential growth phase.
Table 4
EMect of the concentrations of yeast extract and=or tryptone on the growth and hydrogen production by T. eli at the end of the fermentation
Medium components(g=l)
Consumed (mmol=l)
Produced (mmol=l medium)
Glucose
Yeast
extract
Tryptone
OD580
Glucose
Hydrogen
Acetate
—
—
—
—
2
2
5
5
0
2
0
5
0.152
0.171
0.209
0.327
—
—
—
—
13.9
14.8
14.0
28.8
3.5
3.4
—
4.9
10
10
10
10
2
2
5
5
0
2
0
5
0.301
0.783
0.759
0.962
10.3
18.3
13.1
17.9
25.8
78.5
84.9
82.5
10.7
19.7
26.3
21.2
The molar growth yield of C. saccharolyticus on sucrose
(YSX ) was about 45 g DW=mol sucrose. Assuming a value
for YATP of 10 g DW=mol ATP [21], the biomass yield indicates an ATP yield of about 4 mol ATP=mol sucrose. Hence,
the YATP is ca. 11:3 g DW=mol ATP, which agrees well with
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
acetate produced. However, applying the same calculation
to the yield factors obtained with T. eli, an ATP yield of 3.5
– 4 mol=mol glucose is found, which is twice the theoretical
maximum. This implies not only that T. eli uses another
C- and energy source in addition to glucose, but also that the
carbon balances cannot be completed. Determination of the
other carbon sources used and the identity of other products
formed was not possible due to the high background caused
by the myriad compounds in yeast extract and tryptone. It
is known that T. eli can produce high quantities of alanine
[22], and this may very well be the product formed in the
fermentation on glucose in the absence of tryptone.
In this study maximum speci@c hydrogen production rates
of 5 –12 mmol H2 =g DW=h were found. This is about 2–5
times lower than for T. maritima (26:9 mmol H2 =g DW=h),
which was calculated from the data of SchrOoder et al [8]. The
latter culture had a cell density in the order of 108 cells=ml.
It might be possible that such low culture densities can reach
high speci@c rates, because of lower dissolved hydrogen
concentrations. High cell densities are aMected earlier by
dissolved hydrogen, because they produce more hydrogen
per unit volume [13] and may cause retention of hydrogen
in the liquid [23].
Yeast extract was required for the optimal growth of C.
saccharolyticus, but did not act as a primary C- and energy
source. Interestingly, in the absence of the organic nitrogen,
no lactate was produced, which could partially explain the
non-proportional increase of hydrogen production in these
cultures. Since lactate acts as a sink for electrons, the increase in hydrogen production will suMer from this dissipation of reducing power. It is unclear whether the switch to
lactate, often observed at high hydrogen concentrations in
cultures of C. saccharolyticus [13], resulted from the availability of organic nitrogen or from the supplementation with
vitamins.
Cultures of C. saccharolyticus reached identical hydrogen production yields when yeast extract was replaced by
casamino acids provided that in addition either proline or
cysteine was present. The addition of both proline and
cysteine was less successful, probably because of an antagonistic eMect. 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 possibly be further increased by micronutrients such as nucleic
acid bases.
The lack of growth of T. eli on sucrose is in contrast to
the @ndings of Ravot et al. [12], who observed growth on
sucrose in the presence of thiosulfate as electron acceptor.
However, since growth on sucrose was determined by following the increase of OD, the latter @nding may have been
biased by the utilisation of yeast extract by T. eli for growth
and hydrogen production. It was found that growth of T. eli
was completely dependent on enrichment with yeast extract
as a source of carbon, nitrogen and energy. Yeast extract
could not be replaced by amino acids and vitamins alone
(Table 3), which is in agreement with the observations of
Ravot et al. [12]. Apparently, other micronutrients in yeast
extract are essential for growth and hydrogen production of
T. eli. Besides yeast extract, tryptone also improved fermentation. The amino acid composition of the latter nitrogen source is fairly similar to that of yeast extract, except
for proline, which is twice as high in tryptone. Moreover, in
contrast to yeast extract, tryptone is rich in peptides, which
may be, as seen with many lactic acid bacteria [24], the preferred form of amino acids by T. eli. So far, attempts to
elucidate or quantify the amino acid conversion to ammonia,
hydrogen, and acetate did not succeed. The quanti@cation of
ammonia mobilisation has been hampered by the seemingly
instantaneous consumption for biomass production.
Acknowledgements
This work was funded by a grant from the European Commission (Proposal No. QLRT-1999-01267). We thank Wim
Roelofsen and Giel van Noorden for technical assistance
during these investigations.
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