international journal of hydrogen energy 34 (2009) 12331243

Available at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/he

Feasibility of biohydrogen production at low temperatures

in unbuffered reactors

Venkataramana Gadhamshettya,*, David C. Johnsonb, Nagamany Nirmalakhandanc,

Geoff B. Smithd, Shuguang Denge
a
Air Force Research Laboratory, Tyndall AFB, 139 Barnes Drive, Panama City, FL 32403, USA
b
Institute for Energy and Environment, New Mexico State University, Las Cruces, NM 88003, USA
c
Civil Engineering Department, New Mexico State University, Las Cruces, NM 88003, USA
d
Biology Department, New Mexico State University, Las Cruces, NM 88003, USA
e
Chemical Engineering Department, New Mexico State University, Las Cruces, NM 88003, USA

article info abstract

Article history: Feasibility of biohydrogen production by dark fermentation at two temperatures (22  C and
Received 28 August 2008 37  C) in unbuffered batch reactors was evaluated using heat-treated compost as inocula
Received in revised form and sucrose as substrate, without any initial pH adjustment or inorganic nutrient
6 October 2008 supplements. Gas production was quantified by two different pressure release methods
Accepted 9 October 2008 intermittent pressure release (IPR) and continuous pressure release (CPR). Hydrogen
Available online 31 December 2008 production (47.2 mL/g COD/L) and sucrose-to-hydrogen conversion efficiency (53%) were
both found to be highest at the lower temperature and IPR conditions. Hydrogen produc-
Keywords: tion was higher at the lower temperature irrespective of the pressure release condition.
Biohydrogen The high yield of 4.3 mol of hydrogen/mole of sucrose obtained in this study under IPR
Intermittent pressure release conditions at 22  C is equivalent to or better than the literature values reported for buffered
Temperature reactors. Even though literature reports have implied potential inhibition of hydrogen
Anaerobic fermentation production at high hydrogen partial pressures resulting from IPR conditions, our results did
Gibbs free energy not show any negative effects at hydrogen partial pressures exceeding 5.0  104 Pa. While
Unbuffered our findings are contrary to literature reports, they make a strong case for cost-effective
Bioenergy hydrogen production by dark fermentation.
2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights
reserved.

1. Introduction cheaper and environment-friendly approach [2,3]. The dark

Hydrogen has been identified as a renewable, energy-efficient,
and pollution-free energy carrier that has the potential to
replace the nonrenewable fossil fuels of today. Currently,
hydrogen is produced by chemical, thermal, and electrical
processes, which are neither sustainable nor cost-effective [1].
Recent studies have demonstrated biological means of
fermentation process, in particular, has potential for cost-
effective biohydrogen production in that, it can utilize organic
wastes as feedstock [4].
Theoretically, a yield of 8 mol of hydrogen can be obtained
from 1 mol of sucrose according to reactions [5]:
0
C12 H22 O11 9H2 0 / 4AC 8H 4HCO
3 8H2 DG ;

producing hydrogenbiohydrogen may be sustainable, 457:5 kJ=mol (1)

* Corresponding author. Tel.: 1 850 283 6721; fax: 1 850 283 6509.
E-mail address: vgadhamshetty@fairpoint.net (V. Gadhamshetty).
0360-3199/$ see front matter 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.ijhydene.2008.10.037
1234 international journal of hydrogen energy 34 (2009) 12331243
0
C12 H22 O11 5H2 0 / 2BU 6H 4HCO
3 4H2 DG
554:1 kJ=mol (2)
In practice, however, the net yield may be lower due to the
production of other end products such as acetic acid, butyric
acid, propionic acid, etc. When mixed cultures are utilized in
biohydrogen production, the reaction pathways and the
resulting byproducts can change unpredictably depending on
reactor conditions such as pH, temperature, hydrogen partial
pressure, nutrient levels, mixing intensity, etc. [68]. Despite
several experimental investigations [9], effects of some of the
above parameters on biohydrogen production are not fully
documented [10]. The goal of this study was to assess the
impact of the above conditions and evaluate the feasibility of
the hydrogen production by the dark fermentation in unbuf-
fered reactors at ambient temperatures, so that the economics
of the process could be improved.
1.1. Effect of pH
pH can inhibit hydrogenase activity causing shift in metabolic
pathways from hydrogen/acid production phase to a solvent
production phase [11,12]. According to Byung and Zeikus [13]
this shift is supposed to occur at pH of 4.5 while Gottwald and
Gottschalk [14] have suggested a pH of 5.7. Van Ginkel et al.
[15] have suggested an optimum pH of 5.5; while Wu et al. [16]
have recommended a range of 67. Lin and Fang [17] reviewed
more than 160 experiments to summarize 22 optimal pH
values reported in the literature, and attributed these differ-
ences to variations in bacterial strains, types of substrate,
temperature, experiment duration, etc. While most experi-
ments have been carried out with pure cultures and with
initial pH adjustments at higher temperatures, only a limited
number of reports from unbuffered reactors have been pub-
lished [17].
1.2. Effect of temperature
Temperature can affect substrate degradation, hydrogen
production, metabolite product distribution, and bacterial
growth [18]. Even though higher temperatures favor reaction
kinetics, rapid changes in pH may inhibit hydrogen producing
bacteria [5]. While buffers have been used to mitigate these
negative effects, lower temperatures provide ample time for
hydrogen producing bacteria to self adapt to pH dynamics in
unbuffered reactors [19]. Very few studies have been designed
specifically to compare biohydrogen production in unbuffered
reactors at low temperatures vs. high temperatures.
1.3. Effect of partial pressure of hydrogen
Controversies exist on the influence of hydrogen partial
pressure on hydrogen production [20]. It has been reported
that hydrogen accumulations in the reactor headspace may
inhibit hydrogen production [2123]. Attempts have been
made to improve hydrogen production by reducing hydrogen
partial pressures by nitrogen sparging and by maintaining
vacuum in the headspace. Though, increased hydrogen yields
were obtained with these energy-intensive methods [2325], it
was found that hydrogen partial pressure was not a sole factor
affecting hydrogen production [10]. For example, Mandal et al.
[23] reported higher hydrogen yield under lower headspace
pressure using chemically selected high hydrogen producing
mutant strain [26].
Low hydrogen solubility (Henrys constant of 8  104 M/
atm) coupled with shorter experimental duration (typical
batch operations w1530 days), near ambient operating
conditions (30  C, w100200 kPa) and frequent gas pressure
release may limit hydrogen availability to bacteria in the
liquid phase to further weaken the hypothesis about negative
effects of hydrogen partial pressure on biohydrogen produc-
tion. In addition, most of the literature reports claiming
increased hydrogen production under low hydrogen partial
pressure were based on hydrogen analysis in the gas phase
rather than the liquid phase [27].
Redox potential analysis of NADH pathway, in fermenta-
tive bacteria, revealed that hydrogen production can be sus-
tained even under hydrogen partial pressure of 1.0  105 Pa
when operating pH was 6 [28]. Our analysis based on Gibbs
free energies showed no effect of hydrogen partial pressure on
sucrose-to-acetate (DG0 457.5 kJ/mol) or sucrose-to-buty-
rate (DG0 554.0 kJ/mol) fermentation, in the operating
range of 01.0  105 Pa of hydrogen partial pressure at 25  C.
The above result is in concurrence with the suggestion by
Logan et al. [29] that hydrogen partial pressures have to be in
the order of 7.9  1014 Pa to inhibit hydrogen production, not
reported in the literature so far. These calculations were based
on the theoretical assumptions and have not been validated
with experimental results, yet.
1.4. Effect of pressure release
Continuous pressure release (CPR) method has been claimed
to produce higher hydrogen yields than the intermittent
pressure release (IPR) method, a fact that could not be justified
on thermodynamic basis [29]. While influence of pressure
buildup on the biohydrogen production remains controversial
[24], most literature studies still employ the IPR method. For
example, Van Ginkel et al. [15] obtained yields as high as
4.9 mol of hydrogen/mole of sucrose using the IPR method.
While gas phase hydrogen composition and the reactor pres-
sure were not reported here, it is interesting to note that no
inhibitory effects of hydrogen accumulation were observed.
Temporal data on total headspace pressure and its influence
on hydrogen production and associated parameters in IPR
batch reactors are still lacking.
1.5. Scope of this study
This study attempts to extend the literature results from
controlled laboratory experiments (buffered reactors, pure
cultures, high temperatures, with initial pH adjustments/
inorganic nutrient supplements) to more practical conditions
(unbuffered reactors, mixed cultures, ambient temperatures,
without initial pH adjustments/inorganic nutrient supple-
ments). Experiments under the latter conditions were
designed to evaluate the following:
1. biohydrogen production in unbuffered reactors at low
temperature (22  C), vs. high temperature (37  C).
 international journal of hydrogen energy 34 (2009) 12331243
2. effect of pressure buildup and the resulting hydrogen
partial pressure on hydrogen production and sucrose-to-
hydrogen conversion, at 22  C and 37  C
3. the thermodynamic feasibility of sucrose fermentation to
acetate and butyrate under both IPR and CPR conditions
using the experimental data.
2. Materials and methods
2.1. Culture conditions
Compost collected from a nearby composting facility was
heat-shocked by drying w1 cm thick samples at 104  C in an
aluminum pan for 2 h [30]. Sample was cooled to room
temperature and, sieved using a #30 mesh (600 mm), and
stored in aluminum foil in a refrigerator (4  C) [30]. Thirty-five
grams of this dried sample was added to 2 L of deionized water
containing sucrose concentration of 10 g/L COD. While this
solution was being continuously stirred, 175 mL of the media
was transferred to the test reactors (250 mL capacity bottles;
Wheaton Scientific). The reactors were then capped with red
butyl rubber septa and crimp sealed with aluminum crimp
rings. Volume of headspace in the reactors was 75 mL. All the
reactors were continuously stirred at 160 rpm at the test
temperatures (37  C and 22  C). No mineral or vitamin
supplements were added. Initial pH of the media solution was
8.5 without any adjustments. Nothing was done to strip initial
oxygen levels in the reactors.
2.2. Reversible displacement method (RDM) apparatus
The reversible displacement method (RDM) was used to
release gas pressure and measure gas volume. This device can
be utilized for gas measurement by continuous pressure
release (CPR) by maintaining the headspace always subat-
mospheric or by intermittent pressure release (IPR) by allow-
ing the headspace pressure to rise to any predetermined level.
Even though this method has been used previously for
measuring gas evolution [23], it has not been deliberately used
for pressure release studies in biohydrogen production by
dark fermentation. The apparatus consisted of a custom-
blown 1 L glass column, 88 mm diameter and 914 mm tall,
open at one end. The column was filled with distilled water,
and placed vertically with the open end immersed in a beaker.
Special 6.35 mm fitting on the lower section of the column was
used to connect the column to the headspace of the reactor via
a 2.38 mm  3.175 mm in. polypropylene tubing (95875-01,
Cole Parmer) fitted with a 1/8 in. NPT Viton ball valve (01377-
06, Cole Parmer). The purpose of the ball valve was to control
the gas pressure release from the headspace of the reactor.
Gas evolution/pressure release from the reactor could be
visually seen in the form of bubbles in the column when the
valve is opened. Gas volume evolved in the reactor headspace
can be directly measured by observing the amount of water
being displaced from the glass column. In the CPR method, the
ball valve is kept open at all the times, and the gas evolution
noted at specific time intervals. In the IPR method, the gas
builds up in the bottles, and pressure was released every 12 h
until the gas pressure in the reactor equilibrates with the
1235
column pressure. Pressure release using IPR technique is
similar to Owens method [31], except that the latter uses
lubricated syringe for the pressure release.
2.3. Analytical methods
Hydrogen, oxygen, nitrogen, and methane in the headspace of
the reactors were measured every 12 h. Gas samples were
drawn from the reactors using a gastight syringe and analyzed
by gas chromatograph (SRI Instruments, model 8610, Tor-
rence, CA) equipped with a thermal conductivity detector and
a molecular sieve column (Alltech Molesieve 5A 80/100
1.83 m  38 mm  26 mm) with argon as carrier gas. The
injection volume was 0.5 mL. The operational temperatures of
the injection port, the oven, and the detector were 100, 70 and
100  C, respectively. The detection limit of the TCD for
hydrogen and methane was w0.1%. Hydrogen gas production
was calculated from headspace measurements, and the total
volume of biogas produced, for each interval, using the mass
balance equation [10].



VH;i VH;i1 CH;i VG;i  VG;i1 VH CH;i  CH;i1 (3)
where VH,i and VH,i1 are cumulative hydrogen gas volumes at
the current (i) and previous (i  1) time intervals; VG,i and VG,i1
the total biogas volumes in the current and previous time
intervals; CH,i and CH,i1 the fractions of hydrogen gas in the
current and previous intervals, respectively; and, VH is the
total volume of headspace in the reactor. Linear change in
hydrogen concentration was assumed to calculate hydrogen
gas composition between the sampling times. [32].
Samples from the liquid phase were withdrawn on a daily
basis with a gastight syringe to measure pH, volatile fatty
acids, and reducing sugars. The concentrations of the volatile
fatty acids (VFAs) were determined using a gas chromato-
graph (SRI Instruments, model 8610A, Torrence, CA) equip-
ped with a flame ionization detector (Alltech AT-Steel P/W
Haysep Q 80/100, 1.83 m  38 mm  26 mm) with helium as
the carrier gas. The injection volume was 1 mL. The opera-
tional temperatures of the injector port, oven, and the
detector were 200  C, 200  C and 220  C. Reducing sugars
were measured spectrophotometrically (Hach, wave-
length 575 nm) using di-nitro salicylic acid (DNS) assay. pH
was measured using Cole-Palmer pH electrode probe. All gas
production data reported were standardized to standard
temperature (0  C) and pressure (1.01  105 Pa). Sucrose-to-
hydrogen conversion efficiencies were based on stoichio-
metric value of 8 mol of hydrogen per mole of sucrose as
shown in equation (1); further, these conversion efficiencies
were calculated based on sucrose input (10 g COD/L) and not
sucrose consumption [33].
Control and test reactors were set up in triplicates, as
summarized in Table 1. Control reactors were set up to verify
the absence of biotic, abiotic hydrogen production, and also to
verify the absence of background hydrogen production from
compost. One factor analysis of variance (ANOVA) was used to
determine if there were statistically significant differences in
hydrogen production between low temperature and high
temperature reactors, under the two pressure release condi-
tions. The threshold level of statistical significance for this
study was a 0.05.
1236 international journal of hydrogen energy 34 (2009) 12331243

Table 1 Details of test and control reactors for biohydrogen experiments.

Reactor Temperature Substrate Inocula Conditions adapted Purpose

CPR37 37 C Sucrose Compost Continuous pressure release, high Compare with high temperature IPR and
temperature low temperature CPR and IPR
IPR37 37  C Sucrose Compost Intermittent pressure release, high Compare with high temperature CPR and
temperature low temperature CPR and IPR
IPR22 22  C Sucrose Compost Intermittent pressure release, low Compare with high temperature CPR and
temperature IPR and low temperature CPR
CPR22 22  C Sucrose Compost Continuous pressure release, low Compare with high temperature CPR and
temperature IPR and low temperature IPR
Control1 37  C None Compost Without sucrose, autoclaved for To verify absence of biotic hydrogen
30 min production without substrate present
Control2 37  C Sucrose Compost With sucrose, autoclaved for To verify absence of biotic hydrogen
30 min production with substrate present
Control3 37  C None Compost Without sucrose To verify absence of biotic hydrogen
production

reported in the literature with optimized external inorganic

3. Results
There was no measurable hydrogen production in any of the
three control reactors. This confirmed that the hydrogen
production was from the substrate (sucrose) and not the
compost. Methane was not detected in the headspace of any
of the test reactors. This confirmed that heat treatment was
adequate to suppress methane-formers. Other key findings
from our experiments are summarized in Table 2 and dis-
cussed below.
3.1. Role of compost
As demonstrated by other researchers [34] our results confirm
that heat-treated compost is a suitable inoculum source for
biohydrogen production by dark fermentation. Based on the
hydrogen production data from all the test reactors, none of
which were supplemented with any vitamins and minerals, it
is concluded that compost by itself could provide sufficient
inorganic nutrients. For example, the yield of 4.3 mol
hydrogen/mol sucrose achieved in IPR22 without any inor-
ganic nutrient supplement is comparable to the results
nutrient supplements [35]. This result adds credence to the
utility value of compost in practical application of the dark
fermentation process for biohydrogen production.
3.2. Sucrose degradation
Sucrose concentration as a function of time is shown in Fig. 1.
Under both CPR and IPR conditions, the sucrose consumption at
22  C was higher than that at 37  C. Under CPR conditions,
sucrose consumption and sucrose-to-hydrogen conversion at
22  C were 96% and 40% respectively; whereas, those at 37  C
were only 45% and 29% respectively. Similarly, under IPR
conditions, sucrose consumption and sucrose-to-hydrogen
conversion at 22  C were 98% and 53% respectively; whereas
those at 37  C were only 45% and 21% respectively (Table 2). The
results at 22  C under IPR conditions were the best of all four
conditions tested. Conversion efficiencies in high temperature
reactors were significantly lower due to incomplete sucrose
utilization, reasons of which are discussed in later sections.
3.3. Aqueous products

Butyric acid was the major byproduct with negligible pro-

pionic acid in all the reactors. This is in agreement with
Table 2 Summary of experimental results.
CPR method IPR method 12
Sucrose concentration [g COD/L]

22  C 37  C 22  C 37  C 10

Lag phase, h 36 18 36 18
8
Duration of gas production, h 466 79 466 90
Total biogas production, mL 631 406 793 278 IPR37
6
Max. biogas production rate, mL/h 6.4 13 8 9
Total hydrogen produced, mL 356 266 472 196 CPR37
4
Range of hydrogen content, % 4156 5458 4161 4366 CPR22
pH at end of gas production, 4.9 4.0 4.9 3.9 IPR22
2
pH units
Sucrose consumption, % 96 47 98 45 0
Sucrose-to-hydrogen conversion, % 40 29 53 21 0 100 200 300 400 500
Moles hydrogen/mol sucrose, 3.2 2.3 4.3 1.7 Elapsed time [hrs]
mol/mol
Fig. 1 Sucrose consumption in the four reactors. (Error
Best values in each case are in bold.
bars indicate std. dev.; n [ 3, 95% CI).
 international journal of hydrogen energy 34 (2009) 12331243
literature reports of butyrate production in the pH range of
4.04.5 observed in this work [36]. Butyric acid to acetic acid (B/
A) ranged from 1.2 to 2.0, which is similar to the range
reported by Zhang et al. [8]. High yield of 4.3 mol of hydrogen
per mole of sucrose (Table 2) in this work agrees with litera-
ture reports on higher hydrogen production at similar ratios
[37] followed by negligible propionate production [38]. Total
volatile fatty acid (TVFA) accumulation rate at 37  C was twice
as that at 22  C. For example, TVFA accumulation rates in
CPR37 and IPR37 were 44.0 and 42.3 g COD/L/h respectively
(Reactors ceased in 79 h) whereas that in CPR22 and IPR22
were 20.2 and 21.9 g COD/L/h respectively. Effects of higher
TVFA accumulation rate, at the higher temperature, on the
hydrogen production are described in Section 4.
3.4. Gaseous products
Major gases produced in all the test reactors were hydrogen
and carbon dioxide, with similar compositions among the
triplicates (SD < 5%, 95% confidence interval). Maximum
cumulative hydrogen production of 472 mL was recorded at
22  C under IPR conditions, followed by 356 mL under CPR
conditions also at 22  C; cumulative hydrogen production at
37  C was 266 mL under the CPR conditions followed by
196 mL under IPR conditions (Fig. 2). Though hydrogen
compositions are somewhat similar between IPR and CPR
reactors ( p-value 0.07) as shown in Fig. 3, there is significant
difference between biogas production at each temperature ( p-
value 5.0E-11). As shown in Figs. 2 and 3, gas production
began after a short lag of 18 h at 37  C and, after 36 h at 22  C.
However, gas production ceased in just 90 h at 37  C, while gas
production continued for about 466 h at 22  C. It can also be
noted from Fig. 3 that, at 37  C, peak concentrations of 58%
and 66% were achieved in CPR and IPR reactors within 36 h; at
the 22  C, comparable peak concentrations of 56% and 61%
were achieved within 80 h. However, the significant difference
is that at 37  C, the hydrogen concentration declined in both
the reactors to almost half the peak value and hydrogen
production ceased completely; while at 22  C, hydrogen
concentration declined only slightly and hydrogen production
continued at a slower rate. Such decrease in hydrogen
500
Cumulative hydrogen production [mL]
400
300
200
100
0 all the four reactors occurred at pH range of 4.55.5. In
0 100 200
Elapsed time [hrs]
Fig. 2 Average cumulative hydrogen production in the
four reactors.
1237
concentration can occur spontaneously (DG0 166 kJ/mol)
via homoacetogenesis reaction as suggested by Park et al. [10]:
4H2 aq 2CO2 aq / CH3 COOHaq 2H2 Ol (4)
Thus, lower temperatures are necessary to maintain
hydrogen production in unbuffered reactors at peak levels so
as to avoid any inhibition of hydrogen producers.
3.5. Continuous pressure release (CPR) 22  C vs. 37  C
Headspace pressure in the CPR method remained slightly
below atmospheric at the two temperatures (Fig. 4). As
expected, at 37  C, the lag time was shorter than that at 22  C
(Fig. 2) and the maximum hydrogen generation rate was
higher (10 mL/h vs. 5 mL/h, Fig. 4). However, at 37  C, biogas
production stopped much sooner, and the total volumes of
hydrogen generated were lower than those at 22  C (Figs. 2 and
3). Hydrogen production rate began to decline in CPR37 within
36 h as the pH dropped below 4.5, and ceased at 79 h when the
pH reached 4.0. In CPR22, the decline began after 56 h, but
continued, albeit, at a lower rate while the pH remained above
4.0 (Fig. 4).
3.6. Intermittent pressure release (IPR) 22  C vs. 37  C
Headspace pressure in the IPR method reached a maximum of
2.3  103 Pa in IPR37 and 2.1  103 Pa in IPR22. As in the case of
CPR reactors, higher hydrogen production was noted at 22  C
in the IPR reactors too. At 37  C, gas evolution started quickly
yielding higher hydrogen production rates (Figs. 2 and 3). Here
again, hydrogen production stopped much sooner at 37  C,
and the volume of hydrogen generated was lower than that at
22  C. Hydrogen production rate began to decline in IPR37 after
45 h as the pH dropped below 4.0; but, hydrogen production
rate in IPR22 continued for over 466 h while the pH remained
above 4.0 (Fig. 4). Further, hydrogen production ceased at 90 h
in IPR37 while, it continued in IPR22, albeit, at a lower rate,
observations similar to CPR reactors. Maximum hydrogen
production rate in IPR22 reached only 6 mL/h at 56 h at an
overpressure condition of 2.09  103 Pa. Thereafter, pH
declined slightly by 0.7 units, and remained above 4.5 to
sustain hydrogen production for as long as 466 h.
IPR22 4. Discussion
The operating conditions at which maximum sucrose-to-
CPR22 hydrogen conversion was noted in the four cases in Fig. 5 are
tabulated in Table 3. The maximum values of sucrose-
CPR37 hydrogen conversion of 99 mL H2/g COD/L were noted in IPR22
and CPR22 at 22  C, and hydrogen partial pressures of
IPR37
1.25  105 Pa and 0.56  105 Pa respectively. The corresponding
values in IPR37 and CPR37 at 37  C were 44 and 35 mL H2/
g COD/L, at hydrogen partial pressures of 1.53  105 Pa and
0.58  105 Pa respectively. Maximum hydrogen conversions in
300 400 500
summary, irrespective of the test temperature, hydrogen
accumulation in the headspace did not inhibit hydrogen
production. The lower production at 37  C is attributed to
inhibitory pH levels.
1238 international journal of hydrogen energy 34 (2009) 12331243

80 800 80 800
CPR37 IPR37
700 700
60 600 60 600
Hydrogen content
Hydrogen content
of biogas [ ] 500 of biogas [ ] 500
40 400 40 400
300 300
20 200 20 200
Cumulative Cumulative
biogas [mL] 100 biogas [mL] 100
0 0 0 0
0 100 200 300 400 500 0 100 200 300 400 500
Elapsed time [hrs] Elapsed time [hrs]

80 800 80 800
CPR22 IPR22
700 700
Hydrogen content
60 of biogas [ ] 600 60 Hydrogen content 600
of biogas [ ]
500 500
40 400 40 400
Cumulative
300 biogas [mL] 300
20 Cumulative 200 20 200
biogas [mL]
100 100
0 0 0 0
0 100 200 300 400 500 0 100 200 300 400 500
Elapsed time [hrs] Elapsed time [hrs]

Fig. 3 Average hydrogen content in the biogases, and cumulative biogas production in the four reactors (Error bars indicate
std. dev.; n [ 3, 95% CI).

10 500 10 500
CPR37 IPR37

8 400 8 400
H2 production [mL/hr] H2 production [mL/hr]

6 300 6 300
pH pH
4 200 4 200

2 100 2 100
Headspace pressure Headspace pressure
[kPa] [kPa]
0 0 0 0
0 100 200 300 400 500 0 100 200 300 400 500
Elapsed time [hrs] Elapsed time [hrs]

10 500 10 500
CPR22 IPR22

8 400 8 400

6 pH 300 6 300
pH

4 200 4 200
Headspace pressure [kPa] Headspace pressure [kPa]

2 100 2 100
H2 production [mL/hr]
H2 production [mL/hr]

0 0 0 0
0 100 200 300 400 500 0 100 200 300 400 500
Elapsed time [hrs] Elapsed time [hrs]

Fig. 4 Temporal profiles of pH, headspace pressure, and hydrogen production rate (Error bars not shown for clarity).
 international journal of hydrogen energy 34 (2009) 12331243 1239

100 9 100 9
CPR37 IPR37
8 8
80 7 80 7
pH 6 pH 6
60 60
5 5
4 4
40 40
3 3

20 2 20 Sucrose-hydrogen 2
Sucrose-hydrogen
1 [mL H2/g/L COD] 1
[mL H2/g/L COD]
0 0 0 0
0 100 200 300 400 500 0 100 200 300 400 500
Elapsed time [hrs] Elapsed time [hrs]
100 9 100 9
CPR22 IPR22
8 8
80 7 80 7
6 6
60 pH 60 pH
5 5
4 4
40 40
3 3

20 2 20 Sucrose-hydrogen 2
Sucrose-hydrogen [mL H2/g/L COD]
[mL H2/g/L COD] 1 1
0 0 0 0
0 100 200 300 400 500 0 100 200 300 400 500
Elapsed time [hrs] Elapsed time [hrs]

Fig. 5 Temporal profiles of sucrose-to-hydrogen conversion and pH in the four reactors (Error bars indicate std. dev.; n [ 3,
95% CI).

Results (summarized in Table 2) show that sucrose
consumption and its conversion to hydrogen for IPR reactors at
22  C are more than double that at 37  C. One possible reason is
that higher temperatures can inactivate the hydrogenase
enzyme at higher temperature negating the positive kinetic
effect of higher temperature [39]. However, 37  C may not be
high enough to cause pronounced effect of denaturation of
hydrogenase enzyme [40]. Closer examination of Fig. 4
suggests that the lower performance of high temperature
reactors was due to pH inhibition. Temporal pH profiles for all
the four reactors are included in Figs. 4 and 5 for easy
comparison. As seen from these Figures, pressure release
conditions had no significant influence on the differences in
pH profiles at each temperature ( p-value 0.47). However,
a sharp initial decline in pH can be noted at higher temperature
(37  C) in contrast to a gradual decline at 22  C (0.06 vs. 0.01
pH units/h). Higher temperature favored faster biokinetics [39]
and total volatile fatty acids accumulation rate (>42 g TVFA as
COD/L/h) to lower the pH to inhibitive levels (4.0 in 79 h) in
CPR37 and IPR37. Such abrupt pH changes have been reported
to cause a negative shock to hydrogen producers [5], a fact seen
in our high temperature experiments as well. The poor
performance of CPR37 and IPR37, at 37  C, is therefore attrib-
uted to the inability of hydrogen producers to maintain pH
above the inhibition value of 4.0 [41]. This is further confirmed
in our studies by the fact that more than 50% of the sucrose was
still available in both IPR37 and CPR37 reactors when the
biogas production ceased (Figs. 24).
Biohydrogen researchers have overcome pH inhibition by
incorporating initial pH adjustments and external buffers
[5,15,22,42]. While we realize that studies on pH inhibition in
biohydrogen production are fairly established, uninhibited
biohydrogen production at low temperature in the absence of
buffers is a highlight of this research. Hydrogen producers in
reactors IPR22 and CPR22 were adept at self-adjusting to slow
pH changes, even in absence of buffers to achieve sucrose
hydrogen conversion efficiencies >50% (Table 4). As seen in
Figs. 4 and 5, even though pH declined gradually from 8.0 to 4.4

Table 3 Operating conditions for maximum sucrose-to-hydrogen conversion.

Reactor Maximum sucrose-H2 Temperature [ C] Duration [h] Pressure [kPa] H2 content [%] pH range
conversion [mL H2/g COD/L]

IPR22 99 22 5679 208 6061 5.05.5

IPR37 35 37 2436 142231 5666 4.55.0
CPR22 98 22 5679 100 5056 5.05.5
CPR37 44 37 2436 100 5658 4.55.0
1240 international journal of hydrogen energy 34 (2009) 12331243

Table 4 Comparison of productivity and conversion efficiencies.

Source Pressure release Buffer Initial pH adjust. Temperature [ C] Substrate Sucrose-to-hydrogen Notesa
method conversion

Khanal et al. [5] IPR Yes Yes 37 Sucrose 40% a, b

Oh et al. [32] CPR Yes Yes 25 Glucose 24% a, b
Logan et al. [29] CPR Yes Yes 26 Sucrose 23% a, b
Van Ginkel et al. [15] IPR No Yes 37 Sucrose 61% a, b
Wu et al. [16] na No Yes 25 Sucrose 49% b, c, d
Sung et al. [48] IPR Yes Yes 37 Sucrose 19% e
This study IPR No No 22 Sucrose 53% f
This study CPR No No 22 Sucrose 40% f
This study IPR No No 37 Sucrose 29% f
This study CPR No No 37 Sucrose 21% f

a a nitrogen sparging; b nutrient supplements; c argon sparging; d immobilized sewage sludge; e repeated heat treatment; f RDM.

and 4.3 in CPR22 and IPR22, hydrogen producers adapted and
self-adjusted pH to noninhibitory level (>4.0) after 90 h;
further, pH in both IPR22 and CPR22 increased to 4.9 at the end
of 466 h (Fig. 4). While limited buffering capacity of the
compost [15] cannot be attributed as sole reason for this pH
increase, reactor conditions would have triggered domination
of acid consuming bacteria in the bacterial consortium.
Similarly, hydrogen producers are reported to be capable of
decreasing proton concentration by switching to stationary
growth phase (>284 h) for concurrent participation in sucrose
degradation and reassimilation of TVFAs to decrease
hydrogen production by more than 50% [43]. Though analysis
of solvent phase production is beyond the scope of this study,
pH rise in both IPR22 and CPR22 occurred during the last 180 h
when associated hydrogen production in this duration is less
than 20% of the total hydrogen production.
4.1. Pressure-buildup and hydrogen partial
pressure effects
Headspace pressure in the IPR reactor was calculated based on
the volume of gas release from the reactor headspace. Pres-
sure profiles for the four reactors are included in Fig. 4. In the
CPR method, headspace pressure remained slightly below
atmospheric at the two temperatures. As such, performance
of the two CPR reactors is not affected by headspace pressure.
In contrast, headspace pressure in IPR reactors varied signif-
icantly. In IPR37, the headspace pressure ranged from 1.0  105
to 2.32  105 Pa. At the point of maximum overpressure,
hydrogen composition was maximum at 66%, and the pH was
4.3 while the hydrogen production rate was only 1.3 ml/h.
However, following overpressure and the maximum hydrogen
partial pressure, hydrogen production rate continued to
increase to 8.7 mL/h at 45 h, demonstrating no negative effect
of reactor headspace pressure on hydrogen production.
Similarly, in CPR37, maximum hydrogen partial pressure of
5.8  104 Pa had no negative effect on hydrogen production
rate (Figs. 3 and 4). Similarly in IPR22, overpressure of
2.08  105 Pa and highest hydrogen composition of 60% lasted
from 56 to 79 h, with no negative effect on hydrogen produc-
tion rate. In fact, this reactor achieved the highest sucrose
conversion of 99 ml H2/g COD/L in the same period (Fig. 5).
Even though Hallenbeck [44] had suggested hydrogen
partial pressure of 6.0  104 Pa to be inhibitory, we did not
observe any inhibition in our unbuffered reactors. Moreover,
the suggested hydrogen partial pressure of 6.0  104 Pa was
with reference to atmospheric pressure; higher hydrogen
accumulation in IPR37 and IPR22 reactors due to pressurized
conditions (1  1052.32  105 Pa in this study) also did not
impair hydrogen production. This observation further
weakens the hypothesis that hydrogen accumulation may
inhibit hydrogen production. It can also be concluded that the
lower performance of IPR37 cannot be attributed to hydrogen
accumulation, owing to similarities in hydrogen composition
in IPR37 and IPR22 ( p-value 0.46), irrespective of the
temperature differences between IPR37 and IPR22.
The above results seem contrary to literature findings,
because it has been hypothesized that IPR leads to higher
headspace pressure and consequently to higher hydrogen
accumulation, thought to be unfavorable for hydrogen
production. However, Kataoka et al. [45] had found no effect of
pressure release on hydrogen production. A study by Logan
et al. [29] could not justify the high performance of CPR reactor
on thermodynamic basis and pointed out other factors such as
partial inhibition of intermediate steps or enzymes in the
process. The following hypothesis serves to support our
contrary results: hydrogenase enzyme can produce hydrogen
either by pyruvate oxidation or, via NADH oxidation. Hydrogen
producers in our reactor may have followed the pathway
suggested in the former case which is demonstrated not to be
affected by typical hydrogen concentrations observed in
fermentation systems (1.0  1052.32  105 Pa) [27]. Though
hydrogen production (2H/H2, 2e; pH 7; 0.414 V) was
reported to be inhibited at hydrogen partial pressure of
6  104 Pa, in NADH pathway (NAD/NADH, 2e; pH 7;
0.32 V) [27]; microorganisms capable of adjusting potential of
electron carrier (NADH/NAD) to 0.42 V can sustain hydrogen
production even under higher hydrogen partial pressure
(1.62  105 Pa). While thermodynamic feasibility of the NADH
pathway depends on the redox potential of the starting
substrates (Sucrose in this experiment) and reduces with pH of
the culture media, fermentative bacteria can find alternative
electron carriers such as ferredoxin (Fdred, Fdox, e; pH 7;
0.48 V [9]) to improve thermodynamic feasibility of hydrogen
production with hydrogen accumulations.
 international journal of hydrogen energy 34 (2009) 12331243 1241

4.2. Thermodynamic analysis 22  C and 37  C. Fig. 6 demonstrates the spontaneity of

As discussed earlier, equations (1) and (2) represent two
possible hydrogen-forming reactions via sucrose fermenta-
tion to acetate and butyrate, where DG0 is the change of Gibbs
free energy [46] under standard conditions (pH 7, incubation
temperature 298 K and all the solute concentrations are 1 M,
and all the gases have partial pressure of 1 atm). The actual DG
for the reactions (1) and (2) are calculated using the Nernst
equation:

4
4
Ac HCO H2 8
DG DG0 2:303RT log 3
(5)
C12 H22 O11

2
Bu HCO
DG DG0 2:303RT log
C12 H22 O11
where DG0 (kJ/mol) is the value of DG at pH 7.0 under standard
conditions (i.e., all solutes are at the concentration of 1 M and
gases have partial pressure of 1 atm). R is the ideal gas
constant, 8.314 J/K, T is the absolute temperature (K), and the
values in parentheses represent either the molar concentra-
tions of solute or the partial pressure of the gases in
atmosphere.
As illustrated in Fig. 6, the DG values calculated from
Equations (5) and (6) were consistently negative for reactions
(1) and (2) for all the four reactors, demonstrating that
hydrogen production via acetate and butyrate fermentation
mode is independent of pressure release conditions at both
a -600
Changes in free energy [kJ]
hydrogen production under CPR conditions (DG 562.22 kJ/
mol and DG 631.256 at 37  C; and, DG 558 kJ/mol and
DG 623.589 kJ/mol at 22  C, for sucrose-to-acetate and
butyrate fermentation, respectively) with no negative influ-
ence of hydrogen partial pressures (up to 6  104 Pa) on
hydrogen production. It is also interesting to realize that
reactions (1) and (2) are exergonic even under IPR conditions
with DG values of 547.6 kJ and 621.8 kJ at 37  C; and,
543.33 kJ and 618.23 kJ at 22  C, for acetate and butyrate
formation respectively, even with hydrogen partial pressures
as high as 16  104 Pa (Fig. 6). According to this thermody-
namic analysis, then, there is no negative effect of hydrogen
partial pressure and intermittent pressure release conditions

4 on sucrose-to-hydrogen fermentation.
3 H2 4
(6)
4.3. Hydrogen yield and sucrose consumption
Based on the mass of sucrose added to the reactors, yield
values in our experiments are 2.3, 1.7, 3.2, and 4.3 mol
hydrogen per mole of sucrose, for CPR37, IPR37, CPR22, and
IPR22, respectively. The results are compared in Table 4 with
other studies that had used sucrose as the substrate, in both
buffered and unbuffered reactors, at two different temper-
atures. Logan et al. [29] reported a yield of 1.8 was reported
for mixed cultures at a conversion of 23%. Zhang et al. [8]
reported yield values ranging from 1.94 to 2.73 with mixed
cultures. Lee et al. [47] reported a yield of 2.01. Our results
for IPR22, CPR22, and CPR37 are superior to those of Logan
et al. [29], Oh et al. [32], and Sung et al. [48], while that of
IPR22 is better than that of Khanal et al. [5] and Wu et al.
[16]. Sucrose-to-hydrogen conversion of 47.2 mL H2/g COD/L
IPR22
found in IPR22 is comparable to the value of 46.6 mL H2/
g COD/L reported by Van Ginkel et al. [15]. Although the

CPR22
-620
IPR37 reactors in the latter case were buffered and run at 37  C, it
CPR37 is interesting to note the similarity of IPR method used in
-640
both the experiments. Though, the performance of IPR22 is
-660 slightly lower that of Van Ginkel et al. [15], it has to be noted
that, in contrast to our experiments, they had used initial pH
-680 adjustments, buffers, and inorganic nutrient supplements at
Sucrose fermentation to butyrate and hydrogen
Hydrogen partial pressure in CPR reactors = 0 - 0.6 atm. elevated temperature.
-700 Hydrogen partial pressure in IPR reactors = 0 - 1.6 atm.

-720
5. Conclusions
b -500
Changes in free energy [kJ]

IPR22 This study demonstrated the feasibility of biohydrogen

-550 CPR22 production by dark fermentation at 22  C in unbuffered
IPR37
reactors. Sucrose-to-hydrogen conversion efficiencies
CPR37
obtained in our unbuffered systems are comparable to or
-600
better than those reported in buffered systems. The results
of this study can translate to cost savings in large scale
-650 Sucrose fermentation to acetate and hydrogen applications by the elimination of inorganic nutrient
Hydrogen partial pressure in CPR reactors = 0 - 0.6 atm.
supplements, nitrogen sparging, initial pH adjustment, and
Hydrogen partial pressure in IPR reactors = 0 - 1.6 atm.
expensive buffers. Gradual pH changes induced by slower
-700 kinetics at lower temperature were recognized as the reason
20 70 120 170 220 270 320
for sustaining hydrogen production under unbuffered
Elapsed time [hrs]
conditions. The supposition that high hydrogen partial
Fig. 6 Energetics of hydrogen production in the four pressure decreases hydrogen production has been disproved
reactors a) sucrose fermentation to butyrate and hydrogen; through thermodynamic analysis and validated with
b) sucrose fermentation to acetate and hydrogen. experimental results.
1242 international journal of hydrogen energy 34 (2009) 12331243
Acknowledgments
This study was funded in part by the Office of Vice President
for Research at New Mexico State University and by the
National Science Foundations CBET Division, under Grant No.
0607175. We are thankful to the anonymous reviewers for
their valuable comments.
references
[1] Debabrata D, Veziroglu TN. Hydrogen production by
biological processes: a survey of literature. Int J Hydrogen
Energy 2001;26:1328.
[2] Gadhamshetty V, Sukumaran A, Nirmalakhandan N,
Myint MT. Photofermentation of malate for biohydrogen
production a modeling approach. Int J Hydrogen Energy
2008;33(9):213846.
[3] Vijayaraghavan K, Ahmad D. Biohydrogen generation from
palm oil mill effluent using anaerobic contact filter. Int J
Hydrogen Energy 2006;31(10):128491.
[4] Kotay SM, Das D. Biohydrogen as a renewable energy
resource prospects and potentials. Int J Hydrogen Energy
2008;33:25863.
[5] Khanal SK, Chen W-H, Li L, Sung S. Biological hydrogen
production: effects of pH and intermediate products. Int J
Hydrogen Energy 2005;29(11):112331.
[6] Kim IS, Hwang MH, Jang NJ, Hyun SH, Lee ST. Effect of
low pH on the activity of hydrogen utilizing methanogen
in bio-hydrogen process. Int J Hydrogen Energy 2004;
29(11):113340.
[7] Tanisho S, Kuromoto M, Kadokura N. Effect of CO2 removal
on hydrogen production by fermentation. Int J Hydrogen
Energy 2004;23(7):55963.
[8] Zhang Y, Shen G, Shen J. Hydrogen production in batch
culture of mixed bacteria with sucrose under different iron
concentrations. Int J Hydrogen Energy 2005;30(8):85560.
[9] Hallenbeck PC, Benemann JR. Biological hydrogen
production; fundamentals and limiting processes. Int J
Hydrogen Energy 2002;27(11/12):118593.
[10] Park W, Hyun S, Oh SE, Logan BE. Removal of headspace CO2
increases biological hydrogen production. Environ Sci
Technol 2005;39(12):441620.
[11] Davila-Vazquez G, Mondragon FA, Rodrguez AL, Flores ER.
Fermentative hydrogen production in batch experiments
using lactose, cheese, whey and glucose: influence of initial
substrate concentration and pH. Int J Hydrogen Energy 2008;
33(19):498997.
[12] Lay JJ. Modeling and optimization of anaerobic digested
sludge converting starch to hydrogen. Biotechol Bioeng 2000;
68(3):13617.
[13] Byung HK, Zeikus JG. Importance of hydrogen metabolism in
regulation of solventogenesis by Clostridium acetobutylicum
continuous culture system of hydrogen-producing anaerobic
bacteria. In: Proceedings of the eight international
conference on anaerobic digestion, vol. 2;1985. p. 38390.
[14] Gottwald M, Gottschalk G. The internal pH of Clostridium
acetobutylicum and its effect on the shift from the acid to
solvent formation. Arch Microbiol 1985;143(1):426.
[15] Van Ginkel S, Sung S, Lay JJ. Biohydrogen production as
a function of pH and substrate concentration. Environ Sci
Technol 2001;35(24):472630.
[16] Wu SY, Lin CN, Chang JS, Lee KS, Lin PJ. Microbial hydrogen
production with immobilized sewage sludge. Biotechnol Prog
2002;18(5):9216.
[17] Lin CL, Fang HHP. Fermentative hydrogen production from
wastewater and solids wastes by mixed cultures. Critical Rev
Environ Sci Technol 2007;37(1):139.
[18] Mu Y, Han-Quing Y, Wang G. Evaluation of three methods for
enriching hydrogen-producing cultures from anaerobic
sludge. Enzyme Microb Technol 2006;40(4):94753.
[19] Lin CY, Chang RC. Fermentative hydrogen production at
ambient temperature. Int J Hydrogen Energy 2004;29(7):
71520.
[20] Ruzicka M. The effect of hydrogen on acidogenic glucose
cleavage. Water Res 1996;30(10):244751.
[21] Doremus MG, Linden JC, Moreira AR. Agitation and pressure
effects on acetonebutanol fermentation. Biotechol Bioeng
1985;27(6):85260.
[22] Levin DB, Pitt L, Love M. Biohydrogen production: prospects
and limitations to practical application. Int J Hydrogen
Energy 2004;29(2):73186.
[23] Mandal B, Nath K, Debabrata D. Improvement of
biohydrogen production under decreased partial pressure of
H2 by Enterobacter cloacae. Biotechnol Lett 2006;28(11):8315.
[24] 24 Mizuno O, Dinsdale R, Hawkes FR, Hawkes DL, Noike T.
Enhancement of H2 production from glucose by nitrogen gas
sparging. Bioresour Technol 2000;73(1):5965.
[25] Kim S-H, Han S-K, Shin H-S. Effect of substrate concentration
on hydrogen production and 16S rDNA-based analysis of the
microbial community in a continuous fermenter. Process
Biochem 2006;41(1):199207. 14851491.
[26] Kraemer JT, Bagley DM. Improving the yield from
fermentative hydrogen production. Biotechnol Lett 2007;
29(5):68595.
[27] Kraemer JT, Bagley DM. Supersaturation of dissolved H2 and
CO2 during fermentative hydrogen production with N2
sparging. Biotechnol Lett 2006;28(18):148591.
[28] Tanisho S. A scheme for developing the yield of hydrogen by
fermentation. In: Miyake J, Matsunaga T, San Pietro A,
editors. Biohydrogen II. Tokyo: Pergamon; 2001. p. 22943.
[29] Logan BE, Oh SE, Kim IS, Van Ginkel S. Biological hydrogen
production measured in batch anaerobic respirometers.
Environ Sci Technol 2002;36(11):25305.
[30] Van Ginkel S, Sung S, Logan BE. Biohydrogen gas production
from food processing and domestic wastewaters. Int J
Hydrogen Energy 2005;30:153542.
[31] Owen WF, Stuckey DC, Healy JB, Young Jr LY, McCarty PL.
Bioassay for monitoring biochemical methane potential and
anaerobic toxicity. Water Res 1979;13:48592.
[32] Oh SE, Van Ginkel S, Logan BE. The relative effectiveness of
pH control and heat treatment for enhancing biohydrogen
gas production. Environ Sci Technol 2003;37(22):518690.
[33] Venkata Mohan S, Vijaya Bhaskar YH, Murali Krishna P,
Chandrashekara Rao V, Lalit Babu V, Sarma PN. Biohydrogen
production from chemical wastewater as substrate by
selectively enriched anaerobic mixed consortia: influence of
fermentation pH and substrate composition. Int J Hydrogen
Energy 2007;32(13):228695.
[34] Zhu H, Beland M. Evaluation of alternative methods of
preparing hydrogen producing seeds from digested
wastewater sludge. Int J Hydrogen Energy 2006;31(14):19808.
[35] Lin CY, Lay CH. A nutrient formulation for fermentative
hydrogen production using anaerobic sewage sludge
microflora. Int J Hydrogen Energy 2005;30(3):28592.
[36] Hwang MH, Jang NJ, Hyun SH, Kim IS. Anaerobic bio-
hydrogen production from ethanol fermentation: the role of
pH. J Biotechnol 2004;111(3):297309.
[37] Hawkes FR, Dinsdale R, Hawkes DL, Hussy I. Sustainable
fermentative hydrogen production: challenges for process
optimization. Int J Hydrogen Energy 2002;27(1112):133947.
[38] Kawagoshi Y, Hino N, Fujimoto A, Nakao M, Fujita Y,
Sugimara S, et al. Effect of inoculum conditioning on
 international journal of hydrogen energy 34 (2009) 12331243
hydrogen fermentation and pH effect on bacterial
community relevant to hydrogen production. J Biosci Bioeng
2005;100(5):52430.
[39] Nath K, Kumar A, Das D. Effect of some environmental
parameters on fermentative hydrogen production by
Enterobacter cloacae DM11. Can J Microbiol 2006;52(6):5250532.
[40] Fabiano B, Perego P. Thermodynamic study and optimization
of hydrogen production by Enterobacter aerogenes. Int J
Hydrogen Energy 2002;27(2):14956.
[41] Bowles LK, Ellifson WL. Effects of butanol on Clostridium
acetobutylicum. Appl Environ Microbiol 1985;50(5):116570.
[42] Ren NQ, Chua H, Chan SY. Assessing optimal fermentation
type for biohydrogen production in continuous-flow
acidogenic reactors. Bioresour Technol 2007;98(9):177480.
[43] Jones DT, Woods DR. Acetonebutanol fermentation
revisited. Microbiol Rev 1986;50:484524.
1243
[44] Hallenbeck PC. Fundamentals of the fermentative
production of hydrogen. Water Sci Technol 2000;52(12):219.
[45] Kataoka N, Miya A, Kiriyama K. Studies on hydrogen
production by continuous culture system of hydrogen-
producing anaerobic bacteria. Water Sci Technol 1997;
36(67):417.
[46] Thauer RK, Jungermann K, Decker K. Energy conservation in
chemotrophic anaerobic bacteria. Bacteriol Rev 1977;41(1):
10080.
[47] Lee YJ, Miyahara T, Noike T. Effect of iron concentration on
hydrogen fermentation. Bioresour Technol 2001;80(3):22731.
[48] Sung S, Raskin L, Duangmanee T, Padmasiri S, Simmons JJ.
Hydrogen production by anaerobic microbial communities
exposed to repeated heat treatments. In: Proceeding of the
2002 U.S. Doe hydrogen program review. NREL/CO-610-
32405.