international journal of hydrogen energy 34 (2009) 4509–4516

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Biohydrogen production by dark fermentation: Experiences

of continuous operation in large lab scale

M. Krupp*, R. Widmann
Department of Environmental Engineering: Waste and Water, University of Duisburg-Essen, Universitätsstr. 15, 45141 Essen, Germany

article info abstract

Article history: Hydrogen is a clean energy carrier which can be used as fuel in fuel cells. Today, hydrogen
Received 24 March 2008 is produced mainly by steam reforming of fossil fuels like natural gas or oil. But only
Received in revised form hydrogen produced by renewable sources can be called clean energy production. One
14 October 2008 possibility for hydrogen production is the biological fermentation of biogenous wastes by
Accepted 14 October 2008 hydrogen producing bacteria. For the experimental setup four 30-L-working-volume
Available online 28 November 2008 reactors were constructed for continuous biohydrogen production. As inoculum, heat-
treated sludge of a wastewater treatment plant was used. Different hydraulic retention
Keywords: times (HRT) were tested and an organic loading rate (OLR) of 2–14 kg VS/m3*d. As starting
Biohydrogen substrate, waste sugar medium was used. The pH and other parameters were observed to
Anaerobic digestion find boundary conditions for a stable continuous process with a minimum of online-
Dark fermentation control measurements. The high concentration of organic acids in the reactor led to a very
Biowaste low pH, which was controlled manually and online > 4 up to 5.5, otherwise the bio-
Biological waste treatment hydrogen production decreased rapidly. The gas amount varied with the different OLRs,
but could be stabilised on a high level as well as the hydrogen concentration in the gas with
44–52%. No methane was detected in the gas. It turned out, that continuous biohydrogen
production with stable gas amounts and qualities could be achieved at different operation
conditions. The results showed, that the operation of a continuous biohydrogen reactor
has to be observed very carefully to ensure a constant gas production, and that pH-control
is necessary to ensure stable operation conditions.
ª 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights
reserved.

1. Introduction discussed here is only biohydrogen production by dark

Biological hydrogen production can be carried out by a wide
variety of different organisms. The biological processes differ
according to the essential boundary conditions, basically the
distinction into light driven processes such as photolysis or
photo fermentation and light independent processes such as
dark fermentations [1]. Table 1 gives an overview about the
different biohydrogen production methods. Described and
fermentation.
Hydrogen production by dark fermentation is restricted by
the incomplete degradation of organic matter into volatile fatty
acids, hydrogen and carbon dioxide. A complete biological
conversion into hydrogen would be carried out only by
a combined multistep-treatment, for example dark fermenta-
tion as the first step and conversion of the produced volatile fatty
acids into hydrogen by photo fermentative microorganisms as

* Corresponding author. Tel.: þ49 2016302565.

E-mail address: michaela.krupp@uni-due.de (M. Krupp).
0360-3199/$ – see front matter ª 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.ijhydene.2008.10.043
4510 international journal of hydrogen energy 34 (2009) 4509–4516

Table 1 – Processes of biological hydrogen production.

Metabolic process Organisms Enzymes Light Electron source Products

Direct biophotolysis Green algae Hydrogenase Yes H2O H2, O2

Indirect biophotolysis Cyanobacteria Nitrogenase, hydrogenase Yes H2O H2, O2
Photofermentation Phototrophic bacteria Nitrogenase, hydrogenase Yes Organic compounds H2, CO2
Water-gas shift Phototrophic bacteria Hydrogenase No CO H2, CO2
Dark fermentation Fermentative bacteria Hydrogenase, nitrogenase No Organic compounds H2, CO2, VFA

the second step [2]. Another possibility for a complete conver-
sion into energy could be the application of a microbial fuel cell
[3]. For method implementation even combined dark fermen-
tation/methane fermentation plants or material utilisation of
the reactor effluent is conceivable.
All biohydrogen production technologies have so far been
tested on a lab scale. It can be stated, that the feasibility of
a technical implementation for hydrogen producing photo-
systems is currently restricted by space requirements [4]. Up
to now very few technical and economical studies for biolog-
ical hydrogen production systems have been carried out
[3,5,6]. In Table 3 available data for biological hydrogen
production by microalgae (sulphur-deprived system) and
a combined dark/photo-fermentation process are shown.
Considering the hydrogen production yields from the
processes presented in Table 2, at the present time, dark
fermentation and water-gas shift are the only methods that
have feasible reactor dimensions for practical applications [4].
Secondly, biohydrogen production by dark fermentation is
most interesting option for the conversion of organic wastes
because of its analogies to AD (anaerobic digestion).
production of hydrogen occurs in two of the four steps of
anaerobic degradation (hydrolysis and acetogenesis). In the
absence of methanogenic bacteria, a production of bio-
hydrogen can be achieved by degradation of organic matter to
volatile fatty acids (VFA) as liquid compounds and CO2 and H2
as gaseous compounds. Acetate fermentation processes are
well understood with a maximum yield of 4 mol H2/mol
glucose equivalent [9], and butyrate fermentation processes
with a yield of 2 mol H2/mol glucose equivalent.
C6 H12 O6 þ 2H2 O02CH3 COOH þ 2CO2 þ 4H2
C6 H12 O6 þ 2H2 O0C3 H7 COOH þ 2CO2 þ 2H2
In mixed cultures, this reaction often occurs as a combined
production [9], modified:
4C6 H12 O6 þ 2H2 O02CH3 COOH þ 3C3 H7 COOH þ 8CO2 þ 10H2
3. Objectives

3.1. Scale up for technical implementation

2. Biology
Many microorganisms have the potential for hydrogen
fermentation, but microbes of the genus Clostridium are the
most promising due to their high potential for hydrogen yield,
their rapid metabolism and their attribute of spore-forming,
which makes them easy to handle by heat treatment [7]. They
allow hydrogen production with mixed cultures under mes-
ophilic or thermophilic conditions within a pH range of
5.0–6.5. While hydrogen production from specific, sugar-based
substrates using pure bacteria strains have been investigated
in many studies [8], the use of carbohydrate-rich wastes/
wastewater in mixed cultures has gained more attention. The
One important task for continuous biohydrogen production is
the scale up from lab scale into half-technical or pilot scale. It
is well known that for all new researched processes and
methods the scale up represents an essential challenge.
Therefore, continuous biohydrogen production in 35 L reac-
tors (30 L working volume) was investigated to test the bio-
hydrogen production behaviour on this larger lab scale. In
relation to common international research in biohydrogen
fermentation this represents an enlargement of reactor
volume by factor 10. Regarding international approaches in
scale up, only very few studies [10,11] in pilot scale have been
carried out successfully till date. Therefore the operation
experiences achieved by 30 L test reactors should give useful
information for layout and construction of a pilot plant.

Table 2 – Size of bioreactor required to power PEM fuel 3.2. Control mechanisms and boundary conditions
cells [4]. for continuous operation
System Size of bioreactor [m3] required
to power a PEM fuel cell of: Most of international biohydrogen research has been carried
out in sterile, microbiological optimised surroundings. So one
1.0 kW 1.5 kW 2.5 kW 5.0 kW
fundamental question in this study was whether a stable
Direct photolysis 341 512 856 1710 continuous biohydrogen production process could be carried
Indirect photolysis 67.3 101 169 337 out under self-regulating conditions with only a minimum of
Photo-fermentation 149 224 374 758
control mechanisms. The approach to answer this question
Dark fermentation min. 0.198 0.297 0.495 0.989
was to start at the basics; therefore the test program should
Dark fermentation max. 2.91 4.38 7.31 14.62
check first the process changes in an uncontrolled
 international journal of hydrogen energy 34 (2009) 4509–4516 4511

Table 3 – Parameters for biological hydrogen production systems [2,5,6].

Parameter Unit Algae Combined fermentation

Technical maturity – Small labscale Labscale

System availability % n.a. n.a.
Durability a n.a. 8
Space required m2/Nm3 H2/h 864.3 n.a.
Specific Energy demand MJ/Nm3 H2 n.a. 4.47
Hydrogen efficiency factor % n.a. 30
Conversion efficiency % 3–10 80
Specific investment costs (10 V/m2 reactor algae) V/Nm3 H2/h 22,400–24,600 8828
H2 production costs V/Nm3 H2 0.52–1.19 0.25
CO2-emissions kg CO2/Nm3 H2 n.a. n.a.

n.a.: data not available.

environment. Then inevitable regulation methods should be
implemented step by step. During and connected with these
first steps boundary conditions in the form of organic loading
rate, hydraulic retention time and temperature in the system
should be determined. For these trials, the model substrate
waste sugar was used.
4. Experimental setup
Four 35 L-Reactors were used for continuous biohydrogen
production. The working volume was 30 L, reactor material
was acrylic glass. The reactors were fit with a water jacket for
heating by chemostats (Thermo, Karlsruhe, Germany and
Julabo, Seelbach, Germany). In Fig. 1 the general reactor layout
is displayed.
Reactor type was CSTR (continuously stirred tank reactor)
for three reactors, one reactor was layouted as a rotating
biodisc reactor with upstream pump mixing (see Fig. 2). Mix-
ing was carried out with a Masterflex tube pump (Cole Parmer
Instrument Company, Illinois, USA).
The reactors were loaded and emptied with Heidolph
(Schwabach, Germany) tube pumps, feeding intervals were
controlled by timers. pH-regulation was obtained with 4 mol
NaOH, and during all tests a nutrient dilution according to
Ref. [12] was fed additional to the feeding substrate to ensure
the supply with trace elements, nitrogen and phosphorous.
The reactors were maintained daily and several parameters
were recorded manually. Input and output amounts were
noted; the fill level of each reactor was recorded and adjusted
by filling or emptying if necessary. Input and output samples
were taken and measured as well as gas amount and quality.
Each reactor was sparged once a day with 3–5 L nitrogen gas to
reduce the hydrogen partial pressure in the system according
to Ref. [12]. During all test runs the feeding intervals were
adapted to the designated HRT. At HRT of 20 h the feeding
interval was fixed to 2 h and the same amount of input
substrate was fed into the reactors at each pumping proce-
dure. For further reduction of HRT pumping time was
extended. Different reactor test runs should answer different
research questions. One reactor (D) was operated in thermo-
philic mode, one reactor (B) was fed with different organic
materials. The operation of two further reactors (A and C)
should give information about operation modes concerning
minimum retention times and maximum organic loading
rate. Due to the high number of different test runs in the test
period, only the results for two selected test runs of reactors A
and C are presented here. Further information may be found
in Ref. [13]. In Table 4 the boundary conditions for the two
reactors are given. Runs A3 and C1 were conducted in order to

Fig. 1 – Reactor outline for continuous biohydrogen

production. Fig. 2 – Scheme of upstream mixed biodisc reactor.
4512 international journal of hydrogen energy 34 (2009) 4509–4516

Table 4 – Runs A3, C1: boundary conditions for two biohydrogen test runs.
Run no. HRT, h Feeding substrate OLR, kg/m3*d Days, d Days, d Feeding interval, h Temp.,  C Control

A3 20 Waste Sugar 6 15 43 2 35 Comp.

15 8 8 2
15 10 10 2
12.5 12 4 2
12.5 14 6 2

C1 20 Waste Sugar 6 39 69 2 35 Comp.

15 8 8 2
15 10 10 2
12.5 12 4 2
12.5 14 6 2

optimise HRT and OLR in a biohydrogen production system.
Preliminary tests with HRT of 120–40 h showed, that short
HRT improved process stability [13]. Boundary values should
be determined by decreasing HRT and increasing OLR: in both
reactors, the pH was controlled online and HRT and OLR were
changed simultaneously. Due to technical problems, run A3
was started 31 days after run C1, but was operated at the same
boundary conditions.
5. Results and discussion
In Figs. 3 and 4 overall gas production and composition in the
two presented test runs are given. Regarding Fig. 3, very high
gas production rates up to 170 L/d were achieved during test
run A3. The gas quality after the first 20 operation days was
more equalised due to shorter feeding intervals and also the
gas production stayed at high levels.
A very consistent gas production can be found for the test
runs A3 and C1 (HRT 20–12.5 h) after a start-up phase of 10
days. For run C1 this effect is diminished due to minor loading.
The reactors fill level had to be adjusted daily by addition of
5–10 L input substrate which caused non-uniform loading and
a lower hydrogen yield.
5.1. OLR and HRT
In Fig. 5 the planned and the effective organic loading rate
based on VS for the mesophilic test runs A3 and C1 are
pictured. Due to technical problems, run C1 started 32 days
earlier than run A3, but was operated at the same level (HRT
20 h, OLR 6 kg VS/m3*d) for this period. Only values for the
parallel run time of A3 and C1 are pictured.
It can be seen that the organic loading rate showed high
variations. While in run C1 a shock load on days 3–7 gave
important information, run A3 showed a rapid decrease fol-
lowed by a high peak between day 17 and 19. This was caused
by a leakage of the reactor drain. The reactor had to be
emptied and repaired while the content was stored for 24 h in
a cooling chamber until the run was started again.
For run C1 shock load was produced by a feeding failure but
gave important information about the process stability at

Fig. 3 – Run A3: Gas production and composition.

 international journal of hydrogen energy 34 (2009) 4509–4516 4513

Fig. 4 – Run C1: Gas production and gas composition.

shorter HRT. Although OLR was tripled from values of about
4 kg VS/m3*d to 12 kg VS/m3*d, this shock load just caused
a single peak in gas production as seen in Fig. 4. No further
operational interferences were observed. In Fig. 5 it can also be
seen that even if the middle OLR as presented in Fig. 5 meets
the planned values (for example in run A3 during the last
phases), large differences may occur from day to day. Regular
reactor feeding is not easy to achieve due to inexact pumping,
mixing or production of a consistent input mixture.
It can be stated that the continuous reactor operation was
stable over the whole test duration. Critical limits for OLR and
HRT were not reached for both reactors, the biological process
was not disturbed. Overall, run C1 showed lower gas produc-
tion in comparison to run A3, but this was caused by daily fill
level adjustments as described above.
5.2. Hydrogen yields and efficiency
The hydrogen yields were calculated differently according to
the divergent values measured in the analysis. It can be seen
that the calculated hydrogen yield in the run A3 is signifi-
cantly higher than in run C1. Maximum hydrogen yields were

Fig. 5 – Run A3, C1: Organic loading rate - planned and realised values.
4514 international journal of hydrogen energy 34 (2009) 4509–4516

Table 5 – Runs A3, C1: hydrogen yields and carbon degradation.

Parameter, OLR Plan H2-yield, H2-yield, Carbon degradation, COD-degradation, hCOD %
Unit mol H2/mol glucose mol H2/mol glucose hc,gas %
based on VS input based on COD input

A3 6 0.69 0.94 12.63 26.22

8 2.51 1.54 32.14 28.80
10 2.93 1.57 42.01 40.29
12 1.05 1.63 21.62 19.16
14 1.30 1.78 24.01 22.18

C1 6 1.04 1.20 20.33 33.40

8 1.24 1.11 26.02 9.72
10 0.95 0.80 20.68 29.84
12 0.79 1.05 17.35 45.01
14 0.78 1.40 16.16 9.82

achieved in run A3 during OLR phases of 8–10 kg VS/m3*d with
2.51 and 2.93 mol H2/mol glucose (based on VS). Nevertheless,
the data differ widely and are dependent on the different
input parameters, such as VS, COD or the amount of sugar
weighed in the input vessels (sugar input). Here, for the same
loading periods only hydrogen yields of 1.45–1.47 mol H2/mol
glucose (based on sugar input) and 1.57–1.63 mol H2/mol
glucose (based on COD input) were calculated. Reasons for
these variations were not easy to find. Different measure-
ments like COD, TOC and VS were all conducted from one
sample, and standards were measured regularly. So the
quality of analysis data was good. But, it is a well-known
problem that a larger scale causes difficulties in balancing, so
maybe the high substrate flow in combination with slight
inaccuracies in volume and mass measurements are respon-
sible for the correlation problems. Results for the two test runs
can be found in Table 5.
These are calculated based on different input analysis
measurements, input COD and input DOC. From these input
analysis, the amount of glucose fed into the reactors was
calculated and set in relation to the hydrogen production. In
Fig. 6 the hydrogen yields for test run A3 are pictured. The figure
clarifies the strong relationship between HRT and hydrogen
production efficiency. Regarding the peaks between day 20 and
30 it is obvious that the hydrogen yield follows the HRT. A
retention time of 14–15 h seems to provide optimal conditions
for biohydrogen production, but maybe reduced further.
In Fig. 7 the relationship between HRT and hydrogen yield
is less clear than in test run A3, and the achieved hydrogen
yields are lower. This is caused by the fact that the input
feeding had to be corrected each day by addition of a high
amount of input substrate (6–10 L). But also here the hydrogen
yield follows the retention time and HRT < 15 h led to
a significant decrease in hydrogen yields for this reason.
5.3. Reactor design
Based on the results presented above it can be stated that no
significant influence for improved hydrogen production could
be measured by the modified reactor layout of reactor C. After
reactor shutdown it was also found that the biofilm produc-
tion on the biodiscs did not take place as expected, so the
main advantage of the biodisc layout was not used. Reasons
for this may be found in a lack of suitability of the biodisc

Fig. 6 – Run A3: Hydrogen yield and hydraulic retention time.

 international journal of hydrogen energy 34 (2009) 4509–4516
Fig. 7 – Run C1: Hydrogen yield and hydraulic retention time.
material and in the difficult reactor feeding and operation due
to minor loading as described previously.
6. Conclusions
In the test runs A3 and C1 limits of OLR and HRT should be
found. Preliminary test runs with higher HRT showed that
short retention times and high loads could optimise the bio-
hydrogen production process. It can be stated, that the
continuous reactor operation was stable during the whole test
duration. Critical limits for OLR and HRT were not reached for
both reactors, the biological process was not disturbed.
Overall, run C1 showed lower gas production in comparison to
run A3.
Regarding the hydraulic retention time it turned out that
the highest achieved hydrogen yields were found at an
optimum of 14–15 h retention time. A further reduction down
to 12 h led to a decrease in hydrogen yields. The increase of
the organic loading rate affected the hydrogen yields only
slightly. So it can be stated that for this reactor configuration
with organic loading rates up to 14 kg VS/m3*d an optimal
retention time seem to be at around 15 h. Further reduction of
HRT may be possible and is limited at around 2 h due to
washout, as shown in Ref. [14].
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