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International Journal of Hydrogen Energy 29 (2004) 173 – 185

www.elsevier.com/locate/ijhydene

Biohydrogen production: prospects and limitations to practical

application
David B. Levina; b;∗ , Lawrence Pittb , Murray Loveb
a Department of Biology, University of Victoria, Victoria, BC, Canada V8W 3P6
b Institute for Integrated Energy Systems, University of Victoria, Victoria, BC, Canada V8W 3P6

Accepted 24 March 2003

Abstract
Hydrogen may be produced by a number of processes, including electrolysis of water, thermocatalytic reformation of
hydrogen-rich organic compounds, and biological processes. Currently, hydrogen is produced, almost exclusively, by elec-
trolysis of water or by steam reformation of methane. Biological production of hydrogen (Biohydrogen) technologies provide
a wide range of approaches to generate hydrogen, including direct biophotolysis, indirect biophotolysis, photo-fermentations,
and dark-fermentation. The practical application of these technologies to every day energy problems, however, is unclear. In
this paper, hydrogen production rates of various biohydrogen systems are compared by 6rst standardizing the units of hydro-
gen production and then by calculating the size of biohydrogen systems that would be required to power proton exchange
membrane (PEM) fuel cells of various sizes.
? 2003 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.

Keywords: Biological hydrogen production; Dark-fermentation; Fuel cells

1. Introduction with many technical challenges, from the production of suf-

Energy is vital to global prosperity, yet dependence
on fossil fuels as our primary energy source contributes
to global climate change, environmental degradation, and
health problems [1]. Hydrogen (H2 ) o=ers tremendous po-
tential as a clean, renewable energy currency. Hydrogen
has the highest gravimetric energy density of any known
fuel and is compatible with electrochemical and combus-
tion processes for energy conversion without producing
carbon-based emissions that contribute to environmental
pollution and climate change. Hydrogen fuel cells and
related hydrogen technologies provide the essential link
between renewable energy sources and sustainable energy
services. The transition from a fossil fuel-based economy
to a hydrogen energy-based economy, however, is fraught
∗ Corresponding author. Tel.: +1-250-472-4069; fax: +1-250-
472-4075.
E-mail address: dlevin@uvic.ca (D.B. Levin).
6cient quantities of hydrogen to its storage, transmission,
and distribution [2].
Hydrogen may be produced by a number of processes,
including electrolysis of water, thermocatalytic reformation
of hydrogen-rich organic compounds, and biological pro-
cesses. Currently, hydrogen is produced, almost exclusively,
by electrolysis of water or by steam reformation of methane.
Biological production of hydrogen (biohydrogen), using
(micro) organisms, is an exciting new area of technology
development that o=ers the potential production of usable
hydrogen from a variety of renewable resources. Biological
systems provide a wide range of approaches to generate
hydrogen, and include direct biophotolysis, indirect biopho-
tolysis, photo-fermentations, and dark-fermentation [3–5].
While biohydrogen systems can produce H2 , no commer-
cial systems are yet available, and questions concerning the
practical application of biohydrogen loom large. Can bio-
hydrogen systems be developed as practical and commer-
cial applications? For example, can biohydrogen systems be

0360-3199/03/$ 30.00 ? 2003 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/S0360-3199(03)00094-6
174 D.B. Levin et al. / International Journal of Hydrogen Energy 29 (2004) 173 – 185

integrated with hydrogen fuel cell technologies to generate Table 1

electricity at a practical scale? The energy produced by a range of fuel cell sizes as a percentage
One of the major limitations to the practical application of of coastal residential loads in British Columbia, Canada
biohydrogen systems is that scientists who study biohydro- Fuel cell Yearly energy Percentage of BC hydro load
gen systems do not talk to engineers who develop hydrogen output power production
fuel cell technologies (and vice versa). Thus, the rates of hy- kW KWh Non-Electrically Electrically
drogen produced by biological systems are unknown to fuel heated house heated house
cell engineers and the amounts of H2 required for practical (%) (%)
applications, such as fuel cells, are unknown to biohydrogen
0.25 2190 17 11
researchers. Moreover, the rates of hydrogen produced by 0.50 4380 34 22
the various biohydrogen systems are expressed in di=erent 0.75 6570 51 34
units, making it diFcult to assess and compare the rates and 1.00 8760 68 45
amounts of hydrogen synthesized by di=erent biohydrogen 1.50 13,140 101 67
technologies. 2.00 17,520 135 89
In order to assess the potential application of the vari- 2.50 21,900 169 112
ous biohydrogen systems, we have calculated the size of 3.00 26,280 203 134
bioreactors that would be required to supply suFcient H2 to 4.00 35,040 270 179
4.60 40,296 311 206
proton exchange membrane fuel cells (PEMFC) to generate
5.00 43,800 338 223
enough electricity to meet the energy demands of a typi-
cal house located in the Paci6c Northwest of North Amer-
ica (British Columbia, Canada). The choice of a PEMFC is
based on the idea that biohydrogen systems might best be
used as a means of delivering small, distributed power sys- 60-day storage system. Without a storage system, electricity
tems to communities. If a biohydrogen system can deliver would need to be supplemented from a backup supply dur-
enough H2 to power a PEMFC for 24 h, on a continuous ing the times that the fuel cell would fail to meet demand.
basis, and the fuel cell system can produce enough electric- For non-remote locations, the conventional electrical grid
ity to supply the electrical demand year round, then the bio- would be a more economical source than batteries or diesel
hydrogen system could have a truly useful, and potentially generators.
commercial, application. Alternatively, the size of the fuel cells could be increased
In British Columbia, the average non-electrically heated so that suFcient electricity is generated to meet peak load
house uses 12; 971 kWh of electricity every year, while an demands. A 5 kW PEMFC would provide suFcient power
electrically heated house requires 19; 606 kWh (BC Hy- to meet the peak load demand of an electrically heated house
dro, unpublished data). This amount of energy could be without diFculty, but would generate excess energy dur-
produced by PEMFCs with output ratings equal to the ing non-peak load periods. This could create an interest-
average electrical loads, approximately 1.5 and 2:5 kW, re- ing scenario in which a residential fuel cell unit, fuelled by
spectively (Table 1). However, electrical demand varies on hydrogen produced biologically, could have potential as a
diurnal and seasonal bases, and there are large di=erences small-scale distributed power generator. Several utilities in
between the residential demand pro6les of di=erent re- the US are beginning to o=er “net-metering” programs that
gions. In northern latitudes (such as the province of British allow customers to sell unused electricity (from residential
Columbia in Canada, or Canada in general), peak residential solar installations or electric vehicles, for instance) back to
energy demands increase signi6cantly in winter, while in the grid [6].
more southerly latitudes (Southern California, for example) In this paper, several biohydrogen systems and their
peak residential energy demand occurs in summer. rates of H2 production are described. Following this, we
While PEMFCs of 1.5 and 2:5 kW would produce suF- describe several fuel cell systems, their fuel requirements,
cient energy to meet an average residential load, they would and their limitations. We then calculate the size of biore-
fail to meet demand during peak load hours (typically the actors required to supply suFcient H2 to power PEMFCs
late afternoon and early evening) and would produce excess of 1:5 kW (minimum averaged load for non-electrically
energy during periods of low demand, assuming they run heated homes), 2:5 kW (minimum averaged load for elec-
at a constant output. Taking variations in peak residential trically heated homes), and 5:0 kW (excess output to cover
energy demand into account, the 1:5 kW fuel cell system peak load demand without storage or grid supplement) for
would fail to meet the load requirements for 3722 h of the each biohydrogen system.
year (∼ 42%), while the 2:5 kW fuel cell would fail to meet The calculated bioreactor sizes are meant to serve as rela-
load requirements for 3918 hours of the year (∼ 45%). tive measures of scale, so that we may determine if a partic-
To meet demand, the 1:5 kW fuel cell would require an ular biohydrogen system has any hope of practicality. They
energy storage system capacity equivalent to approximately are not intended as de6nitive or absolute values. Much of
20 days load, and the 2:5 kW fuel cell would require a the data used in our analysis was obtained in laboratory
 D.B. Levin et al. / International Journal of Hydrogen Energy 29 (2004) 173 – 185
bench-scale experiments with pure, de6ned substrates, and
in some cases with pure cultures of known bacteria. We ac-
knowledge that scaling up biohydrogen systems to 1000 l or
more will be a great challenge with respect to substrate com-
position and supply, culture impurities, and removal of gas
products from the aqueous phase, as well as H2 separation,
puri6cation, and storage. The intent of this paper is, there-
fore, to provide a benchmark by which we may determine
which biohydrogen system(s) o=er the greatest potential for
practical applications.
2. Methods
Descriptions of the biohydrogen systems and their rates
of H2 production were gathered from the literature and from
direct, personal communications with scientists. One major
obstacle to comparison of di=erent biohydrogen systems is
that di=erent units of H2 synthesis rate are used for di=er-
ent biohydrogen technologies. Rates of H2 production are
reported variously as ml of H2 =ml of culture/h, l of H2 =l of
culture/h, mol of H2 =l of culture/h, nmol of H2 =g of pro-
tein in the culture/h, or mol of H2 =mg of chlorophyll (chl)
a=h. In the following analysis, rates of H2 production by the
various biohydrogen systems were converted to a standard
format; mmoles of H2 =l of culture/h, and is expressed as
175
The anode reaction (1) generates two electrons for every
molecule of H2 reacted. The charge transfer for the reaction
is given by
charge = zF × amount of reactant; (4)
where z is the number of electrons transferred per molecule
of fuel (z = 2 for H2 ) and F is the Faraday constant,
96; 485 Coulombs (C). The maximum thermodynamic
e6ciency (max ) of a fuel cell is de6ned as the ratio of the
electrical energy produced in the cell (PgQf = change in
Gibbs free energy) to the energy that would be produced by
burning the fuel (PhQf = change in enthalpy of formation):
PgQf
max = : (5)
PhQf
The maximum cell voltage (Emax ) is the voltage that would
result if all the fuel’s energy were transformed into electrical
energy:
−PhQf
Emax = : (6)
zF
For H2 fuel, PhQf is −285:85 kJ=mol (HHV) and z = 2. Emax
is, therefore, 1:48 V. The actual cell e6ciency can be related
to the cell voltage Vc as follows:
Vc
cell eFciency = : (7)
Emax
Since part of the fuel entering the fuel cell always passes
mmol H2 =(l × h). through unused, a fuel utilization coe6cient (f ) is de6ned
For systems that reported the rate of H2 production as as
ml of H2 =ml=h or l of H2 =l culture/h, the rates were con- mass of fuel reacted in cell
f = :
verted to the standard format by dividing the number of l mass of fuel input to cell
(or the fraction of a l in the case of ml of H2 =ml of cul- Hydrogen fuel cells often have a fuel utilization coe6cient
ture/h) by 24.8 to derive the number of moles (1 mole H2 (f ) approaching 0.95 [7]. The overall cell e6ciency () is
occupies 24:8 l at 303 K(=30◦ C), a common temperature at therefore
which mesophilic bacteria are cultured) and standard pres- Vc
sure (1 atm = 760 mm=Hg = 101:3 kPa). For systems that  = f : (8)
Emax
reported the rate of H2 production as nmoles of H2 =g of
The cell voltage, if not known, can be calculated by rear-
protein in the culture/h, the rates were converted to the stan-
ranging Eq. (8):
dard format by multiplying the concentration of protein in
 Emax
the culture (usually given as mg protein/ml of culture) by Vc = : (9)
f
the nmoles of H2 =mg of protein in the culture times 1000,
giving the moles of H2 =l of culture. For systems that re- To calculate the rate of H2 consumption (rate of fuel use) of
ported the rate of H2 production as mol of H2 =h=mg of chl a PEMFC, the charge is divided by time yielding the current
a, the rates were converted to the standard format using the (I ), in amperes. The rate of fuel use can be calculated for a
number of mg of chl a=l of culture. Because the protein and single cell, where z = 2:
chlorophyll content can vary for each type of culture, con- I
fuel usage = (mole=s):
centrations used in each calculation were based on values 2F
reported by authors. For a stack of n cells:
The rate of H2 consumption by PEMFCs was calculated In
fuel usage = (mole=s): (10)
as described below based on well established equations [7,8] 2F
The anodic, cathodic, and overall reactions in a PEMFC are The output power (Pe ) of the stack with cell voltage Vc
(calculated using Eq. (9), above) is given by
H2 (g) → 2H+ (aq) + 2e− ; (1)
Pe = Vc In;
1 + −
O (g)
2 2
+ 2H (aq) + 2e → H2 O(l); (2) therefore,
Pe
I= :
H2 (2) + 12 O2 (g) → H2 O(l): (3) Vc n
176 D.B. Levin et al. / International Journal of Hydrogen Energy 29 (2004) 173 – 185

Table 2 Green algae, under anaerobic conditions, can either use H2

Flow rates of H2 required to power PEM fuel cells as an electron donor in the CO2 -6xation process or evolve
Size of PEMFC H2 Uow rate required
H2 . Hydrogen production by green microalgae requires sev-
eral minutes to a few hours of anaerobic incubation in the
(kW) (g/h) (mol/h) (SL/h) dark to induce the synthesis and/or activation of enzymes
involved in H2 metabolism, including a reversible hydroge-
1.0 49 23.9 577.7
1.5 73 35.9 866.6
nase enzyme. The hydrogenase combines protons (H+ ) in
2.5 121 59.9 1444.3 the medium with electrons (donated by reduced ferredoxin)
5.0 243 119.7 2888.5 to form and release H2 . Thus, green microalgae possess the
genetic, enzymatic, metabolic, and electron-transport ma-
50% eFciency, 95% H2 utilization rate, average cell voltage chinery to photoproduce H2 gas. The synthesis of H2 per-
0:779 V. mits sustained electron Uow through the electron-transport
chain, which supports synthesis of ATP [9].
The process of algal photosynthesis oxidizes H2 O and
Substituting this into Eq. (10) gives the fuel usage as a evolves O2 . Light energy absorbed by photosystem II (PSII)
function of output power and cell voltage: generates electrons which are transferred to ferredoxin,
Pe using light energy absorbed by photosystem I (PSI). A
fuel usage = (mole=s): (11)
2Vc F reversible hydrogenase accepts electrons directly from the
To express fuel usage as a mass 8ow rate (kg=s), it is nec- reduced ferredoxin to generate H2 [10]. Because the hy-
essary to multiply Eq. (11) by the molar mass of the fuel drogenase enzyme responsible for evolution of molecular
(2:02 × 10−3 kg=mol for H2 ). Similarly, to express fuel us- H2 is highly sensitive to O2 , photosynthetic production of
age as a volumetric 8ow rate (SL/s), the density of the fuel H2 and O2 must be temporally and/or spatially separated.
is required (8:4 × 10−5 kg=SL at NTP for H2 ). Substituting In a two-phase process, CO2 is 6rst 6xed into H2 -rich sub-
the above 6gures into Eq. (11) yields the following relations: strates during normal photosynthesis (Phase 1), followed
by light-mediated generation of molecular H2 when the mi-
2:02 × 10−3 kg=mol Pe
H2 usage = croalgae are incubated under anaerobic conditions (Phase
2Vc F 2). Phase 2 of the two-stage process can be achieved by
Pe incubating the microalgae in medium that does not contain
= 1:05 × 10−8 × (kg=s)
Vc sulfur-containing nutrients [9].
When culture of the green alga Chlamydomonas rein-
Pe
= 1:25 × 10−4 × (SL=s): (12) hardtii are deprived of inorganic S, the rates of O2 synthesis
Vc and CO2 6xation decline signi6cantly within 24 h (in the
For hydrogen fuel cells, the storage volume (Vs ) required light). The reason for this loss of activity is due to the need
at a temperature Ts of 25◦ C (298 K) and a given pressure for frequent replacement of the H2 O-oxidizing protein D1 in
Ps is estimated by assuming hydrogen to be an ideal gas, the PSII reaction centre [11]. Depletion of sulfur blocks the
following the relation: Ps Vs = nRTs . synthesis of the D1 polypeptide chain (32 kDa), which con-
This was rearranged to solve for volume and convert to tains many sulfur containing amino acids, such as cysteine
standard units (litres): and methionine. While photosynthetic capability declines
nRTs greatly, respiration continues, and after approximately 22 h
Vs = 1000 ; (13) of sulfur deprivation, C. reinhardtii cultures maintained in
Ps
the light become anaerobic, and begin to synthesize H2 [9].
where Ps is storage pressure, in kPa, n is the number of Based on this phenomenon, systems for sustained H2
moles of H2 required, and R is the universal gas constant gas production in C. reinhardtii have been developed
8:314 × 10−3 kJ mol=K. The Uow rates of H2 required to [12,13]. The rate of H2 production by C. reinhardtii re-
power PEMFCs of various sizes were calculated using the ported by Kosourov et al. [12] was 7:95 mmol H2 =l of
above equations and are presented in Table 2. culture after 100 h, which corresponds to approximately
0:07 mmole H2 =(l × h). Melis et al. [13] reported similar
values.
3. Biohydrogen systems
3.2. Indirect biophotolysis
3.1. Direct biophotolysis
Cyanobacteria can also synthesize and evolve H2 through
Photosynthetic production of hydrogen from water is a bi- photosynthesis via the following processes:
ological process that can convert sunlight into useful, stored Light energy
chemical energy by the following general reaction: 12H2 O + 6CO2 −−−−−→ C6 H12 O6 + 6O2 ; (15)
Light energy Light energy
2H2 O −−−−−→ 2H2 + O2 : (14) C6 H12 O6 + 12H2 O −−−−−→ 12H2 + 6CO2 : (16)
 D.B. Levin et al. / International Journal of Hydrogen Energy 29 (2004) 173 – 185
Cyanobacteria (also know as blue-green algae, cyano-
phyceae, or cyanophytes) are a large and diverse group of
photoautotrophic microorganisms, which evolved and di-
versi6ed early in Earth’s history [14]. Cyanobacteria con-
tain photosynthetic pigments, such as chl a, carotenoids, and
phycobiliproteins and can perform oxygenic photosynthe-
sis. They are a morphologically diverse group that includes
unicellular, 6lamentous, and colonial species. Within the 6l-
amentous cyanobacteria, vegetative cells may develop into
structurally modi6ed and functionally specialized cells, such
as the akinetes (resting cells) or heterocysts (specialized
cells that perform nitrogen-6xation; [15]). The nutritional
requirements of cyanobacteria are simple: air (N2 and O2 ),
water, mineral salts, and light [16].
Species of cyanobacteria may possess several enzymes
directly involved in hydrogen metabolism and synthesis of
molecular H2 . These include nitrogenases which catalyze
the production of H2 as a by-product of nitrogen reduction
to ammonia, uptake hydrogenases which catalyze the oxida-
tion of H2 synthesized by the nitrogenase, and bi-directional
hydrogenases which have the ability to both oxidize and
synthesize H2 (reviewed by Tamagrini et al. [15]).
Hydrogen production by cyanobacteria has been stud-
ied for over three decades and has revealed that eFcient
photoconversion of H2 O to H2 is inUuenced by many fac-
tors [17]. Hydrogen production has been assessed in a
very wide variety of species and strains, within at least 14
genera, under a vast range of culture conditions [17]. The
rates of H2 evolution are as wide-ranging as the species
and conditions used in these studies. Rates of H2 pro-
duction by non-nitrogen-6xing cyanobacteria range from
0:02 mol H2 =mg chl a=h (Synechococcus PCC 6307) to
0:40 mol H2 =mg chl a=h (Aphanocapsa montana) [18].
These rates are very low compared with those of hetero-
cystous cyanobacteria, which range from 0:17 mol H2 =mg
chl a=h (Nostoc linckia IAM M-14) to 4:2 mol H2 =mg
chl a=h (Anabaena variablilis IAM M-58) [19].
Because of the higher rates of H2 production by Anabaena
species and strains, these have been subject to intense study
for the past several years. Mutant strains of A. variabilis
have demonstrated signi6cantly higher rates of H2 produc-
tion compared with wild-type strains. A. variabilis PK84,
for example, produced H2 at a rate of 6:91 nmol=g of pro-
tein/h (in 350 ml cultures). When A. variablis PK84 was
cultured under conditions of nitrogen starvation, the rate
of H2 synthesis was 12:6 mol=g of protein/h (in 350 ml
cultures). The concentration of total protein in the culture
was 28:2 g=ml of culture [20]. Assuming no change as the
culture volume is scaled up, a 1 l culture would contain
28; 200 g protein and would produce 355; 320 nmol H2 =h
or approximately 0:355 mmol H2 =(l × h).
3.3. Photo-fermentation
Purple non-sulfur bacteria evolve molecular H2 catalyzed
by nitrogenase under nitrogen-de6cient conditions using
177
light energy and reduced compounds (organic acids).
Light energy
C6 H12 O6 + 12H2 O −−−−−→ 12H2 + 6CO2 : (17)
These photoheterotrophic bacteria have been investigated
for their potential to convert light energy into H2 using waste
organic compounds as substrate [21–25] in batch processes
[26], continuous cultures [27,28], or cultures of bacteria im-
mobilized in carrageenan [29], in agar gel [30], on porous
glass [24], on activated glass [31], or on polyurethane foam
[23].
In general, rates of hydrogen production by photo-
heterotrophic bacteria are higher when the cells are im-
mobilized in or on a solid matrix, than when the cell are
free-living. Continuous cultures of Rhodopseudomonas
capsulata and Rhodobacter spheroides were reported to
produce H2 at rates that range from 40 to 50 ml H2 =l of
culture/h [21,32,33], 80 ml to 100 ml H2 =l of culture/h
[25]. Continuous cultures of Rhodospirillum rubrum were
reported to produce H2 at a rate of 180 ml H2 =l of culture/h
[28]. Cultures of Rb. spheroides immobilized on porous
glass, on the other hand, were reported to produce H2 at
a rate of 1:3 ml H2 =ml=h (1:3 l H2 =l of immobilized cul-
ture/h) [24]. Rates of H2 production by Rb. spheroides GL1
immobilized on activated glass were 3.6 –4:0 ml H2 =ml=h
[31,34]. If the system of culturing Rb. spheroides on porous
glass could be scaled-up without compromising the rate of
H2 synthesis, this would result in rates of 3.6 –4:0 l H2 =l
of immobilized culture/h, which would correspond to
0:145 mmol H2 =(l × h) to 0:161 mmol H2 =(l × h).
3.4. Hydrogen synthesis via the water–gas shift reaction
of photoheterotrophic bacteria
Certain photoheterotrophic bacteria within the superfam-
ily Rhodospirillaceae can grow in the dark using CO as the
sole carbon source to generate ATP with the concomitant
release of H2 and CO2 [35–37]. The oxidation of CO to CO2
with the release of H2 occurs via a water–gas shift reaction:
CO(g) + H2 O(l) → CO2 (g) + H2 (g); PG◦
= − 20 (kJ=mol): (18)
In these organisms, however, the reaction is mediated by
proteins coordinated in an enzymatic pathway. The reaction
takes place at low (ambient) temperature and pressure.
Thermodynamics of the reaction are very favorable to
CO-oxidation and H2 synthesis since the equilibrium is
strongly to the right of this reaction.
Stoichiometric amounts of CO2 and H2 are produced dur-
ing CO-oxidation [37]. The enzyme that binds and oxidizes
CO, carbon monoxide:acceptor oxidoreductase (carbon
monoxide dehydrogenase = CODH) is part of a membrane
bound enzyme complex [37,38]. Rubrivivax gelatinosus
CBS is a purple non-sulfur bacterium that not only performs
178 D.B. Levin et al. / International Journal of Hydrogen Energy 29 (2004) 173 – 185
the CO–water–gas shift reaction in darkness, convert-
ing 100% CO in the atmosphere into near stoichiometric
amounts of H2 , it also assimilates CO into new cell mass in
the light (via CO2 6xation) when CO is the sole source of
carbon [39,40]. Even when an organic substrate is available
with CO, R. gelatinosus CBS will utilize both substrates
simultaneously, indicating that the CO-oxidation pathway
is fully functional even when a more favorable substrate is
included [41].
R. gelatinosus CBS exhibits a doubling time of 7 h in
light when CO serves as the only carbon source [42,41].
The mass transfer of CO may be enhanced by a high ratio
of gas phase to liquid bacterial suspension, and by stirring
the culture vigorously. The hydrogenase from this organism
is tolerant to O2 , exhibiting a half-life of 21 h when whole
cells were stirred in full air [43].
A speci6c rate of CO oxidation to H2 production of
0:8 mmol=min=g of cells, dry weight (cdw) [41] was mea-
sured using a low-density culture (6nal OD660 ¡ 0:2),
stirred at a high rate (250 rpm), and supplemented with
20% CO in the gas phase. Because the conversion of CO
to H2 is stoichiometric, this corresponds to a rate of CO
uptake and conversion of approximately 1:34 g CO=h=g
cdw, or 48 mmol CO=h=g dcw. An OD660 of 2.0 yields
2:0 g R. gelatinosus CBS cdw/l. This corresponds to
a H2 production rate of 96 mmol H2 =2 g cdw/h or
96 mmol H2 =(l × h).
3.5. Dark-fermentation
Hydrogen can be produced by anaerobic bacteria,
grown in the dark on carbohydrate-rich substrates. Fer-
mentation reactions can be operated at mesophilic (25
–40◦ C), thermophilic (40 –65◦ C), extreme thermophilic
(65 –80◦ C), or hyperthermophilic (¿ 80◦ C) temperatures.
While direct and indirect photolysis systems produce pure
H2 , dark-fermentation processes produce a mixed biogas
containing primarily H2 and carbon dioxide (CO2 ), but
which may also contain lesser amounts of methane (CH4 ),
CO, and/or hydrogen sul6de (H2 S). The gas composition
presents technical challenges with respect to using the
biogas in fuel cells (see below).
Bacteria known to produce hydrogen include species of
Enterobacter, Bacillus, and Clostridium. Carbohydrates are
the preferred substrate for hydrogen-producing fermenta-
tions. Glucose, isomers of hexoses, or polymers in the form
of starch or cellulose, yield di=erent amounts of H2 per
mole of glucose, depending on the fermentation pathway
and end-product(s). When acetic acid is the end-product, a
theoretical maximum of 4 mole H2 per mole of glucose is
obtained:
C6 H12 O6 + 2H2 O
→ 2CH3 COOH + 4H2 + 2CO2 : (19)
When butyrate is the end-product, a theoretical maximum
of 2 moles H2 per mole of glucose is obtained:
C6 H12 O6 + 2H2 O
→ CH2 CH2 CH2 COOH + 2H2 + 2CO2 : (20)
Thus, the highest theoretical yields of H2 are asso-
ciated with acetate as the fermentation end-product. In
practice, however, high H2 yields are associated with a
mixture of acetate and butyrate fermentation products,
and low H2 yields are associated with propionate and
reduced end-products (alcohols, lactic acid). Clostridium
pasteurianum, C. butyricum, and C. beijerinkii are high
H2 producers, while C. propionicum is a poor H2 producer
[44].
Hydrogen production by these bacteria is highly de-
pendent on the process conditions such as pH, hydraulic
retention time (HRT), and gas partial pressure, which a=ect
metabolic balance. Thus, fermentation end-products pro-
duced by a bacterium depend on the environmental condi-
tions in which it grows. Reduced fermentation end-products
like ethanol, butanol, and lactate, contain hydrogen that has
not been liberated as gas. To maximize the yield of H2 , the
metabolism of the bacterium must be directed away from
alcohols (ethanol, butanol) and reduced acids (lactate)
towards volatile fatty acids (VFA). C. pasteurianum is a
classic H2 and VFA producer, but its metabolism can be
directed away from H2 production and towards solvent
production by high glucose concentrations (12.5% w/v), by
CO (which inhibits Fe-hydrogenase), and by limiting Fe
concentrations [45].
The partial pressure of H2 (pH2 ) is an extremely impor-
tant factor for continuous H2 synthesis. Hydrogen synthesis
pathways are sensitive to H2 concentrations and are subject
to end-product inhibition. As H2 concentrations increase,
H2 synthesis decreases and metabolic pathways shift to pro-
duction of more reduced substrates such as lactate, ethanol,
acetone, butanol, or alanine. As the temperature increases,
however, conditions that favor reaction (15), shown above,
are less a=ected by H2 concentration. Continuous H2 syn-
thesis requires pH2 of ¡ 50 kPa at 60◦ C [46], ¡ 20 kPa at
70◦ C [47], and ¡ 2 kPa at 98◦ C [48].
Some pure cultures of Clostridium species can degrade
insoluble starch without pre-treatment, while some pure cul-
tures of Enterobacter species can degrade soluble starch
[49]. Species of Clostridium generally yield high rates of H2
synthesis using xylose as a substrate. For example, Taguchi
et al. [50] found that a pure strain of Clostridium (species
No. 2) could produce H2 in a continuous culture, at pH 6.0,
using xylose (a major product of hemicellulose hydrolysis)
as substrate. In other studies with a variety of natural sub-
strates, the rate of H2 production varied with the speci6c
substrate used [51–53]. The best rate obtained with a pure
culture of Clostridium (species No. 2) was 21:03 mmol H2 =
 D.B. Levin et al. / International Journal of Hydrogen Energy 29 (2004) 173 – 185
(l × h) using 3% xylose as substrate [53]. The yield,
after 5 h HRT, was 2.36 mol of H2 per mole of
xylose.
Stable fermentation processes capable of using non-sterile
feedstocks, however, require mixed cultures of bacteria en-
riched from natural sources. Such mixed cultures are re-
ported to contain mostly species of Clostridium. Lay et al.
[54] observed a rate of 1600 l H2 =m3 =day (66:7 ml H2 =l of
culture/h), and a yield of 2:14 mol of H2 per mol of hex-
ose using a 0.75% solution of soluble starch. H2 synthesis
was obtained with a HRT of 17 h, at an organic loading
of 6 g of total organic sludge per litre of culture, at 37◦ C,
and a pH of 5.2. This system produced H2 at a rate of
64:5 mmol H2 =(l × h).
Using 6xed-bed bioreactors containing mesophilic bacte-
ria (predominantly Clostridium spp.) derived from domes-
tic sewage sludge, Chang et al. [55] compared the use of
expanded clay matrices or activated carbon as support ma-
trixes for the bacteria within the bioreactors. The HRT was
varied from 0.5 to 5 h. At an HRT of 1 h, with sucrose
as substrate (20 g=l of culture), the activated carbon ma-
trix bioreactor produced 1:3 l H2 =l of culture/h. The use of
a hollow-6ber micro6ltration membrane increased the rate
of H2 production to 3:0 l H2 =l of culture/h. H2 production
began 2–3 days post-inoculation and was continuous over a
2-week period. This corresponds to a rate of approximately
121 mmol H2 =(l × h).
Ueno et al. [56] observed rates of H2 production of up
to 198 mmol H2 =l of culture after 24 h, using an unde-
6ned population of bacteria, enriched for spore-forming
Clostridium species, at thermophilic temperatures (60◦ C).
With sugar factory wastewater as the substrate, a yield of
2:52 moles of H2 per mole of glucose was observed. Max-
imum H2 synthesis was obtained with a HRT of 12 h, at
an organic loading of 5 g of total organic sludge per litre
of culture, at a pH of 6.8. The bioreactor was mixed at
a rate of 200 rpm and argon gas was used to replace the
gas phase. H2 synthesis did not begin until the 26th day
of operation, but once H2 production began, gas evolution
was observed for at least 20 days for each HRT cycle, and
was continuous for a total of 212 days [56]. This system
produced H2 at a rate of 8:2 mmol H2 =(l × h).
Hydrogen production by the extreme thermophiles
Caldicellulosiruptor saccharolyticus and Thermotoga el@i
is currently under investigation [57]. The maximum rate
of H2 production by C. saccharolyticus on sucrose (10 g=l of
culture) was 8:4 mmol H2 =(l × h). The maximum rate of
H2 production by T. el@i on glucose (10 g=l of culture) was
2.7–4:5 mmol H2 =(l × h). Maximal H2 production rates
were observed during exponential growth phase, which
was in the order of 8–12 h, within a total growth cycle
(lag phase to stationary phase) of approximately 2 days.
The total amount of H2 produced during these batches
were 110 mmol H2 for C. saccharolyticus cultured on su-
crose (at 10 g=l), and 76 mmol H2 for T. el@i cultured on
glucose.
179
3.6. Fuel cell technologies
Fuel cells are electrochemical devices that create an elec-
tron Uow using charged ions. A variety of di=erent fuel cells
systems have been developed. They di=er in the type of
electrolyte used, in the operating conditions, in their power
density range, in their application, and each has its advan-
tages and disadvantages (reviewed by Larminie et al. [7];
Table 3).
Alkaline fuel cells (AFC) utilize hydroxyl ions (OH− ) as
the mobile ion (derived from potassium hydroxide, KOH),
operate in the 50 to 200◦ C range, and are extremely sensitive
to the presence of CO2 . Phosphoric acid fuel cells (PAFC)
utilize protons (H+ ) as the mobile ion and operate at ap-
proximately 200◦ C. PAFC systems were the 6rst fuel cells
produced commercially and are used as stationary power
sources, generating up to 200 kW of electricity. Many are
in use in the USA and in Europe. The high operating tem-
perature and corrosive nature of the electrolyte makes them
unsuitable for use in mobile and transportation applications.
Molten carbonate fuel cells (MCFC) utilize carbonate ions
(CO2− ◦
3 ) as the mobile ion, operate at approximately 650 C,
and can take H2 , CO2 , CO, and/or CH4 as fuel, which means
they can use natural gas, coal gas, or biogas as fuel sources.
Like PAFC, MCFC are used as stationary power sources,
generating electricity in the MW range. Solid oxide fuel
cells (SOFC) utilize oxygen radicals (O2− ) as the mobile
ion and operate between 500◦ C and 1000◦ C. Like MCFC,
SOFC can utilize H2 , CO, and/or CH4 as fuel, which means
they can use methane, coal gas, or biogas as fuel sources.
Carbon dioxide is not utilized as a fuel and is discharged as
a waste gas. Like other high-temperature fuel cell systems
(PAFC and MCFC) SOFC systems are used as stationary
power sources, generating electricity from the low kW to
the MW range.
PEMFC utilize hydrogen protons (H+ ) as the mobile ion,
operate in the 50 –100◦ C range, require pure H2 and are
extremely sensitive to the presence of CO. Of all the fuel
cell systems that are available, PEMFC systems are espe-
cially suitable for mobile and transportation applications,
and PEMFC engines have been demonstrated successfully in
both cars and buses. Small PEMFCs, in the 1–10 kW range,
are also under commercial development as small stationary
power units to provide electricity to homes and small busi-
nesses. Because of their imminent commercial applications,
PEMFC technologies are under intense research and devel-
opment. The rate of H2 consumption by PEMFCs is usually
expressed in kg of H2 =s or in mol of H2 =s. We have cal-
culated the rates of H2 consumption by PEMFCs of several
sizes and expressed them as g H2 =h or mol H2 =h (Table 2).
3.7. Rates of biohydrogen synthesis
A comparison of H2 production rates reported for several
biohydrogen systems is presented in Table 4. Conversion
of reported units of H2 production to the standardized unit
180 D.B. Levin et al. / International Journal of Hydrogen Energy 29 (2004) 173 – 185

Table 3
The fuel constraints for di=erent fuel cell types (adapted from [30])

Fuel cell type PEMFCa AFCb PAFCc MCFCd SOFCe

(operating tempt.) (80◦ C) (200◦ C) (220◦ C) (600 –700◦ C) (800 –1100◦ C)

Component in fuel gas

H2 Fuel Fuel Fuel Fuel Fuel
CH4 Diluent Diluent Diluent Diluent Diluent
CO Poison if ¿ 10 ppm Poison Poison if ¿ 0:5% Fuel Fuel
CO2 Diluent Poison Diluent Diluentf Diluent
S (H2 S and COS) Poison if ¿ 10 ppm Poison Poison ¿ 50 ppm Poison ¿ 1 ppm
a PEMFC—proton exchange membrane fuel cell.
b AFC—alkaline fuel cell.
c PAPF—phosphoric acid fuel cell.
d MCFC—molten carbonate fuel cell.
e SOFC—solid oxide fuel cell.
f CO must be supplied to cathode of MCFC.
2

Table 4
Comparison of the rates of H2 synthesis by di=erent technologies

BioH2 System H2 synthesis rate H2 synthesis rate References

(reported units) (converted units)

Direct photolysis 4:67 mmol H2 =l=80 h 0:07 mmol H2 =(l × h) [29]

Indirect photolysis 12:6 nmol H2 =g protein/h 0:355 mmol H2 =(l × h) [51]
Photo-fermentation 4:0 ml H2 =ml=h 0:16 mmol H2 =(l × h) [60,61]
CO-oxidation by R. gelatinosus 0:8 mmol H2 =g cdw/min 96:0 mmol H2 =(l × h) [71]

Dark-fermentations
Mesophilic, pure straina 21:0 mmol H2 =1 l=h 21:0 mmol H2 =(l × h) [56]
Mesophilic, unde6nedb 1; 600:0 l H2 =m3 =h 64:5 mmol H2 =(l × h) [32]
Mesophilic, unde6ned 3:0 l H2 =l=h 121:0 mmol H2 =(l × h) [8]
Thermophilic, unde6ned 198:0 mmol H2 =l=24 h 8:2 mmol H2 =(l × h) [66]
Extreme thermophilic, pure strainc 8:4 mmol H2 =l=h 8:4 mmol H2 =(l × h) [67,68]
a Clostridium species #2.
bA consortium of unknown microorganisms cultured from a natural substrate and selected by the bioreactor culture conditions.
c Caldicellulosiruptor saccharolyticus.

(mmol H2 =(l × h)) reveals the wide range of H2 synthesis
by di=erent biohydrogen systems. Light-dependent biohy-
drogen systems (direct photolysis, indirect photolysis, and
photo-fermentation) all have rates of H2 synthesis well be-
low 1 mmol H2 =(l × h). dark-fermentation systems, all pro-
duce H2 at rates that are well above 1 mmol H2 =(l × h). The
rates of H2 synthesis by an unde6ned consortium of ther-
mophilic Clostridium [56] and by the extreme thermophilic
Caldicellulosiruptor saccharolyticus [47,57] are very sim-
ilar (8.2 and 8:4 mmol H2 =(l × h), respectively). A pure
strain of mesophilic Clostridium [53] demonstrated very
good rates of H2 synthesis with xylose as a substrate (21.0
mmol H2 =(l×h), and two dark-fermentation systems that uti-
lized unde6ned consortia of mesophilic bacteria [55,54,58]
had impressively higher rates of H2 synthesis (64.5 and
121:0 mmol=H2 =(l × h), respectively).
3.8. Biohydrogen: prospects for practical application
Our analyses indicate that photosynthesis-based systems
do not produce H2 at rates that are suFcient to meet the goal
of providing enough H2 to power even a 1 kW PEMFC on
a continuous basis (Table 5). This does not mean that these
systems should be abandoned. There may be applications
other than our hypothetical objective to which they may be
more suited. Moreover, continued research will no doubt
result in signi6cant improvements in their respective tech-
nologies, and thus in the rates of H2 production (see below).
Thermophilic and extreme thermophilic biohydrogen sys-
tems would require bioreactors in the range of approximately
2900 –14; 600 l to provide suFcient H2 to power PEMFCs
of 1.5 –5:0 kW, and a bioreactor of approximately 5700 l
would be required to power the 5:0 kW fuel cell using the
 D.B. Levin et al. / International Journal of Hydrogen Energy 29 (2004) 173 – 185 181

Table 5
Is Biohydrogen practical? The size of bioreactor required to power PEM fuel cells of di=erent output

BioH2 system H2 synthesis rate Size of bioreactor requiredb to power a:

(mmol H2 (l × h))a 1:0 kW FC(l) 1:5 kW FC (l) 2:5 kW FC (l) 5:0 kW FC (l)

Direct photolysis 0.07 3:41 × 105 5:12 × 105 8:56 ×105 1:71 × 106
Indirect photolysis 0.355 6:73 × 104 1:01 × 105 1:69 × 105 3:37 × 105
Photo-fermentation 0.16 1:49 × 105 2:24 × 105 3:74 × 105 7:58 × 105
CO-oxidation by R. gelatinosus 96.0 2:49 × 102 3:74 × 102 6:24 × 102 1:25 × 103

Dark-fermentations
Mesophilic, pure strainc 21.0 1:14 × 103 1:71 × 103 2:85 × 103 5:70 × 103
Mesophilic, unde6nedd 64.5 3:71 × 102 5:57 × 102 9:29 × 102 1:86 × 103
Mesophilic, unde6ned 121.0 1:98 × 102 2:97 × 102 4:95 × 102 9:89 × 102
Thermophilic, unde6ned 8.2 2:91 × 103 4:38 × 103 7:31 × 103 1:46 × 104
Extreme thermophilic, unde6nede 8.4 2:85 × 103 4:28 × 103 7:13 × 103 1:43 × 104
a Conrested units.
b Approximate volumes. Calculated volumes were rounded up to nearest whole value.
c Clostridium species #2.
d A consortium of unknown microorganisms cultured from a natural substrate and selected by the bioreactor culture conditions.
e Caldicellulosiruptor saccharolyticus.

pure culture of mesophilic Clostridium sp. (strain No. 2).
The size of bioreactors required for these systems are very
large, and thus these systems may be considered impractical
for our hypothetical application at this time.
Some dark-fermentation systems and the CO–water shift
reaction of R. gelatinosus CBS, however, appear promis-
ing. Bioreactors of reasonable size would be suFcient to
power the 5.0 kW fuel cell using unde6ned consortia of
mesophilic bacteria, enriched for Clostridium species. The
system reported by Chang et al. [55] in particular appears
most promising. A bioreactor of approximately 500 l (495 l,
in Table 5) would provide enough H2 to power a 2:5 kW
PEMFC, while a bioreactor of approximately 1000 l (989 l,
Table 5) would provide suFcient H2 to power a 5:0 kW
PEMFC. The CO–water shift reaction of R. gelatinosus CBS
is intriguing as it o=ers the potential to capture and reform
CO, and produce H2 . A bioreactor of approximately 624 l
would be required provide enough H2 to power the 2:5 kW
PEMFC, while a bioreactor of approximately 1250 l (1247 l,
Table 5) would provide suFcient H2 to power a 5:0 kW
PEMFC.
By way of comparison, the current state-of-the-art for
distributed, on-site production of hydrogen is via station-
ary electrolyzers which can generate H2 from H2 O at a rate
of 1000 l=h, or 40:3 mol=h (see www.stuartenergy.com).
Equivalent rates of H2 synthesis could be achieved by a
bioreactor of approximately 334 l using a dark-fermentation
system, or by a bioreactor of approximately 420 l contain-
ing R. gelatinosus CBS.
The CO-water shift reaction of R. gelatinosus CBS of-
fers an additional advantage. The CO–water shift reaction
also yields equi-molar amounts of CO2 , which R. gelati-
nosus CBS can assimilate into new cell biomass using
organic compounds, such as butyrate or propionate, as a
carbon source and ATP as an energy source [59]. Thus, in
addition to reformation of CO and H2 synthesis, this pro-
cess could be incorporated into an integrated energy system
to sequester CO2 in light, after H2 is recovered from the
system. Bioreactors of 1000 –1500 l in the basement of a
home is not unthinkable. Before electrical and natural gas
heating, it was normal for North American homes located
in northern latitudes to have, in their basements, tanks of
heating oil that were approximately this size.
While dark-fermentation systems and the CO-water shift
reaction may have practical applications, there are a number
of technical challenges that must be considered and over-
come before these systems can be used to produce H2 to
power a PEMFC. The most signi6cant of these problems
is whether the systems can be scaled up to volumes large
enough to generate the required Uow rate (22:1 mol H2 =h
for the 5:0 kW fuel cell). The working volume of the biore-
actor described that produced 121 mmol H2 =(l × h) was 3 l,
and used sucrose (at 20 g=l of culture) as the carbon source
for bacterial growth [55]. The biogas produced was 25 –35%
H2 and 65 –75% CO2 . Further research is required to deter-
mine if the rate of H2 production will remain at high levels
if these systems are scaled up to much larger volumes (183 l
or more), and if carbon sources other than pure sucrose can
be used. The major challenge for the CO–water shift reac-
tion is the problem of mass transfer: the gas must be avail-
able to the bacteria, in solution, at a suFcient concentra-
tion that the bacteria can absorb and metabolize eFciently.
This may require radically new technologies and bioreactor
designs.
182 D.B. Levin et al. / International Journal of Hydrogen Energy 29 (2004) 173 – 185
3.9. Biohydrogen: future directions
Both light-dependent (direct photolysis, indirect photol-
ysis, and photo-fermentation) and dark-fermenation biohy-
drogen systems are under intense investigation to 6nd ways
to improve both the rates of H2 production and the ultimate
yield of H2 . Hydrogen production by direct photolysis using
green algae is currently limited by three parameters [60]: (i)
solar conversion eFciency of the photosynthetic apparatus;
(ii) H2 synthesis processes (i.e. the need to separate the pro-
cesses of H2 O oxidation from H2 synthesis); and (iii) biore-
actor design and cost. A number of approaches to improve
H2 production by green algae are currently under investi-
gation. These include genetic engineering of light gathering
antennae [61], optimization of light input into photobioreac-
tors [62], and improvements to the two-phase H2 production
systems used with green algae [63,64].
Hydrogen production via indirect photolysis using
cyanobacteria can be improved by screening for wild-type
strains possessing highly active hydrogen evolving enzymes
(nitogenases and/or hydrogenases), in combination with
high heterocyst formation [17]. Genetic modi6cation of
strains to eliminate uptake hydrogenases and increase levels
of bidirectional hydrogenase activity may yield signi6cant
increases in H2 production. For example, a mutant strain of
Anabaena (AMC 414), in which the large subunit of the
uptake hydrogenase (hupL) was inactivated by a deletion
event [65], produced H2 at a rate that was more than twice
that of the parent wild-type strain, Anabaena PCC 7120
[66]. Finally, optimization of cultivation conditions such
as light intensity, pH, temperature, and nutrient content, as
well as maintaining low partial pressures of H2 and CO2
(see below) will contribute to increased H2 production.
Many of the parameters that limit H2 production
by green algae and cyanobacteria also apply to photo-
heterotrophic bacteria used in photo-fermentation systems.
E=orts to improve H2 production in these bacteria also
include elimination of competing microorganisms, such
as microalgae, using light 6lters [67], co-cultures of pho-
toheterotrophic bacteria with di=erent light utilization
characteristics [68], two-phase fermentation systems in
which photoheterotrophic bacteria utilize substrates pro-
duced by anaerobic bacteria during dark-fermentation pro-
cesses [69], novel photobioreactor designs [70,68], and use
of speci6c waste streams as substrate for photo-fermentation
[71].
Dark-fermentation systems also appear to have the great
potential to be developed as practical biohydrogen systems.
Substantial improvements, however, can be made through
rapid gas removal and separation, bioreactor design, and
genetic modi6cations in the microorganisms.
Improvements in gas separation will contribute to signi6-
cant increases in H2 production. As discussed above, the pH2
is an extremely important factor for continuous H2 synthesis.
As H2 concentrations increase, H2 synthesis decreases and
metabolic activity shifts to pathways that synthesize more
reduced substrates. The concentration of CO2 also a=ects
the rate of synthesis and 6nal yield of H2 . Cells synthesize
succinate and formate using CO2 , pyruvate, and reduced
nicotinamide adenine dinucleotide (NADH) via the hexose
monophosphate pathway [3]. This pathway competes with
reactions in which H2 is synthesized by NADH-dependent
hydrogenases (which oxidize NADH to NAD+). EFcient
removal of CO2 from the fermentation system would reduce
competition for NADH and thus result in increased H2 syn-
thesis.
In dark-fermentation processes, this problem is com-
pounded by the fact that the gas produced is a mixture of
primarily H2 and CO2 , but may also contain other gases
such as CH4 , H2 S, or ammonia (NH4 ). Moreover, the H2
content of the gas mixture may be low (¡ 50%). PEMFCs
require high-purity H2 (¿ 99%) and cannot tolerate CO at
concentrations ¿ 10 ppm [7]. To both maintain continu-
ous H2 synthesis and remove diluting (CO2 ; CH4 ) and/or
contaminating (CO) gases, rapid removal of the gases and
puri6cation of the H2 are essential.
Methods to enhance H2 production by removal of H2 and
CO2 include sparging with N2 or argon (Ar) gas and ap-
plying a vacuum to the head space to reduce the H2 partial
pressure [72]. An increase in H2 production of over 50%
was obtained after periodic sparging with N2 [73]. Too much
sparging, however, dilutes the H2 and creates a serious prob-
lem with respect to separation of the H2 from the sparging
gas.
Removal and selective puri6cation of H2 has been
demonstrated using membrane technologies. A hollow
6ber/silicone rubber membrane e=ectively reduced biogas
partial pressure in a dark-fermentation system, resulting
in a 10% improvement in the rate of H2 production and a
15% increase in H2 yield [74], and a non-porous, synthetic
polyvinyltrimethylsilane (PVTMS) membrane was used
for production of high-purity H2 from three di=erent H2
producing bioreactor systems [75].
Substantial gains in H2 production can also be
achieved through optimization of bioreactor designs. Using
6xed-bed bioreactors containing an unde6ned consor-
tium of mesophilic bacteria, Chang et al. [8] observed
rates of H2 synthesis far greater than other studies
(121 mmol H2 =(l × h)). This remarkable rate of H2 pro-
duction was achieved using activated carbon as a support
matrix that allowed retention of the H2 producing bacteria
within the bioreactor, plus a membrane 6lter system to
remove the biogas and maintain low gas partial pressures.
A theoretical study of H2 production by (hyper)thermo-
philic bacteria in a high rate bioreactor was conducted by van
Groenesttijn et al. [76]. In this system, (hyper)thermophilic
bacteria would be growing as a bio6lm within an anaerobic
trickling 6lter containing packing material with a very high
surface area. The liquid-suspended biomass substrate would
be passed continuously through the 6lter, so that the biomass
substrate, the H2 producing bacteria, and the resulting gas
phase would be in close proximity. Low H2 and CO2 partial
 D.B. Levin et al. / International Journal of Hydrogen Energy 29 (2004) 173 – 185
pressures would be maintained by stripping the H2 gas from
the bioreactor using steam within the 6lter.
Finally, gains in H2 production may be achieved through
genetic modi6cation of H2 producing bacteria. Hydrogen
producing strains of bacteria can be genetically modi6ed
in several ways to increase H2 synthesis, including (1)
over-expression of cellulases, hemi-cellulases, and lignases
that can maximize substrate (glucose) avaliability; (2)
elimination of uptake hydrogenases; (3) over-expression of
H2 evolving hydrogenases that have themselves been modi-
6ed to be oxygen tolerant; and (4) elimination of metabolic
pathways that compete for reducing equivalents required
for H2 synthesis.
4. Concluding remarks
Biohydrogen technologies are still in their infancy. Ex-
isting technologies o=er potential for practical application,
but if biohydrogen systems are to become commercially
competitive they must be able to synthesize H2 at rates that
are suFcient to power fuel cells of suFcient size to do
practical work. Further research and development aimed at
increasing rates of synthesis and 6nal yields of H2 are essen-
tial. Optimization of bioreactor designs, rapid removal and
puri6cation of gases, and genetic modi6cation of enzyme
pathways that compete with hydrogen producing enzyme
systems o=er exciting prospects for biohydrogen systems.
Even a 10-fold increase in the rate of H2 synthesis by
some dark-fermentation systems would reduce bioreactor
size dramatically. This would greatly facilitate overcom-
ing the engineering challenges of scale up, and create new
opportunities for practical applications.
Acknowledgements
We wish to thank the following people for their encour-
agement and helpful comments during the preparation of
this manuscript: Maria Ghirardi, Freda Hawkes, Pin-Ching
Maness, and Ed van Niel.
References
[1] Bockris JO’M. The origin of ideas on a hydrogen economy and
its solution to the decay of the environment. Int J Hydrogen
Energy 2002;27:731–40.
[2] Dunn S. Hydrogen futures: toward a sustainable energy
system. Int J Hydrogen Energy 2002;27:235–64.
[3] Das D, Nejat Veziroglu T. Hydrogen production by biological
processes: a survey of literature. Int J Hydrogen Energy
2001;26:13–28.
[4] Hallenbeck P, Benemann JR. Biological hydrogen production:
fundamentals and limiting processes. Int J Hydrogen Energy
2002;27:1185–94.
[5] Nandi R, Sengupta S. Microbial production of hydrogen: an
overview. Crit Rev Microbiol 1998;24:61–84.
183
[6] Kempton W, Tomic J, Letendre S, Brooks A, Lipman
T. Vehicle-to-grid power: battery, hybrid, and fuel cell
vehicles as resources for distributed electric power in
California. Working Paper UCD-ITS-RR-01-03, Institute of
Transportation Studies, 2001.
[7] Larminie J, Dicks A. Fuel cell systems explained. New York:
Wiley, 2000.
[8] Moran M, Shapiro H. Fundamentals of engineering
thermodynamics, 3rd ed. New York: Wiley, 1996.
[9] Ghirardi ML, Zhang L, Lee JW, Flynn T, Seibert M,
Greenbaum E, Melis A. Microalgae: a green source of
renewable H2 . Trends Biotechnol 2000;18:506–11.
[10] Adams MWW. The structure and mechanism of iron-
hydrogenases. Biochem Biophys Acta 1990;1020:115–45.
[11] Wyko= DD, Davies JP, Melis A, Grossman AR. The
regulation of photosynthetic electron-transport during nutrient
deprivation in Chlamydomonas reinhardtii. Plant Physiol
1998;117:129–39.
[12] Kosourov S, Tsygankov A, Seibert M, Ghirardi ML. Sustained
hydrogen photoproduction by Chlamydomonas reinhardtii:
e=ects of culture parameters. Biotechnol Bioeng 2002;78:
731–40.
[13] Melis A, Zhang L, Forestier M, Ghirardi ML, Seibert M.
Sustained photobiological hydrogen gas production upon
reversible inactivation of oxygen evolution in the green alga
Chlamydomonas reinhardtii. Plant Physiol 2000;122:127–35.
[14] Schopf JW. The fossil record. Tracing the roots of the
cyanobacterial lineage. In: Whitton BA, Potts M, editors.
The ecology of cyanobacteria. Dordrecht, The Netherlands:
Kluwer Academic Publishers, 2000.
[15] Tamagnini P, Axelsson R, Lindberg P, Oxelfelt F, Wunschiers
R, Lindblad P. Hydrogenases and hydrogen metabolism in
cyanobacteria. Microbiol Mol Biol Rev 2002;66:1–20.
[16] Hansel A, Lindblad P. Mini-review: toward optimization
of cyanobacteria asbiotechnologically relevant producers of
molecular hydrogen, a clean energy source. Appl Environ
Microbiol 1998;50:153–60.
[17] Pinto FAL, Troshina O, Lindblad P. A brief look at three
decades of research on cyanobacterial hydrogen evolution. Int
J Hydrogen Energy 2002;27:1209–15.
[18] Howarth DC, Codd GA. The uptake and production of
molecular hydrogen by unicellular cyanobacteria. J Gen
Microbiol 1985;131:1561–9.
[19] Masukawa H, Nakamura K, Mochimaru M, Sakurai H.
Photohydrogen production and nitrogenase activity in some
heterocystous cyanobacteria. BioHydrogen 2001;II:63–6.
[20] Sveshnikov DA, Sveshnikov NV, Rao KK, Hall DO.
Hydrogen metabolism of Anabaena variabilis in continuous
cultures and under nutritional stress. FEBS Lett 1997;147:
297–301.
[21] Arik T, Gunduz U, Yucel M, Turker L, Sediroglu V, Eroglu
I. Photoproduction of hydrogen by Rhodobacter sphaeroides
OU001. In: Viroglu TN, Winter CJ, Baselt JP, Kreysa G,
editors. Proceedings of the 11th World Hydrogen Energy
Conference, Stuttgart, Germany. Frankfurt: Scon & Wetzel
GmbH, 1996. p. 2417–26.
[22] Bolton JR. Solar photoproduction of hydrogen. Sol Energy
1996;57:37–50.
[23] Fedorov AS, Tsygankov AA, Rao KK, Hall DO. Hydrogen
photoproduction by Rhodobacter sphaeroides immobilised on
polyurethane foam. Biotechnol Lett 1998;20:1007–9.
184 D.B. Levin et al. / International Journal of Hydrogen Energy 29 (2004) 173 – 185
[24] Tsygankov AA, Hirata Y, Miyake M, Asada Y, Miyake J.
Photobioreactor with photosynthetic bacteria immobilized on
porous glass for hydrogen photoproduction. J Ferment Bioeng
1994;77:575–8.
[25] Tsygankov AA, Fedorov AS, Laurinavichene TV, Gogotov
IN, Rao KK, Hall DO. Actual and potential rates of
hydrogen photoproduction by continuous culture of the purple
non-sulphur bacteria Rhodobacter capsulatus. Appl Microbiol
Biotechnol 1998;49:102–7.
[26] Zurrer H, Bachofen R. Hydrogen production by the
photosynthetic bacterium, Rhodospirillum rubrum. Appl
Environ Microbiol 1979;37:789–93.
[27] Fascetti E, Todini O. Rhodobacter sphaeroides RV cultivation
and hydrogen production in a one- and two-stage chemostat.
Appl Microbiol Biotechnol 1995;22:300–5.
[28] Zurrer H, Bachofen R. Aspects of growth and hydrogen
production of the photosynthetic bacterium Rhodospirillum
rubrum in continuous culture. Biomass 1982;2:165–74.
[29] Francou N, Vignais PM. Hydrogen production by
Rhodopseudomonas capsulata cells entrapped in carrageenan
beads. Biotechnol Lett 1984;6:639–44.
[30] Vincenzini M, Materassi R, Sili C, Florenzano G. Hydrogen
production by immobilized cells III. Prolonged and stable
H2 photoevolution by Rhodopseudomonas palaustris in
light-dark-cycles. Int J Hydrogen Energy 1986;11:623–6.
[31] Tsygankov AA, Fedorov AS, Talipova IV, Laurinavichene
TV, Miyake J, Gogotov IN, Rao KK, Hall DO. Application
of immobilized phototrophic bacteria for simultaneous waste
water treatment and hydrogen photoproduction. Appl Biochem
Microbiol 1998;34:1–5 (in Russian).
[32] Jouanneau Y, Lebecque S, Vignais PM. Ammonia and light
e=ect on nitrogenase activity in nitrogen-limited continuous
cultures of Rhodopseudomonas capsulata: role of glutamate
synthetase. Arch Microbiol 1984;119:326–31.
[33] Willison JC, Jouanneau Y, Michalski WP, Colbeau A,
Vignais PM. Nitrogen 6xation and H2 metabolism in
the photosynthetic bacterium Rhodopseudomonas capsulata.
In: Papageorgiou GC, Packer L, editors. Photosynthetic
prokaryotes: cell di=erentiation and function. New York:
Elsevier, 1983. p. 333–52.
[34] Tsygankov AA, Fedorov AS, Talipova IV, Laurinavichene
TV, Miyake J, Gogotov IN. Use of immobilized
phototrophic microorganisms for waste water treatment
and simultaneous production of hydrogen. Appl Biochem
Microbiol 1998;34:362–6 (in Russian).
[35] Champine JE, U=en RL. Membrane topography of anaerobic
carbon monoxide oxidation in Rhodocyclus gelatinosus.
J Bacteriol 1987;169:4784–9.
[36] Kerby RL, Ludden PW, Robert GP. Carbon monoxide-
dependent growth of Rhodospirillum rubrum. J Bacteriol
1995;177:2241–4.
[37] U=en RL. Metabolism of carbon monoxide by
Rhodopseudomonas gelatinosa: cell growth and properties of
the oxidation system. J Bacteriol 1983;155:956–65.
[38] Wakim BT, U=en RL. Membrane association of the
carbon monoxide oxidation system in Rhodopseudomonas
gelatinosa. J Bacteriol 1982;153:571–3.
[39] Maness PC, Weaver PF. A potential bioremediation role for
photosynthetic bacteria. In: Sikdal SK, Irvine RL, editors.
Bioremediation: principles and practice, vol II. Technomic
Publishing Co, Inc., Lancaster PA, 1997.
[40] U=en RL. Anaerobic growth of a Rhodopseudomonas species
in the dark with carbon monoxide as sole carbon and energy
substrate. Proc Natl Acad Sci USA 1976;73:3298–302.
[41] Watt AS, Huang J, Smolinski S, Maness PC, Wolfrum EJ.
The water–gas shift pathway In: Rubrivivax gelatinosus. In
preparation.
[42] Maness PC, Weaver PF. Hydrogen production from
a carbon-monoxide oxidation pathway in Rubrivivax
gelatinosus. Int J Hydrogen Energy 2002;27:1407–11.
[43] Maness PC, Smolinski S, Dillon AC, Heben MJ, Weaver PF.
Characterization of the oxygen tolerance of a hydrogenase
linked to a carbon monoxide oxidation pathway in Rubrivivax
gelatinosus. Appl Environ Microbiol 2002;68:2633–6.
[44] Hawkes FR, Dinsdale R, Hawkes DL, Hussy I.
Sustainable fermentative biohydrogen: challenges for process
optimization. Int J Hydrogen Energy 2002;27:1339–47.
[45] Dabrock B, Bahl H, Gottschalk G. Paramenters a=ecting
solvent production by Clostridium pasteurianum. Appl
Environ Microbiol 1992;58:1233–9.
[46] Lee MJ, Zinder SH. Hydrogen partial pressures in a
thermophilic acetate-oxidizing methanogenic co-culture. Appl
Environ Microbiol 1988;54:1457–61.
[47] van Niel EWJ, Claassen PAM, Stams AJM. Substrate and
product inhibition of hydrogen production by the extreme
thermophile Caldicellulosiruptor saccharolyticus. Biotechnol
Bioeng 2002;81:255–62.
[48] Adams MWW. The metabolism of hydrogen by extremely
thermophilic sulphur-dependent bacteria. FEMS Microbiol
Rev 1990;75:219–38.
[49] Taguchi F, Yamada K, Hasegawa K, Saito-Taki T, Hara K.
Continuous hydrogen production by Clostridium sp. No. 2
from cellulose hydrolysate in an aqueous two-phase system.
J Ferment Bioeng 1996;82:80–3.
[50] Taguchi F, Mizukami N, Yamada K, Hasegawa K, Saito-Taki
T. Direct conversion of cellulosic materials to hydrogen by
Clostridium sp. strain No. 2. Enzyme Microbiol Biotechnol
1995;17:147–50.
[51] Taguchi F, Mizukami N, Hasegawa K, Saito-Taki T. Microbial
conversion of arabinose and xylose to hydrogen by a newly
isolated Clostridium sp. No. 2. Can J Microbiol 1994;40:228
–33.
[52] Taguchi F, Mizukami N, Saito-Taki T, Hasegawa K. Hydrogen
production from continuous fermentation of xylose during
growth of Clostridium sp. strain No. 2. Can J Microbiol
1995;41:536–40.
[53] Taguchi F, Hasegawa K, Saito-Taki T, Hara K. Simultaneous
production of xylanase and hydrogen using xylan in batch
cultures of Clostridium sp. strain X 53. J Ferment Bioeng
1996;18:178–80.
[54] Lay JJ. Modeling and optimization of anaerobic digestion
sludge converting starch to hydrogen. Biotechnol Bioeng
2000;68:269–78.
[55] Chang J-S, Lee K-S, Lin P-J. BioHydrogen Production
with 6xed-bed bioreactors. Int J Hydrogen Energy 2002;27:
1167–74.
[56] Ueno Y, Otauka S, Morimoto M. Hydrogen production from
industrial wastewater by anaerobic microUora in chemostat
culture. J Ferment Bioeng 1996;82:194–7.
[57] van Niel EWJ, Budde MAW, de Haas GG, van
der Wal FJ, Claassen PAM, Stams AJM. Distinctive
properties of high hydrogen producing extreme thermophiles,
 D.B. Levin et al. / International Journal of Hydrogen Energy 29 (2004) 173 – 185
Caldicellulosiruptor saccharolyticus and Thermotoga el@i.
Int J Hydrogen Energy 2002;27:1391–8.
[58] Lay JJ. Biohydrogen generation by mesophilic anaerobic
fermentation of microcrystalline cellulose. Biotechnol Bioeng
2001;74:280–7.
[59] Madigan MT, Martinko JM, Parker J. Biology of
microorganisms. Upper Saddle River, NJ: Prentice-Hall, 1996.
p. 649.
[60] Melis T. Green alga production: process, challenges, and
prospects. Int J Hydrogen Energy 2002;27:1217–28.
[61] Polle JEW, Kanakagiri S, Jin E, Masuda T, Melis A.
Truncated chlorophyll antenna size of the photosystems—
a practical method to improve microalgal productivity and
hydrogen production in mass culture. Int J Hydrogen Energy
2002;27:1257–64.
[62] Gordon J. Tailoring optical systems to optimized
photobioreactors. Int J Hydrogen Energy 2002;27:1175–84.
[63] Laurinavichene TV, Tolstygina IV, Galiulina RR, Ghirardi
ML, Seibert M, Tsygankov AA. Dilution methods to
deprive Chlamydomonas reinhardtii cultures of sulphur for
subsequent hydrogen photoproduction. Int J Hydrogen Energy
2002;27:1245–50.
[64] Tsygankov AA, Kosourov S, Seibert M, Ghirardi ML.
Hydrogen photoproduction under continuous illumination by
sulphur-deprived, synchronous Chlamydomonas reinhardtii
cultures. Int J Hydrogen Energy 2002;27:1139–44.
[65] Carrasco CD, Buettner JA, Golden JW. Programmed DNA
rearrangement of a cyanobacterial hupL gene in heterocysts.
Proc Natl Acad Sci USA 1995;92:791–5.
[66] Lindblad P, Christensson K, Lindberg P, Federov A, Pinto F,
Tsygankov A. Photoproduction of H2 by wildtype Anabaena
PCC 1720 and a hydrogen uptake de6cient mutant: from
laboratory to outdoor culture. Int J Hydrogen Energy
2002;27:1271–81.
[67] Ko I-B, Noike T. Use of blue optical 6lters for suppression
of growth of algae in hydrogen producing non-axenic cultures
of Rhodobacter sphaeroides RV. Int J Hydrogen Energy
2002;27:1297–302.
185
[68] Kondo T, Arawaka M, Wakayama T, Miyake J. Hydrogen
production by combining two types of photosynthetic
bacteria with di=erent characteristics. Int J Hydrogen Energy
2002;27:1303–8.
[69] Lee C-M, Chen P-C, Wang C-C, Tung Y-C. Photohydrogen
production using purple non-sulfur bacteria with hydrogen
fermentation reactor e[uent. Int J Hydrogen Energy
2002;27:1308–14.
[70] Hoekema S, Bijmans M, Janssen M, Tramper J, Wij=els
RH. A pneumatically agitated Uat-panel photobioreactor with
gas recirculation: anaerobic photoheterotrophic cultivation
of a purple non-sulfur bacterium. Int J Hydrogen Energy
2002;27:1331–8.
[71] Zhu H, Ueda S, Asada Y, Miyake J. Hydrogen production
as a novel process of wastewater treatment—studies on tofu
wastewater with entrapped R. sphaetoides and mutagenesis.
Int J Hydrogen Energy 2002;27:1349–58.
[72] Kataoka N, Miya A, Kiriyama K. Studies on hydrogen
production by continuous culture of hydrogen producing
anaerobic bacteria. Proceedings of the Eighth International
Conference on Anaerobic Digestion, vol 2. Sendai, Japan,
1997. p. 383–90.
[73] Mizuno O, Dinsdale R, Hawkes FR, Hawkes DL, Noike
T. Enhancement of hydrogen production from glucose by
nitrogen gas sparging. Bioresource Technol 2000;73:59–65.
[74] Liang T-M, Cheng S-S, Wu K-L. Behavioral study on
hydrogen fermentation reactor installed with silicone rubber
membrane. Int J Hydrogen Energy 2002;27:1157–65.
[75] Teplyakov VV, Gassanova LG, Sostina EG, Slepova
EV, Modigell M, Netrusov AI. Lab-scale bioreactor
integration with active membrane system for hydrogen
production: experience and prospects. Int J Hydrogen Energy
2002;27:1149–55.
[76] van Groenestijn JW, Hazewinkel JHO, Nienord M, Bussmann
PJT. 2002. Energy aspects of biological hydrogen production
in high rate bioreactors operated in the thermophilic
temperature range. Int J Hydrogen Energy 2002;27:1141–7.