© 1996 Nature Publishing Group http://www.nature.com/naturebiotechnology
FE A TU R E H YD R O GE N B I O TE CH N OL O G Y
Hydrogen biotechnology:
Progress and prospects
Whether one considers the “light side” or the “dark side” of hydrogen
production, significant progress is being made.
John Benemann
The popularity of hydrogen as a fuel source
follows the “crises” resulting from the use of
nonrenewable fuels. During the energy crisis of
the 1970s, hydrogen was touted as the “fuel of
the future”—and a great deal of time and money
were put into researching its possible sources
and applications. After the price of oil dropped,
hydrogen and other alternative energy
technologies were no longer pursued on various
national agendas. With the 1990s concerns about
the “greenhouse effect,” a new crisis reignited
interest in hydrogen as a fuel: Hydrogen would
not contribute directly to global climate change
and it would also reduce air pollution. Given the
economic uncertainties and environmental
hazards of fossil fuels, working out the technical
and economic feasibility of hydrogen production
is of major importance as we enter the 21st
century.
Biotechnology is uniquely poised to make a
significant contribution to this effort. The
groundwork for creating renewable hydrogen
production systems through either the dark
fermentations of low-cost substrates or wastes or
through “biophotolysis”—hydrogen production
through photosynthetically splitting water—have
already been demonstrated in scientific
publications in this journal1 and others. This
article gives an overview of the problems and
possibilities for biotechnology in developing a
renewable hydrogen industry.
Biological hydrogen production
Starting in the late 1800s2, basic research
established that algae and bacteria could produce
hydrogen. But it wasn’t until the early 1970s that
biological hydrogen production was first
seriously considered as a practical possibility.
This was largely the result of several National
Science Foundation (NSF, Washington, DC)
sponsored meetings on biological hydrogen
production1. The focus of these early meetings
was chiefly on
John Benemann is a consultant to the
environmental biotechnology and energy
industry, 343 Caravelle Dr., Walnut Creek, CA
94598 (jbenemann@aol.com).
hydrogen production through photosynthetic
processes.
Encouraging
initial
results
demonstrated that hydrogen could be produced
from water by illuminating a mixture of spinach
chloroplast membranes and two bacterial conversion efficiencies—about 10%—and then
proteins—a hydrogenase, to produce the efficiently transferred to hydrogenase. Currently,
photosynthetic organisms like higher plants
hydrogen, and a ferredoxin, to shutcapture only 3-4% of sunlight’s available energy
most. Additional technical challenges are
Interest in hydrogen resurfaced at
presented
in
engineering
low-cost
photobioreactors that can simultaneously provide
in the 1990s when it became
an environment conducive to the efficient
apparent that atmospheric
microbial catalysis of hydrogen production from
water and light as well as hydrogen capture.
pollution by fossil fuels is not
only unhealthy locally but might
also cause significant climate
changes globally. Once again,
biological hydrogen production
became a focus of governmental support.
tie electrons from the photosynthetic membranes
to the hydrogenase (see Fig. 1A)4. Experiments
with Anabaena cylindrica—a nitrogen-fixing
cyanobacteria (blue-green alga)—demonstrated
that in vivo hydrogen production was also
possible5. With the promise of practical
photobiological hydrogen production on the
horizon, a great deal of research was conducted
in this area up through the early 1980s 6,7, when
the energy crisis was declared over.
Interest in hydrogen resurfaced in the
1990s“,£ when it became apparent that
atmospheric pollution by fossil fuels is not only
unhealthy locally but might also cause
significant climate changes globally. Once again,
biological hydrogen production became a focus
of governmental support, particularly in
Germany and Japan, with a smaller effort in the
United States.
At present, these research efforts target the
splitting of water into hydrogen and oxygen by
photosynthetic systems as the ultimate goal for
hydrogen biotechnology. This approach, if
successful, would allow virtually unlimited
production of hydrogen from the earth’s most
plentiful available resources— water and light.
There are, however, tremendous biological
and engineering challenges to be overcome in
realizing this goal: The reducing power
generated by photosynthesis must be produced as
close as possible to the maximal possible solar
NATURE BIOTECHNOLOGY VOLUME 14 SEPTEMBER 1996
The light side
A major obstacle to overcoming the biological
challenges is the fact that photosynthetic
hydrogen production is a two-step process with
incompatible reaction steps: In the first step,
water is split to produce oxygen. In the second,
the reducing power of electrons is passed to
protons to make hydrogen through a
hydrogenase. Since oxygen is a strong inhibitor
of hydrogenase activity, a feedback inhibition
mechanism is inherent in the system.
One way around this dilemma is to use algae
that compartmentalize the two separate reactions
and use C02 as an intermediate to shuttle
between the two compartments (see Fig. IB). For
example, Anabaena cylindrica, a filamentous
cyanobacteria, compartmentalizes these into
vegetative cells, which generate the oxygen from
water and fixing C02, and specialized
nitrogenase-containing heterocyst cells, which
evolve hydrogen when N2 reduction is blocked.
An alternative is to use nonheterocystous
nitrogen-fixing cyanobacteria that separate the
H2 and Oz evolution steps temporally, such as a
day-night cycle, or spatially,
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GE N B I O TE C H N OL O G Y
FEATURE HYD
through separate bioreactions rather than through
two cell types (see Fig. 1C). Here too, COz acts
as an intermediate in the process. The problem
with using nitrogenfixing bacteria for hydrogen
production is that nitrogenase has a high ATP
requirement. This high metabolic energy
requirement lowers potential solar-energy
conversion efficiencies to unacceptable levels.
Since both cyanobacteria and green algae
are also able to evolve hydrogen by means of a
reversible hydrogenase—which requires much
less metabolic energy—the preferred direction
for research is to use this pathway. Two fundamentally different approaches are being studied.
In “direct biophotolysis” the reductant generated
by photosynthesis is directly transferred to
hydrogenase via reduced ferredoxin (see Fig.
1A). In “indirect biophotolysis,” the two
processes are kept apart in separate stages that
are joined through C02 fixation and release—
similar to non-heterocystous cyanobacteria (see
Fig. 1C).
The direct biophotolysis approach has
yielded some impressive results. Under labo
ratory conditions at low light intensities, it has
been demonstrated that the green alga
Chlamydomonas converts up to 22% of light
energy into hydrogen energy, equivalent to a
10% solar-energy conversion efficiency".
Moreover, some recent experiments in this area
appear to refute one of the longest held
Despite the excitement over
these developments, major
obstacles in applying this
process to real-world
applications remain.
dogmas of photosynthesis: the requirement for
two coupled photosystems—photosystem I and
photosystem II. Experimentally, photosystem Idefective mutants of Chlamydomonas were
found to be capable of carrying out both direct
biophotolysis and CO2
A. Direct biophotolysi
4
► Photosystems
► Hydrogenase
► Ferredoxin
B. Heterocystous nitrogen- fixing cyanobacteria
Recycle
°2
CO,
A
*
CO,
4
NADPH
t
>|CH,0],
11,0 ►Photosystems Vegetative
cell
*■ Ferredoxin
■ Nitrogenase
Heterocysr
C. Indirect biophotolysis: Non-heterocystous nitrogen- fixing cyanobacteria
Recycle
C02 -*■ t
• [CH201 ,
H,0
4
► Ferredoxin
► Photosystems
Nitrogenase * or
hydrogenase
The dark side
A nearer-term approach to practical biological
hydrogen production is the conversion of organic
substrates and wastes to hydrogen with anaerobic
hydrogen-fermenting bacteria. The problem with
D. Photofermentation: Photosynthetic bacteria NADPH
this concept is that in the dark, fermentative
bacteria produce only relatively small amounts of
[CH,0| ,
. ►Ferredoxin
■ Nitrogenase
► H,
hydrogen, typically only 10-20% stoichioIt
metrically6As
yields
increase,
hydrogen
Bacterial
photosystem
fermentations
become
thermodynamically
unfavorable. This led to the still widely held view
that “hydrogen production from wastes via
E. Microbial shift reaction: Photosynthetic bacteria C0+H20
fermentation is not at the present time feasible on
Greenbaum, E., Lee, J.W., Tevault.C.V., Blankinship, S.L., and Mets, L.J. 1995. Nature 376:438-441.
►H,+CO,
an industrial scale”7.
Boichenko, V. A.,
1996. Photosyn. Res. 47:291-292.
Ghirardi, M.L., Toon, S.R. and Seibert, M. 1995. Proceedings of the Annual Review Meeting of the DOE Office of Utility Technologies
Program
Miami,
OneHydrogen
way around
thisReview,
difficulty
is FL,
to use
1 cl s| age
photosynthesis)
1
2
3
”2
C02
* NADPH
fixation reactions1. Although this most surprising
finding remains controversial2, it raises the
possibility that photosynthetic efficiencies by
these mutants could be twice that of wild-type
algae.
Despite the excitement over these developments, major obstacles in applying this process
to real-world applications remain. As mentioned,
the oxygen produced by photosynthesis during
this reaction inhibits the hydrogenase enzyme
responsible for hydrogen production. Laboratory
experiments have shown that this inhibition can
be overcome by either consuming or sweeping
out the oxygen as it is produced. However, this
would not be practical for large-scale operations.
One promising solution is to develop microalgae
with an 02-insensitive hydrogenase reaction3. In
addition, direct biophotolysis approaches of this
type (as with heterocystous cyanobacterial
systems)
require
additional
engineering
considerations in that the entire solar-energy
capture area needs to be enclosed in a
photobioreactor.
The alternative “indirect biophotolysis”
concept is further along in demonstrating realworld applicability. In Japan, a two- stage,
indirect biophotolysis system is being tested at an
Osaka power plant4. Although the system is small
in scale, and uses photosynthetic bacteria rather
than algae in the hydrogen producing stage, it
demonstrates “proof of principle” and provides a
pathway for future development of this
technology.
In indirect processes of this type, a major
advantage is that the C02 fixation stage—
representing up to 90% of the total area
required—would be open ponds, which are much
cheaper than the closed photobioreactors required
for the H2 evolution stage. A preliminary
economic feasibility analysis— based on many
favorable assumptions—of a two-stage indirect
biophotolysis concept projects hydrogen costs as
low as $10 per million British thermal units
(MMBTU)'5. However, development of practical
biophotolysis processes, whether direct or
indirect, will require long-term R&D.
2nd stage
(hydrogen production!
Photosystem I ► ATP
1995.
F. DarkT.,
fermentations
Akano,
Miura, Y., Fukatsu, K., Miyasaka, H.,
[CH,0]Y.,
►Hydrogenase
► H,
2 ►Ferredoxin
Ikuta,
Matsumoto,
H. et al. 1996.
App. Biochem Biotechnol. 57/58:677-688.
5 Benemann, J.R. 1994. Hydrogen. Energy Progress X, Proceedings of the 10th World Hydrogen Energy Conference, Cocoa Beach, FL, June 20-24, 1994, pp. 931-940.
Figure
1. Biological
hydrogen
6 Solomon,
B.O., Zeng, A.P.,
Biebl, H.,production
Schlieker, H., processes.
Posten, C., and Deckwer, W.D. 1995. J. Biotechnol. 39:107-117.
7 Archer, D.B., and Thompson, L.A. 1987. J. App. Bacteriol. (Symp. Suppl.) 59s-70s.
4
1102
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GE N B I O TE C H N OL O G Y
photosynthetic bacteria which, in the light, can
convert organic substrates, including many
wastes, quantitatively into hydrogen and carbon
dioxide8 9 10 11 12 13 14 15 16 17 18 12 13 14 15 16 17 19 19 20
(Fig. ID). In principle, relatively little lightenergy
input—and
thus
only
small
photobioreactors—should be required to drive
this reaction, as most of the hydrogen energy is
derived from the organic substrates. So far,
however, measured photosynthetic efficiencies
have been disappointing—not much higher than
those obtained in biophotolysis reactions.
Reasons for this include the high-energy
demands by the nitrogenase-catalyzing hydrogen
evolution in these bacteria, and the relatively low
light intensities at which these bacteria operate—
preventing efficient use of full sunlight
intensities.
One exciting opportunity, with potential for
near-term practical applications of photosynthetic
bacteria, is their use as catalysts in the dark
conversion of carbon monoxide to hydrogen
(Fig. IE). Such a microbial “shift reaction” can
accomplish this conversion at room temperature
and in one pass, in contrast to chemical catalysts
that require high temperatures and multiple
stages. One problem that must be overcome to
make this process economically feasible is the
mass transfer limitation in such a process. One
enticing suggestion is that this could be overcome with gas phase bioreactors21. The microbial
shift reaction could be particularly useful for
small-scale applications that could most
effectively employ biomass gasification to
produce hydrogen.
Another potentially economically viable
approach to dark fermentative hydrogen
production, recently demonstrated in Nature
Biotechnology1, is to look for processes where
hydrogen would be the byproduct of a highvalue biotechnology product, such as gluconic
8
9
10
11
12
13
14
15
16
17
18
19
20
21
Woodward, J., Mattingly, S.M., Danson, M., Hough,
D., Ward, N., and Adams, M. 1996. Nature Biotechnology 14:872-874.
Jackson, D.D. and Ellms, J.W. 1996. Reports Mass.
State Board Health p. 410.
Gibbs, M., Hollaender, A., Kok, B., Krampitz, L.O., and
San Pietro, A. 1973. Proceedings of the Workshop
on Bio Solar Hydrogen Conversion. September 5-6
1973, Bethesda, MD.
Benemann, J.R., Berenson, J.A., Kaplan, N.O., and
Kamen, M.D. 1973. Proc. Nat Acad. Sci. USA
70:2317-2320.
Benemann, J.R. and Weare, N.M. 1974. Science
184:1917-175.
Weaver, P.F., Lien, S., and Seibert, M. 1980. Solar
Energy 24:3-45.
Benemann, J.R., Miyamoto, K., and Hallenbeck, P.C.
1980. Enzyme Microb. Technol. 2:103-111.
Smith, G.D., Ewart, G.D., and Tucker, W. 1992. Int.
J. Hydrogen Energy 17:695-698.
Boichenko, V.A. and Hoffman, P. 1994. Photosynthetica 30:527-552.
Markov, S.A., Bazin, M., and Hall, D.O. 1995. Adv.
Biochem. Eng. /Biotechnol. 52:61-86.
Greenbaum, E. 1988. Biophys. J. 54:365-368.
Ueno, Y, Morimoto, M., Ootsuka, S., Kawai, T., and
Satou, S. 1995. US Patent 5,464,539.
Sasikala, K., Ramana, C.V., Rao, P.R., and Kovacs,
K.L. 1993. Adv. Appi. Microbiol. 38:211-295.
Markov, S.A., Weaver, R., and Seibert, M. 1996.
Presentation at the Hydrogen 96 Meeting, Stuttgart,
Germany, June 1996.
1104
acid. The major problem with these types of
schemes is finding a high-value product for
which there is also a large need. In the case of
gluconic acid, hydrogen represents only about
1% of the total weight and value of the products
derived from the input glucose. With a total US
market of only about 50,000 tons of gluconic
acid per year this means this process would
produce only 500 tons of hydrogen. Since this is
rather a small amount of hydrogen—worth less
than $1 million annually—it is difficult to make
convincing economic arguments on its behalf.
Nevertheless, it could be of interest in this
industry, especially if other fermentations could
also be configured to yield hydrogen as a
byproduct.
More promising would be to convert lowercost substrates at high yields. Specifically,
deriving hydrogen from organic wastes using
dark fermentative processes (Fig. IF). The model
for such a process is the fermentation to methane
of waste sludges, animal manures, and food
process
ing wastes. Hydrogen fermentations would use
similar hardware to that used currently in
industrial methane fermentations. The economics
of hydrogen fermentations could be favorable at
even less than stoichiometric yields: Production
costs of methane fermentations range from about
$3-8 per MMBTU, whereas hydrogen produced
by the same types of hardware could be sold for
as much as $15 per MMBTU—depend-
At the moment, the United
States Department of Energy is
currently spending about $1
million per year for biohydrogen
production.
ing on location, scale, purity, and other factors.
In addition, waste treatment credits could cover
much of the costs of waste handling and
processing, just as they do in most methane
fermentations.
One near-term option in this regard is to
produce a mixture of hydrogen and methane in a
two-stage process. The first step would produce
hydrogen and organic acids, which would be
converted to methane in a second fermentation
stage. A selling point for this mixture is that
hydrogen-methane mixtures significantly reduce
air pollutants in internal combustion engines,
compared with using pure methane as a fuel.
The ultimate goal—and challenge—for dark
hydrogen fermentation R&D is to achieve high
yields of hydrogen. At present, yields of 10-20%
are currently reported. Economic feasibility will
not be sustainable until these yields reach the 6080% mark. The hope is that experimental
approaches to increase the yields of dark
anaerobic hydrogen fermentations, such as the
use of thermophilic bacteria, nutrient limitations,
and, most importantly, genetic and metabolic
engineering to redirect metabolic pathways, will
lead to these high levels of hydrogen production.
The far side
Concerns about global warming have increased
interest in hydrogen as a fuel. Biohydrogen
production will play an important role in making
this interest feasible. However, to accomplish
this goal will require sustained R&D support by
all the major countries that can benefit from it. At
the moment, the US Department of Energy
(Washington, DC) is currently spending about $1
million per year for biohydrogen production
research. In contrast, Japan spends about five
times as much in this area. These sums are far
below
what
both
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FE A TU R E H YD R O GE N B I O TE CH N OL O G Y
the long-term promises and near-term opportunities for practical applications call for: In
addition to delivering a sustainable fuel source,
this type of research will, no doubt, spawn other
commercial technologies. For example, bacterial
hydrogen metabolism is a key process in many
fermentations, is central to biocorrosion, and is
being studied in the bioremediation of hazardous
wastes. However, at present, the promise of
biological hydrogen production providing
efficient and economical energy conversion
processes will remain the primary justification
for this research.
It is still too early to predict which of the
many possibilities summarized in Figure 1 will
be ultimately successful, or how they would
appear in practice—as large-scale production
processes or small-scale roof-top conversion
devices. But when one considers the spectrum of
possibilities for hydrogen biotechnology, and the
likelihood of continued “crises” arising from our
use of non-renewable resources, there is little
doubt that this field will be important in shaping
the new, clean, energy and environmental
technologies that will be required in the 21st
century.
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