Journal of Applied Phycology 12: 291–300, 2000.

© 2000 Kluwer Academic Publishers. Printed in the Netherlands.

291

Hydrogen production by microalgae

John R. Benemann
Department of Plant and Microbial Biology, University of California Berkeley, Berkeley, CA 94720, USA

(for correspondence; phone +1-925-939-5864; fax +1-925-944-1205; e-mail jbenemann@aol.com)

Received 7 February 2000; revised 16 February 2000; accepted 17 February 2000

Key words: biophotolysis,fermentations, hydrogen, microalgae, photobioreactors, photosynthetic efficiencies

Abstract
The production of H2 gas from water and sunlight using microalgae, ‘biophotolysis’, has been a subject of
applied research since the early 1970s. A number of approaches have been investigated, but most proved to
have fundamental limitations or require unpredictable research breakthroughs. Examples are processes based on
nitrogen-fixing microalgae and those producing H2 and O2 simultaneously from water (‘direct biophotolysis’).
The most plausible processes for future applied R & D are those which couple separate stages of microalgal pho-
tosynthesis and fermentations (‘indirect biophotolysis’). These involve fixation of CO2 into storage carbohydrates
followed by their conversion to H2 by the reversible hydrogenase, both in dark and possibly light-driven anaerobic
metabolic processes. Based on a preliminary engineering and economic analysis, biophotolysis processes must
achieve close to an overall 10% solar energy conversion efficiency to be competitive with alternatives sources of
renewable H2 , such as photovoltaic-electrolysis processes. Such high solar conversion efficiencies in photosyn-
thetic CO2 fixation could be reached by genetically reducing the number of light harvesting (antenna) chlorophylls
and other pigments in microalgae. Similarly, greatly increased yields of H2 from dark fermentation by microalgae
could be obtained through application of the techniques of metabolic engineering. Another challenge is to scale-
up biohydrogen processes with economically viable bioreactors. Solar energy driven microalgae processes for
biohydrogen production are potentially large-scale, but also involve long-term and economically high-risk R&D.
In the nearer-term, it may be possible to combine microalgal H2 production with wastewater treatment.

Introduction get for renewable H2 fuel. Thus there is considerable

interest in alternative concepts, including proposed
biological processes using microalgae and other mi-
Hydrogen is starting to move from a fuel of the fu-
crobes (Benemann, 1996, 1998a). Since the early
ture to an energy carrier of the present, promising
1970s, biological H2 production has been the subject
greatly reduced pollution and increased fuel efficien-
of extensive applied research and development efforts,
cies. A major goal is the production of renewable H2
with major programs in Japan, Europe and the USA
fuel at affordable costs. Renewable H2 production was
during the past decade and a subject of several recent
made less urgent by the fossil fuel glut starting in
international conferences (Zaborsky, 1998; Miyake &
the early 1980s, but is now again of increasing pri-
San Pietro, 2000). Here the microalgae-based pro-
ority as the specter of global warming moves from
cesses are reviewed (for recent reviews see Boichenko
prediction to reality and as long-term supplies and
& Hoffman, 1994; Miyamoto, 1994, Schulz, 1996,
costs of fossil fuels are again of concern. However,
Nandi & Sengupta, 1998; Hansel & Lindblad, 1998).
none of the renewable H2 production processes cur-
In the late 19th century it was reported that a natural
rently available, such as photovoltaics-electrolysis and
bloom of the cyanobacterium Anabaena, when placed
gasification of biomass, can yet deliver H2 fuel for
into a glass jar, rapidly started to evolve hydrogen gas
less than US $20/GJ, a reasonable maximal cost tar-
292
(Jackson & Ellms, 1896). The first scientific investiga-
tion of H2 evolution by microalgae was the classic pa-
per by Gaffron and Rubin (1942), who demonstrated
that after a period of dark anaerobic ‘adaptation’, the
green alga Scenedesmus obliquus produces H2 in the
dark at low rates, with H2 production greatly stim-
ulated in the light, though only for relatively brief
periods. Other noteworthy observations were that un-
couplers and low CO2 concentrations stimulated light-
driven H2 production in green microalgae. Although
Gaffron & Rubin (1942) considered both water and
carbohydrates as the source of electrons for H2 evolu-
tion, they favored the latter as the main source. Spruit
(1958) found evidence supporting water as the main
electron donor in such a reaction. As pointed out by
Bishop (1966), the latter studies ‘clearly demonstrated
the incompatability’ of water as direct electron donor,
because ‘hydrogen evolution continued only so long
as the partial pressure of oxygen remained low’. The
reductant source for H2 evolution and its inhibition by
photosynthetically produced O2 have been the cent-
ral issues of this now extensive field of research for
the past half century. Kessler (1966) concluded that
H2 light-driven evolution was of adaptive value to
microalgae during the transition from dark anaerobic
conditions to oxygenic photosynthesis, as a means to
re-oxidize the electron transport pathway. Indeed, un-
der most conditions light-driven H2 production origin-
ates not from water, but from endogenous reductants
that feed into the electron transport chain between PSII
and PSI. Both dark and light-driven H2 production
allow for increased substrate and electron transport-
coupled phosphorylation under anaerobic conditions.
(For reviews of early literature, see: Hallenbeck &
Benemann, 1978; Weaver et al., 1980.)
This early basic research laid the foundations for
the applied research in photobiological H2 produc-
tion initiated after the energy crisis of the early 1970s
(Gibbs et al., 1973). The conceptually simplest pro-
cess for biohydrogen production was the direct trans-
fer of electrons from water to protons, by coupling
the water splitting and ferredoxin reducing reactions of
photosynthesis to a H2 evolving hydrogenase, a mech-
anism now termed ‘direct biophotolysis’. An alternat-
ive approach, ‘indirect biophotolysis’, was suggested
from work with anaerobically adapted green algae as
well as from studies of cyanobacteria: a temporal or
spatial separation of the O2 evolving and H2 pro-
ducing reactions, coupled through intermediate CO2
fixation into carbohydrates. A variation to this scheme
is to use microalgae to produce fermentatively organic
substrates that are then converted to H2 by photosyn-
thetic bacteria. These two central approaches to the
development of microalgae H2 production, direct and
indirect biophotolysis, are reviewed below. Two ad-
ditional issues that must be addressed in this context
are the efficiency of photosynthesis and the economics
of photobioreactors, both subjects of wider interest to
applied phycology.
Direct photobiolysis
Arnon et al. (1961) reported that the photosynthetic
apparatus of green plants could be coupled in vitro to
bacterial hydrogenases through the action of artificial
and natural electron donors, such as plant ferredoxin.
In these experiments the water splitting reaction (PSII)
was inhibited and electrons were transferred to PSI
from ascorbic acid by means of a coupling dye. In
the same laboratory, Benemann et al. (1969) used a
similar system with nitrogenase as electron acceptor
to assay for low redox potential electron carriers.
Thus it was a relatively small step to demonstrate a
similar in vitro reaction in which the water splitting
reaction of chloroplasts was used to demonstrate light-
driven H2 production from water as the electron source
(Benemann et al., 1973).
Experimentally this is a rather simple system: fresh
spinach leaves are blended, filtered through cheese
cloth, the green juice centrifuged and the green chloro-
plast fragments resuspended in 1 ml of buffer and
incubated in a small sealed flask in the presence of
a bacterial hydrogenase enzyme and the electron car-
rier ferredoxin. H2 can be detected in the gas phase
upon shining light on the flasks. Blocking of the water
splitting reaction (PSII) completely inhibits H2 evol-
ution. These experiments demonstrated that a direct
biophotolysis, could take place through action of the
complete photosynthetic apparatus in green plants –
the water splitting PSII and the ferredoxin-reducing
photosystem PSI:
H2 O −→ PSII −→> PSI −→ Ferredoxin
−→ Hydrogenase −→ H2
It was, however, also clear that this process was rather
feeble, fragile and fraught with difficulties. The rates
were very low, less than one-tenth those of other
photosynthetic reactions, and very short-lived, lasting
only about 15 minutes. This was mainly due to O2
accumulation, as demonstrated by the over six-fold
stimulation of rates and longevity of the reaction in

the presence of an O2 absorber or alternative electron
donor (Benemann et al., 1973).
Despite extensive research and improvements of
this in vitro system by many workers, the problem
of O2 inhibition has never been adequately addressed.
Nor is it plausible that the complex and fragile pro-
cess as photosynthesis could be used at an industrial
scale outside of living organisms. To cope with the
limited life-time of the photoactive centers, in particu-
lar PSII, microalgae have evolved very efficient repair
mechanisms (Melis, 1991) that could not be repro-
duced in vitro by any plausible extrapolation of known
technologies. Microalgae cultures are much more re-
silient and stable than subcellular chloroplast prepar-
ations and much cheaper to produce, and, therefore,
could at least in principle be considered in practical
applications.
As already discussed, early research suggested that
a direct biophotolysis process can take place in an-
aerobically adapted green microalgae. Much work has
been carried out in the past half century on such sys-
tems, generating a vast literature not easily summar-
ized. In brief, in green microalgae direct biophotolysis
is essentially a transitional reaction, which generally
takes place for only short periods of time, serving to
re-oxidize the electron transport chain from PSII to
ferredoxin and thus allowing oxygenic photosynthesis
to start. This accounts for the H2 ‘burst’ observed
in the first few minutes after shining light on dark
anaerobic cultures. Under some experimental condi-
tions, specifically when O2 concentrations are kept
very low (achieved through efficient gas sparging or
O2 scavenging reactions) and at relatively low light
intensities and/or absence of CO2 , a direct water-to-
hydrogen electron transfer process can be extended
for relatively long periods, essentially indefinitely. Us-
ing the microalga Chlamydomonas reinhardtii, the
light conversion efficiency of such a sustained direct
biophotolysis process was, under carefully controlled
laboratory conditions, determined to be as high as
22%, essentially the theoretical maximum (Green-
baum, 1988). This would extrapolate to about a 10%
solar conversion efficiency, if one could indeed extra-
polate to sunlight from the low light intensities used in
such laboratory experiments. However, in these exper-
iments O2 partial pressures were extremely low, only
a few parts per million in the gas phase, and thus prob-
ably only a few parts per billion in the liquid. This does
not allow the scale-up of such a process.
There is no apparent solution to the problem of
O2 inhibition of the hydrogenase reaction. Use of
293
O2 consuming reactions are not practical, and neither
are reversible oxygen binders, due to the difficulty
of regenerating these. Chlamydomonas mutants have
been isolated exhibiting H2 production at O2 par-
tial pressures somewhat higher than those tolerated
by the parent strains (McBride et al., 1977; Ghirardi
et al., 1998). However, it appears likely that these
are respiratory mutants, that more rapidly reduce O2
concentrations in the liquid culture, something coun-
terproductive for H2 production. Protein engineering
has been used to demonstrated that hydrogenase re-
actions can be made less sensitive to O2 inhibition
(McTavish et al., 1997). However, these are the so-
called uptake hydrogenases, which evolve only little
H2 and generally are not O2 sensitive. Recent work
on the mechanism and three-dimensional structure of
the reversible hydrogenases useful in H2 production,
(Happe et al., 1997; Peters et al., 1998), provide scant
hope for any eventual development a reversible hy-
drogenase reaction sufficiently resistant to O2 to be
able to operate under practical conditions of direct
biophotolysis (e.g. O2 partial pressures at or above
atmospheric). Thus, direct biophotolysis is still a basic
research problem.
Hydrogen production by nitrogen-fixing
microalgae
To avoid the difficulty of O2 inhibition in direct bio-
photolysis, so called ‘indirect biophotolysis’ processes
have been proposed in which first CO2 is fixed into
carbohydrates that are then used, in a separate step, to
generate H2 . This was first investigated with nitrogen-
fixing cyanobacteria, which produce H2 in the absence
of N2 , due to action of nitrogenase. It was relatively
easy to demonstrate active hydrogen production by
such organisms, as long as the uptake hydrogenase,
present in most nitrogen-fixing bacteria and a con-
founding variable in much of this research, is not
very active. Initially the filamentous Plectonema bory-
anum was used (Benemann, 1973), which turned out
to alternate periods of O2 evolution and CO2 fixation
into carbohydrates with periods of N2 fixation under
anoxic conditions, using the stored carbohydrates as
reductant sources (Weare & Benemann, 1974). In the
absence of N2 , such cyanobacteria evolve H2 , an indir-
ect biophotolysis process. An important finding was
that O2 production by photosynthesis was inhibited
by the N-limitation status of the cultures, avoiding
the problem of inactivation by simultaneously PSII-
294
mediated O2 evolution, at least until the carbohydrate
supply is exhausted and a new cycle of CO2 fixation
could start.
However, another type of cyanobacterium ap-
peared to be, at the time, potentially more prom-
ising: the filamentous heterocystous cyanobacteria.
The heterocyst is the site of nitrogen-fixation, with the
vegetative cells carrying out normal photosynthesis
(e.g. CO2 fixation and O2 evolution) and providing
the heterocyst with reduced carbohydrates (Weare &
Benemann, 1973). A heavy cell wall that reduces
diffusion of gases in combination with high respir-
ation rates allowed the heterocysts to maintain an
essentially anaerobic internal environment. Under an
inert gas, such as argon, such cyanobacteria simultan-
eously evolve H2 and O2 (Benemann & Weare, 1974).
Subsequent work demonstrated the long-term sustain-
ability of such a reaction (Weissman & Benemann,
1977), even in an outdoor demonstration of H2 pro-
duction using solar energy (Hallenbeck et al., 1978).
However, the efficiencies of light energy conversion
into H2 were disappointing, at most 1 to 2% in labor-
atory experiments at low light intensities and less than
0.3% outdoors with sunlight.
Extensive work from many laboratories has ex-
panded on this research (for reviews, see Lambert
& Smith 1981, Mitsui, 1992; Markov et al., 1993)
without resolving the fundamental problem of such
systems: the very low efficiency of solar energy con-
version into H2 . Although in part this also reflects
limitations of photosynthesis, discussed below, the
main reason for the low efficiencies was the very high
metabolic cost of nitrogenase-based, and in particular
heterocyst-based, H2 production. Heterocysts biosyn-
thesis and maintenance account for about half of the
energy metabolism of these cyanobacteria (Turpin et
al., 1987), without even considering the actual nitro-
genase reaction, which requires at least 4 ATP per H2
evolved. This metabolic energy must be provided by
light through PSI mediated cyclic phosphorylation or
by respiration, in either case reducing H2 production
and achievable solar conversion efficiencies to well
below half those expected from simpler, hydrogenase-
based, indirect biophotolysis processes. And the sim-
ultaneous production of H2 and O2 by heterocystous
cyanobacteria requires gas separation, at significant
economic cost, as would also be the case for direct
biophotolysis processes. For all these reasons, hetero-
cystous cyanobacteria specifically, and nitrogen-fixing
cyanobacteria in general, can no longer be recommen-
ded as subjects for applied microalgal H2 production
R&D.
H2 production by nitrogen-fixing photosynthetic
bacteria was proposed (Benemann, 1977) and extens-
ively studied for the past two decades as a method
for converting organic wastes to H2 fuel (Sasikala et
al., 1993). These bacteria can exhibit a light-driven
near-stoichiometric conversions of organic substrates
to H2 . However, solar energy conversion efficien-
cies proved to be rather low, not much better than
with cyanobacteria, both because the H2 -producing
enzyme is nitrogenase and also due to inherent in-
efficiencies of bacterial photosynthesis. Thus, as for
the nitrogen-fixing cyanobacteria, photosynthetic bac-
teria do not appear promising in applied biohydrogen
R&D. One approach, studied by several groups, is to
combine microalgae cultures that fix CO2 with photo-
synthetic bacteria that produce H2 . A small pilot plant
of such a process was operated in Japan for several
years, using two 2 m2 of open ponds for producing
a green algal (Chlamydomonas reinhardtii) biomass
and about 2 m2 of closed photobioreactors, with an
intermediate dark fermentation stage. In an impressive
demonstration of the largest and longest-duration mi-
croalgal biohydrogen production project carried thus
far, these researchers successfully operated this pro-
cess on a continuous basis, producing several liters of
H2 per day (Ikuta et al., 1998). They demonstrated the
ability to recycle algal cultures repeatedly through the
process, which included a separate dark fermentation
stage and a hollow fiber ultrafiltration unit to separate
the algal fermentation products and feed these to the
photosynthetic bacterial photobioreactors. However,
the process exhibited only low overall solar conversion
efficiencies, less than 0.3%, in large part due to the
inefficiency of the photosynthetic bacterial stage. As
many microalgae contain a reversible hydrogenase, it
should be possible to use only microalgae in such a
two-stage process.
Hydrogenase-based indirect biophotolysis
processes
Reversible hydrogenase-based indirect biophotolysis
processes have, at least conceptually, major advant-
ages over the nitrogenase-based systems: The specific
H2 evolution activities of reversible hydrogenases are
almost a thousand-fold higher than those of nitro-
genase and, most importantly, require no ATP. Of
course, there are also some drawbacks. Reversible hy-

drogenases in microalgae are generally expressed at
relatively low activities, resulting in H2 production
rates typically much lower than those observed with
nitrogenase-based processes. Also H2 production by
reversible hydrogenases is typically inhibited by rel-
atively low (<10%) partial pressures of H2 . However,
on balance and in principle, the advantages outweigh
the disadvantages, which could be plausibly overcome
by application of modern biotechnology techniques.
Thus, indirect biophotolysis systems based on revers-
ible hydrogenases present the most likely, at least
presently plausible, approach to practical development
of microalagal H2 production systems.
The first, and most difficult, step in any biophoto-
lysis process is to produce efficiently and store large
amounts of carbohydrates in the algae (see below).
After carbohydrate accumulation, the cells would be
removed from the growth pond or photobioreactor,
concentrated if required, and, through respiratory
O2 uptake, allowed to become anaerobic and activ-
ate or induce the hydrogenase enzyme. H2 evolution
would then commence, first in the dark through en-
dogenous fermentations, and then in a light-driven
(PSI-mediated) electron transport to convert remain-
ing stored carbohydrates and fermentation products
(e.g. acetate) to H2 . The depleted cells would be
re-used for additional cycles of CO2 fixation-H2 pro-
duction. That is, in brief, the concept of indirect
biophotolysis with microalgae (Benemann, 1998b).
Much information is available on each of these
steps, but significant gaps remain in our knowledge.
For one example only, at present the light-driven
step of H2 production has been demonstrated only
in green algae. For unknown reasons cyanobacteria,
which also have reversible hydrogenases, only seem
to produce H2 fermentatively in the dark, not in a
light-driven or stimulated reaction (Aoyama et al.,
1997). For green algae hydrogenase levels are often
not the limiting factor and it is possible to obtain
rather high initial rates for short periods (seconds to
minutes). However, sustained high rates have not been
achieved, possibly due to competition with other re-
actions. Progress in the genetics of the hydrogenases,
both reversible and uptake, in cyanobacteria and green
algae has been rapid in recent years (Hansel & Lind-
blad 1998, Schulz, 1996). However, important reac-
tions of practical interest remain to be demonstrated,
such as a light-driven dissimilation to H2 of the acet-
ate and other metablites excreted during anaerobic
fermentations by microalgae.
295
A major uncertainty is the extent to which microal-
gae (or, for that matter, other microbes) can generate
H2 from carbohydrates in dark fermentations. Dark
fermentations can be carried out in conventional fer-
menters, which are much cheaper than the closed pho-
tobioreactors required for light-driven processes (see
below). At present it is believed that at most one-third
of the H2 stored in carbohydrates could be recovered
in dark fermentations (Thauer, 1977), and the best H2
yields achieved thus far in such processes are about
20% of stoichiometric (Ueno et al., 1995). However, it
appears likely that applications of modern techniques
of molecular biology, specifically metabolic engin-
eering, could result in much higher yields, possibly
avoiding entirely the need for a light-driven H2 evolu-
tion stage (Keasling et al., 1998). The challenge is to
drive this reaction to near-completion against the large
overpressures of H2 encountered in typical anaerobic
fermentations due to mass transfer limitations (Pauss
et al., 1994).
Compared to the relatively extensive research on
direct biophotolysis and nitrogen-fixing cyanobac-
teria, hydrogenase-based indirect biophotolysis pro-
cesses have been somewhat neglected. Perhaps be-
cause the direct process was conceptually simpler
and the nitrogen-fixing cyanobacteria systems exper-
imentally easier for demonstrating H2 evolution. In-
deed, the rates of H2 production observed in the light
with anaerobically adapted green algae are generally
much lower than achievable with nitrogen fixing cy-
anobacteria, and generally of much shorter duration.
However, applying similar approaches as used with
cyanobacteria, that is nutrient limitation to increase
carbohydrate storage and repress photosynthetic O2
production, should also allow an improvement in
rates and longevity (Benemann, 1998b). Following-
up on this suggestion, Melis et al. (2000) used 1-
L cultures of sulfur-limited, mixotrophically (acetate
and light) grown Chlamydomonas reinhardtii and ob-
served about 2 mL H2 h−1 L−1 over two days. This
is only about one-tenth the rate and longevity ob-
tained with autotrophic heterocystous nitrogen-fixing
cyanobacteria under comparable conditions (Weiss-
man & Benemann, 1977). More problematically,
Melis et al. (2000) did not actually demonstrate an in-
direct biophotolysis process. Nevertheless, the funda-
mental advantages of indirect hydrogenase-mediated
processes suggest these as the main future direction
for applied R&D in this field.
Of course, enormous research challenges will be
faced in advancing from the present conceptual sys-
296
tems and low efficiency laboratory-scale experiments
to the large-scale, outdoor, high efficiency sustained
processes required in practical applications. Indeed,
this will require a complete genetic and biochemical
make-over of the algal cell, to redirect essentially the
entire metabolism of these organisms from cell-growth
to cycles of carbohydrate storage and H2 release. Al-
though each step in the process is based on known
enzymatic capabilities of microalgae, these must still
be joined together into one process in a single organ-
ism, operating at near maximal efficiency in outdoor
systems under full sunlight and subject to contamin-
ation and diurnal temperature fluctuations. And even
achieving these ambitious goals is not enough: the pro-
cess must also be contained within a very low capital
cost apparatus requiring only minimal operating costs.
These dual challenges, of very high efficiencies and
very low costs are, of course, also faced by other prac-
tical applications of microalgae, of which biohydrogen
production is but the most challenging example.
Photosynthetic solar conversion efficiencies
Since the earliest days of applied microalgal R&D
(Burlew, 1953), a basic assumption has been that
microalgae are more productive than higher plants.
However, the process of photosynthesis is similar in
both groups and there is no fundamental reason for
assuming that the former are more efficient. It can be
argued that as microalgae do not produce supporting
structures such as roots, their overall productivity can
be higher. However, microalgae have to contend with
other problems, such as the very high O2 tensions in
mass cultures.
Most importantly, photosynthesis by microalgae
cells saturates at rather low light intensities, typic-
ally less than one-tenth full sunlight. That is because
of their high contents of light gathering (‘antenna’)
chlorophyll and other pigments absorb, when exposed
to full sunlight, more photons than can be processed by
the photosynthetic machinery. The excess photons are
wasted as heat and fluorescence. Cells near the surface
of an algal culture receiving full sunlight waste up to
90% of the photons they capture, while those a few
cell layers below are starved for light. Averaged over
the culture depth, microalgae cultures waste roughly
70% of the total incoming sunlight, reducing solar
conversion efficiencies from a theoretical near 10%,
assuming no light saturation, to a maximum of about
3% of solar energy fixed into biomass energy.
This fundamental problem of applied microalgae
culture productivity was already recognized almost 50
years ago (Myers, 1957) and discussed by Kok (1973)
in the context of microalgal hydrogen production. As
was the solution: find or develop algal strains with
fewer antenna chlorophylls per reaction center. Such
cultures would exhibit a higher saturating light in-
tensity for photosynthesis but otherwise should exhibit
similarly high photosynthetic efficiency at all light
levels up to saturating intensities. Thus, fewer photons
would be captured, and wasted, at the surface of the
culture, resulting in an overall increase in culture pro-
ductivity. However, algal strains with small antenna
sizes have not been found in nature. The reason is that
large antenna are required for survival at low light in-
tensities, while wasting photons in full sunlight is no
great loss to the individual algal cell, pursuing its own
selfish objective of replication rather than the human
desire to maximial culture productivity. Thus evolu-
tion has favored large antenna sizes. In any event, not
finding algae in nature with small antenna sizes led to
a neglect of this approach to increasing photosynthetic
productivities (Benemann, 1990).
Nevertheless, under certain condition small an-
tenna sizes are observed in natural strains: when the
algal cells are stressed by a combination of high light
intensities particularly in combination with other en-
vironmental factors, such as high O2 concentrations
and/or CO2 limitation. In such environments the pho-
tosynthetic machinery is damaged beyond the capacity
of the endogenous repair mechanisms, and the cells re-
duce their antenna sizes to limit the damage and avoid
death. When such stressed cells, with small antenna
sizes are transferred to low light the damaged photo-
synthetic apparatus is first repaired and only then are
additional antenna chlorophylls added. Taking advant-
age of the brief time (less than one hour) while the
photosynthetic centers are mostly repaired but antenna
sizes are still small, it was recently demonstrated, us-
ing the green alga Dunaliella salina, that such cultures
can indeed exhibit both high rates and high efficiencies
of photosynthesis at high light intensities (Neidhardt et
al., 1998; Melis et al., 1998, 1999).
The next step is to demonstrate this phenomenon
on a sustained basis with algal mutants with con-
stitutively small low-antenna sizes. Nakajima and
Ueda (1997, 1999, 2000) used pigment deficient
mutants of a cyanobacterium (Synechocystis) and a
green alga (Chlamydomonas perigranulata) to demon-
strate increased productivities in continuous cultures
at high light intensities, some 25 to 50% higher for

the mutants than the wild type. Thus, the fundamental
validity of this approach has now been demonstrated,
even though the increases in productivities noted thus
far are only modest compared to the well over 200%
increase theoretically achievable. However, not all
pigment deficient mutants will exhibit increased ef-
ficiencies at high light intensities. A chlorophyll-less
mutant of the green alga Chlamydomonas reinhardtii
had reduced antenna size for PSII (but not PSI) but
also exhibited reduced photosynthetic efficiency and
growth rates at low light intensities (Polle et al.,
1999) which would also then be the case at high light
intensities.
Although this research is only in its initial stages,
both theory and experimental data suggest that rather
large improvements in productivities in outdoor algal
mass cultures are possible. The goal of a sustained
10% solar conversion efficiency into H2 by algal cul-
tures is considered both achievable and the maximum
feasible (Bolton, 1996), and must be the main re-
search objective in this field. Indeed, this research will
have applications well beyond hydrogen production,
as photosynthetic productivity by outdoor cultures is
the single most important factor in autotrophic mi-
croalgae production economics. Indeed, this research
has applications for higher plant agriculture, where
light saturation is less severe as in microalgal mass
cultures but still is a very significant factor in limiting
crop yields.
Photobioreactors and process economics
After several decades of basic and applied research mi-
croalgae biohydrogen production is still far away from
developing a practical process, or even demonstrat-
ing even a conceptual process at a laboratory scale.
And there has been, for the most part, a lack of even
preliminary analysis of how such processes could be
scaled-up and operated under realistic economic con-
straints. Competitive economics would most likely be
dictated by photovoltaic-electrolysis systems, which
are projected to still be above US $ 20/GJ even with
continued technological advances over the next decade
or longer (Block & Melody, 1991).
Indirect biophotolysis has recently been subjected
to a preliminary conceptual economic analysis (Be-
nemann, 1998b). This assumed that the first stage,
CO2 fixation into storage carbohydrates, would be car-
ried out in large (several hectares) unlined, paddle
wheel mixed, raceway-type ponds, similar to those
297
proposed for microalgal production of high lipid (oil)
biomass suitable for conversion to biodiesel (Bene-
mann, 1993). And such pond systems are used in both
wastewater treatment and for commercial microalgae
production. The algal culture would then be concen-
trated ten- to twenty-fold by settling, and held in a
large anaerobic fermenter (small, deep, covered la-
goons) to induce the hydrogenase enzyme and initiate
H2 production, along with other metabolites, such as
acetate. It was assumed that one-third of the H2 stored
in the carbohydrates would be released by anaerobic
fermentations. Then the culture would be transferred
to closed photobioreactors that expose the cells to
light, converting acetate and remaining carbohydrates
to H2 . Finally the depleted cells would be recycled to
the open ponds to repeat this cycle, for a total of ten
times in this conceptual analysis.
The conceptual engineering design and cost ana-
lysis of this concept was mostly based on prior work
(Benemann, 1993), which projected open pond costs
of US $5/m2, including earthworks, mixers, CO2 sup-
ply and transfer, and ancillary systems. The photobi-
oreactors were simply assumed, without any support-
ing analysis, to cost US $130/m2, including an overall
30% engineering and contingency factor. The CO2
fixation into carbohydrates was assumed to achieve
the above predicted 10% solar conversion efficiency
and the photohydrogen production stage assumed to
require only one photon per H2 evolved, a very min-
imal light input. Overall, the photobioreactor area was
only 10% of that of the open pond area, but repres-
ented about half the total system capital costs, with
the remainder divided about equally between the open
pond system and the gas handling subsystems (H2 col-
lection, clean-up and compression). Capital charges
(25% per annum, for depreciation, maintenance, re-
turn on capital, taxes etc.) amounted to almost 90% of
total costs. The final H2 costs were about US $10/GJ
for a favorable Southwestern site in the USA (Bene-
mann, 1998b). This did not, however, include some
ancillary systems, such as CO2 recovery and recyc-
ling. And, of course, such analyses are based on many
other very favorable assumptions and extrapolations.
Thus actual costs likely would increase significantly
in any detailed engineering design and cost estim-
ate. Therefore such early ‘study level’ analyses should
be used mainly to help focus R&D and engineering
designs on limiting factors. In this case the focus
clearly fell on the high cost of the photobioreactors,
a topic requiring more attention.
298
Subsequently Tredici et al. (1998) estimated that
a simple, slightly inclined internal gas exchange tu-
bular photobioreactor may cost as little as $50/m2,
based on costs of materials and estimated fabrication
and installation costs (without any engineering or con-
tingency allowance). This photobioreactor consisted
of forty 50 m long, 4.2 cm diameter tubes (glass or
teflon, at US $1/m), ganged together with a bottom
manifold and top degasser, positioned on a slightly in-
clined (about 10% slope) earthen platform and mixed
by airlift. The low cost projected for this design, which
nearly maximizes light interception, would allow both
stages of an indirect biophotolysis process (CO2 fix-
ation and H2 production) to be carried out in such
closed photobioreactors, dispensing with the need for
open ponds and the intermediate concentration step.
This could make such a process applicable to much
smaller scales than possible with the above two-stage
open pond /photobioreactor design. The total cost of
H2 was estimated at US $15/GJ H2 . As above, these
cost projections are does not include some critical
components, such as CO2 separation and recycling,
and make many favorable assumptions. Indeed, re-
cent work with such tubular photobioreactors (Radway
et al., 1999) suggests that the mass transfer assump-
tions, on which these economic analyses were based
were too optimistic, requiring design modifications
that would likely significantly increase both capital
and operating costs.
In any event, closed photobioreactors suitable for
H2 capture are likely to prove a major limiting design
and economic factor in any photobiological H2 pro-
duction process. This makes the development of dark
fermentation processes to generate H2 from stored car-
bohydrates of fundamental importance (Keasling et
al., 1998). This is because fermentation bioreactors
would be of much lower cost than photobioreactors.
However, closed photobioreactors, even if not needed
or affordable for photobiological H2 production itself,
would still be required in the initial build-up of the
culture to start-up the large open ponds. The highly
metabolically engineered strains that would be used in
such a process would require a relatively protected ve-
getative growth phase to avoid contamination, before
operating in the nutrient restricted environment of the
CO2 fixation systems. Of course, the problem of cul-
ture stability and contamination are major issues in the
development of this technology, as it is the case for all
algal mass culture processes. Alternative designs are
possible, such as a day-night cycle of CO2 fixation and
H2 production, plausibly carried out in closed photobi-
oreactors, with microalgal respiration and metabolism
creating the anaerobic conditions for hydrogenase ac-
tivation and providing the bioenergetic driving force
required for efficient dark H2 production.
Conclusions
This brief overview has inevitably had to omit many
important studies, such as those dealing with quantit-
ative rates of H2 production. The data available, even
when translatable into common units, are of limited
value in comparing different processes or even strains,
or projecting their future potential. Thus, rather than
extrapolating from laboratory data, the feasibility of
microalgae hydrogen production must presently be as-
sessed based on conceptual engineering designs and
plausible cost estimates. These, in turn, must be
derived from fundamental knowledge of microalgae
photosynthesis and metabolism, assuming the suc-
cessful application of the now available technologies
of molecular biology. Indeed, based on such analyses,
the processes that currently exhibit the highest and
most sustained rates of microalgal hydrogen produc-
tion, nitrogen-fixing cyanobacteria can be dismissed
as potential candidates for future development. That
is because they could not, even with additional R&D,
achieve the necessary efficiency goals mandated by
the economic analyses. Conversely, presently less well
developed systems, such as indirect biophotolysis and
dark fermentations approaches, must be favored in any
future applied R&D efforts, as they have, at least thus
far, potential for long-term development.
It would be most desirable to have processes that
could be developed in the near-term, without requir-
ing the very high efficiencies and low costs demanded
by the above cost analyses. One plausible approach
would be to combine microalgae wastewater treatment
with H2 production. Microalgae are already utilized
in wastewater treatment processes, and methane re-
covery through anaerobic digestion (fermentations)
is widely practiced at wastewater treatment facilit-
ies. The combination of these technologies into an
integrated microalgae waste treatment and methane
production process has been proposed for over 40
years (Oswald & Golueke, 1960). In such wastewater
treatment processes CO2 is often the limiting factor,
suggesting that CO2 be recycled internally and H2 be-
come a major fuel output. In this case, H2 would be
a by-product of the waste treatment process, greatly
reducing the economic constraints and performance

criteria, such as photosynthetic efficiencies. This could
plausibly allow for some nearer-term, even if of more
limited potential, applications of hydrogen production
from microalgae.
Over the past quarter century, microalgae biohydro-
gen production R&D has achieved little progress to-
wards the goals of practical and commercial processes.
This is not for the lack of interest or even R&D in-
vestments, as demonstrated by the extensive literature
and cumulative expenditure of well over a hundred
million dollars. Lack of progress was due to the con-
centration of efforts on approaches that no longer ap-
pear promising, specifically direct biophotolysis and
nitrogen-fixing cyanobacteria, together with a lack of
focus on engineering and economic realities. Research
must now concentrate on the key limiting factors: solar
conversion efficiencies for CO2 fixation by microalgae
cultures, the engineering of low-cost photobioreact-
ors and the development of high yielding dark H2
fermentations. The first two topics are also central
to the entire field of applied phycology, which can
both benefit from and contribute to this area of applied
R&D. Indeed, by exploring the limits of the possible
and even plausible, microalgae biohydrogen R&D can
make significant contributions to applied phycology.
Even a cursory analysis would conclude that any
direct biopotolysis process requiring low O2 tensions
is not be practical due to very high gas transfer require-
ments, without even considering the need for gas (O2
and H2 ) separations.
Acknowledgements
The work was supported by the US DOE Hydrogen
R&D Program under DOE-UC Berkeley Cooperat-
ive Agreement Number DE-FC36-98GO10278. The
views expressed are solely those of the author.
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