1. Biological conversion of carbon dioxide and hydrogen into liquid
fuels and industrial chemicals
Aaron S Hawkins1
, Patrick M McTernan2
, Hong Lian1
, Robert M Kelly1
and
Michael WW Adams2
Non-photosynthetic routes for biological fixation of carbon
dioxide into valuable industrial chemical precursors and fuels
are moving from concept to reality. The development of
‘electrofuel’-producing microorganisms leverages
techniques in synthetic biology, genetic and metabolic
engineering, as well as systems-level multi-omic analysis,
directed evolution, and in silico modeling. Electrofuel
processes are being developed for a range of
microorganisms and energy sources (e.g. hydrogen, formate,
electricity) to produce a variety of target molecules (e.g.
alcohols, terpenes, alkenes). This review examines the
current landscape of electrofuel projects with a focus on
hydrogen-utilizing organisms covering the biochemistry of
hydrogenases and carbonic anhydrases, kinetic and
energetic analyses of the known carbon fixation pathways,
and the state of genetic systems for current and prospective
electrofuel-producing microorganisms.
Addresses
1
Department of Chemical and Biomolecular Engineering, North Carolina
State University, Raleigh, NC 27695-7905, United States
2
Department of Biochemistry and Molecular Biology, University of
Georgia, Athens, GA 30602, United States
Corresponding author: Adams, Michael WW (adams@bmb.uga.edu)
Current Opinion in Biotechnology 2013, 24:376–384
This review comes from a themed issue on Energy biotechnology
Edited by Eric Toone and Han de Winde
For a complete overview see the Issue and the Editorial
Available online 16th March 2013
0958-1669/$ – see front matter, # 2013 Elsevier Ltd. All rights
reserved.
http://dx.doi.org/10.1016/j.copbio.2013.02.017
Introduction
There are intrinsic limitations to the production of biofuels
derived from photosynthetic organisms that impede the
development of a renewable liquid fuel industry at large-
scale. In particular, these include low efficiency of solar
energy conversion (Figure 1) and competition for agricul-
tural resources. Recent initiatives in the U.S. and else-
where have the objective of harnessing the molecular
mechanisms of non-photosynthetic organisms that can
utilize CO2 directly for the production of energy-dense
liquid fuels, which are now referred to as ‘electrofuels’
[1
,2,3
]. Electrofuel-producing microorganisms are being
developed that require the complementary expertise of
synthetic biologists, metabolic engineers, and microbiolo-
gists to equip native CO2-fixing species or autotrophs with
pathways fortargeted fuel production,or confer autotrophy
on heterotrophic host organisms, or both. The range of
possible sources currently being explored for low-poten-
tial, high-energy electrons to power an electrofuel process
includes hydrogen gas, formate, carbon monoxide and
electricity. This review will focus on electrofuel strategies
that use hydrogen gas as source of reducing power for CO2
fixation. Microorganisms that are able to use other sources
of electrons including electricity directly are discussed in
an accompanying review by Lovely [4]. A general scheme
for electrofuel productionfromhydrogenandCO2 isshown
in Figure 2.
CO2 fixation
The reduction of CO2, the most oxidized form of carbon,
into technologically useful organic compounds remains a
daunting task for abiological chemical catalysis. There
are, currently, six naturally occurring biological pathways
for carbon fixation, and these have been reviewed exten-
sively in recent years [5
,6,7,8]. Each pathway has
unique features arising from the ecological and molecular
context in which it evolved. Although there are many
examples of CO2-fixing carboxylases that are utilized for
metabolic purposes other than carbon assimilation, such
as energy conservation, anaplerosis, and redox-balancing
[1
,2,3
,9
,10], this review will focus on the autotrophic
CO2 fixation pathways that are relevant for electrofuel
production in addition to the primary host microorgan-
isms that are currently being considered that use hydro-
gen (Table 1).
The most ubiquitous CO2-fixation pathway is the Calvin–
Benson–Bassham (CBB) cycle found in plants, algae,
cyanobacteria, purple bacteria, and also in some proteo-
bacteria, such as Ralstonia eutropha. R. eutropha is a meta-
bolically diverse, facultatively autotrophic bacterium that
can grow on sugars, fatty acids, amino acids, triacylglycer-
ides as well as on H2/CO2 [5
,6,7,8,11
]. Previous work
on R. eutropha has focused on its ability to store excess
carbon as polyhydroxyalkanoates [12,13] (PHAs) and now
efforts seek to divert carbon flux away from PHA storage
and into other molecular targets. For example, R. eutropha
is a proposed host for isobutanol production via the 2-
ketoisovalerate pathway for branched chain amino acid
synthesis [11
] from H2 and CO2. This strategy has
already been successfully used in E. coli to produce
Available online at www.sciencedirect.com
Current Opinion in Biotechnology 2013, 24:376–384 www.sciencedirect.com

2. Biological conversion of carbon dioxide and hydrogen Hawkins et al. 377
Figure 1
(a)
(b)
CH2OH
H2/CO2
H2/CO2
OH
OH
OH
OH
O
CBP
TBD
TBD
0.18-0.2%c1.8-2.4%b
10-13%e,f
50-70%f
50-70%f
15-20%d
PV
PS
4.5-6%
a
70-85%f
Wet-milling, EH
WEHP
WEHP
WEHP
SMR
Current Opinion in Biotechnology
Electrofuel production from H2 and CO2. (a) Comparison of overall photon-to-fuel efficiency of biofuels versus electrofuels. Percentages represent the
cumulative efficiency of solar energy conversion at different stages. Although the conversion efficiency of an electrofuel process is currently unknown,
the improved efficiency of solar hydrogen compared to photosynthetic sugars indicates that a solar electrofuel process would require less land area
than current biofuels. (b) Hydrogen inputs for electrofuels are highly flexible and generation strategies can include electrolysis of water by renewable
wind power, electrolysis of water by conventional electricity (e.g. coal), or by steam reformation of methane, shown with conversion efficiencies for
each process. Abbreviations: PS – photosynthesis, EH – enzymatic hydrolysis, PV – photovoltaic, WEHP – Water Electrolysis Hydrogen Production,
SMR – steam methane reformation, TBD – To be determined. a
Zhu et al. [76], b
Assuming grain starch represents 40% of total corn biomass, c
Conrado
et al. [1
], d
Parida et al. [77], e
Assuming 65% overall electrolysis efficiency, f
Holladay et al. [78].
Figure 2
H
+
H
2 NADP
+
NADPH
(1) Generating Reducing Power (2) Incorporation of Inorganic Carbon
Acetyl-CoA
Pyruvate
CO2
Fixation
(3) Microbial Synthesis
n-butanol
farnesene
long chain alkenes
isobutanol
HCO
3
_
CO
2
Current Opinion in Biotechnology
Schematic drawing of the primary biochemical modules involved in electrofuel formation. Intracellular reducing power is generated from hydrogen gas
via hydrogenase enzymes. Carbon fixation cycle incorporates inorganic carbon into central metabolism via key intermediates. Microbial synthesis of
the target fuel molecule proceeds via endogenous and/or engineered metabolic pathways.
www.sciencedirect.com Current Opinion in Biotechnology 2013, 24:376–384

3. isobutanol and other higher alcohols from sugars [14,15],
as well as in Clostridium cellulolyticum to produce isobu-
tanol from cellulose [16]. R. eutropha can also use electro-
chemically generated formate as the electron donor for
the production of isobutanol and 3-methyl-1-butanol
[1
,2,3
,17
]. In addition, R. eutropha is being used, in
conjunction with in situ electrochemical generation of
hydrogen via molybdenum polypryridyl-oxo catalysts
tethered to the cell surface, for production of various
molecules including n-butanol (via PHA biosynthesis),
farnesene (via isoprenoid biosynthesis) and long chain
alkanes (via fatty acid biosynthesis) [1
,2,3
,4,18
].
Anaerobic microorganisms that are known to fix CO2
typically use either the reductive TCA (rTCA) cycle
or the Wood–Ljungdahl (W–L) pathway. The most
energetically efficient pathway for CO2 fixation is the
W–L pathway, which is found exclusively in anaerobic,
acetogenic bacteria and methanogenic archaea
[5
,6,7,8,14,15]. Acetogenic bacteria are well-known
for their ability to grow on H2/CO2 or synthesis gas
(CO/H2) and produce acetate, but various species also
naturally generate ethanol, butyrate, butanol and 2,3-
butanediol as end-products [1
,2,3
,9
,10,16,19,20].
These organisms are obvious hosts for electrofuel
development, and already butanol production
has been engineered into Clostridium ljungdahlii
[5
,6,7,8,11
,17
,21
]. Acetogens, such as Sporomusa
ovata, are also being explored for their ability to grow
directly on graphite cathodes and accept electrons for
microbial synthesis [12,13,22].
The most recently discovered pathways for biological
CO2 fixation involve the production of 3-hydroxypropio-
nate (3HP), dicarboxylates (DC) and/or 4-hydroxybuty-
rate (4HB). One version is found in members of the
thermoacidophilic archaeal order Sulfolobales, which
includes species in the genera Sulfolobus and Metallo-
sphaera [5
,6,7,8,11
,23]. One turn of the cycle converts
two molecules of CO2 to acetyl-CoA using NADPH as the
electron donor with 3HP and 4HB as intermediates,
although studies using isotopically labeled substrates
showed that most of the carbon flux enters central metab-
olism via succinyl-CoA rather than acetyl-CoA
[9
,10,14,15,24]. The enzymatic details of this pathway
have recently been completed with the biochemical
characterization of an epimerase and a mutase
[11
,16,25], and the identification of the gene encoding
a CoA-ligase [26]. The 3HP/4HB pathway is being engin-
eered into a hyperthermophilic host, Pyrococcus furiosus,
for the production of n-butanol from H2/CO2 [3
,12,13],
where the nearly 308C difference between the optimal
growth temperature of P. furiosus (Topt = 988C) and M.
sedula (Topt = 738C) is being exploited for a temperature-
dependent production strategy. Around 708C, where the
enzymes from M. sedula are most active, P. furiosus
metabolism is virtually quiescent, thereby minimizing
the maintenance energy requirements for the host.
Recently, this temperature-dependent strategy was used
to produce 3HP in P. furiosus using the first three enzymes
of the 3HP/4HB pathway [27
].
One important characteristic intrinsic to the various CO2-
fixing pathways is the tolerance of the enzymes and redox
carrier molecules to oxygen. The CBB cycle, the 3HP
bicycle, and the 3HP/4HB cycle (with the exception of a
single enzyme, 4-hydroxybutyryl-CoA dehydratase) are
all oxygen-tolerant pathways that operate in aerobic
organisms. The rTCA cycle, the W–L pathway, and
the DC/4HB cycle are all found in anaerobic organisms;
these all utilize oxygen-sensitive reduced ferredoxin as an
electron carrier and contain oxygen-sensitive enzymes,
for example, CO dehydrogenase/acetyl-CoA synthase and
pyruvate synthase [6]. Some species of aerobic H2-oxidiz-
ing bacteria, such as Hydrogenobacter thermophilus, utilize
the rTCA cycle; however, they have special biochemical
adaptations to protect against oxic conditions [28]. In
practical terms, the requirement of O2 to support
microbial growth presents explosion safety challenges
to large-scale electrofuel production using hydrogen gas
as substrate. There are some reactor designs that try to
minimize the risk with aerobic electrofuels hosts (such as
Ralstonia) by keeping oxygen and hydrogen gases separ-
ate [11
]. Utilizing anaerobic hosts, such as Clostridium
spp. and P. furiosus, avoids the issue altogether. However,
a recent study of oxygen detoxification in P. furiosus
showed that the organism is remarkably aero-tolerant
(up to 8% O2 vol/vol). This property would be advan-
tageous in bioprocessing, especially under conditions
where maintaining strict anoxia is costly [11
,29].
378 Energy biotechnology
Table 1
Electrofuel producing hosts in development. The organisms listed utilize hydrogen or formate for the delivery of low-potential electrons,
and have either been reported to produce an electrofuel or are currently in development. R = reported, D = development
Organism Type Electron donor(s) CO2 fixation cycle Target products Status References
Ralstonia eutropha H16 Betaproteobacteria Electrochemically
generated H2
CBB cycle PHB, butanol, farnesene,
long-chain alkenes
D [18
]
Ralstonia eutropha H16 Betaproteobacteria H2 CBB cycle Isobutanol D [11
]
Pyrococcus furiosus Archaeon H2 3HP/4HB cycle from
M. sedula
n-butanol D [3
]
Clostridium ljungdahlii Acetogenic bacteria H2 W–L pathway n-butanol, ethanol R [21
]
Current Opinion in Biotechnology 2013, 24:376–384 www.sciencedirect.com

4. Computational analyses of CO2 fixation
The growing interest in biological carbon fixation for
microbial production of fuels and commodity chemicals
has led to extensive analysis of the thermodynamic and
kinetic characteristics of the six known pathways. Boyle
and Morgan performed flux analysis to determine the
carbon flux and energy demand for biomass synthesis
from CO2, using maximum biomass production as the
objective function [30
]. They found that the energetic
cost of common core metabolites varied across the differ-
ent routes for fixing CO2, indicating that certain pathways
may be more efficient at producing a given product from a
single precursor. On average, they found that the W–L
pathway and the rTCA cycle were the most energy
efficient routes, followed by the DC/4HB cycle, the
3HP/4HB cycle, the 3HP-bicycle, and lastly the CBB
pathway. The same trend has been observed in other flux
balance analyses [31].
Another thermodynamic analysis of carbon fixation reac-
tions examined the ATP requirement and the reduction
potential of electron carriers, as well as other factors,
including the form and concentration of inorganic carbon,
and the associated effects of cellular pH and ionic
strength [32
]. Several possible explanations were put
forward to account for the differences in the ATP
required by the various pathways that are, in most cases,
higher than that required to make the net reaction ther-
modynamically feasible. One common explanation for
this is based on the different electron carriers used. For
example, ferredoxin (Eo’ À400 mV) has a lower
reduction potential than NAD(P)H (Eo’ = À320 mV),
but the extra energetic contribution of oxidizing two
ferredoxin molecules compared to one NADPH molecule
under standard conditions ($20 kJ molÀ1
) does not equal
the energy released by ATP hydrolysis (over 50 kJ molÀ1
)
[33]. Moreover, this effect is not enough to account for the
large differences in ATP cost for pyruvate formation
between, for example, the rTCA cycle (2 ATP) and
the 3HP bicycle (7 ATP) [5
].
A closer look at the thermodynamics of the individual
reactions in carbon fixation pathways helped to further
elucidate the local thermodynamic constraints that shape
the overall energetics and revealed a characteristic pat-
tern in the reaction profile of carbon fixation enzymes
[34
]. Inspection of the reduction potentials of half-
reactions in carbon fixation pathways reveals generalized
groupings of reactions based on reaction type. It is
specifically the carboxylation and carboxyl reduction
reactions that are the most energetically unfavorable in
the pathway and thus require most of the energetic push
to overcome. This is accomplished either directly by
coupling the reaction to energy release from ATP
hydrolysis, or indirectly by coupling hydrolysis of another
high-energy bond (e.g. thioester) to the energetically
unfavorable reaction. Almost all of the ATP investments
in each carbon fixation pathway are coupled directly or
indirectly to carboxylation and carboxyl reduction reac-
tions. Some pathways manage to reduce the ATP require-
ments of carbon fixation by coupling unfavorable
reactions to exergonic reactions other than ATP hydroly-
sis. In the case of the W–L pathway in acetogens, this
strategy is used to reduce the ATP investment to a single
ATP molecule during the formation of pyruvate [5
].
Synthetic pathways are not limited by the enzymatic
profile of naturally occurring pathways. Rather, the pro-
spect of combining enzymes in novel ways holds promise
for improving the efficiency of the pathway or reducing
the energetic cost. Using a constraint-based modeling
approach and drawing from the entire set of approxi-
mately 5000 known metabolic enzymes, Bar-Even et al.
predicted in silico pathways with faster kinetic rates than
naturally occurring ones [35]. They describe a family of
synthetic pathways that utilize PEP carboxylase and the
core of the C4 cycle in plants, which they term the ‘C4-
glyoxylate cycles’. These pathways all produce glyoxy-
late, which is assimilated to central metabolism via the
bacterial-like glycerate pathway, and are predicted to
have overall rates of CO2 to product formation that are
2–3 times faster than that of the CBB pathway. However,
these rates are still lower than those reported for the
rTCA, DC/4HB, and 3HP/4HB cycles.
In addition to the enzymes that catalyze carboxylations
and the carboxyl reduction reactions, the conversion of
CO2 and hydrogen to electrofuels is also dependent on
the two enzymes that activate these two gases, namely,
carbonic anhydrase and hydrogenase, respectively. In the
following the properties of these two enzymes relevant to
electrofuel production will be considered.
Hydrogen utilization
Hydrogen is used as a source of energy by microorganisms
from all three domains of life [36] and is activated by
hydrogenase, which catalyzes the reversible interconver-
sion of molecular hydrogen and protons in the presence of
a suitable electron carrier. Based on structural and bio-
chemical analysis, hydrogenases can be categorized by
the metal atoms in their active sites: [NiFe]-hydroge-
nases, [FeFe]-hydrogenases, and [Fe]-hydrogenases
[37,38]. Since electrofuel hosts containing CO2 fixation
pathways need to coordinate acquisition of reductant with
carbon flux, the types of hydrogenase present in the host
microorganism and the electron carriers that they use are
of paramount importance.
Extensive work on [NiFe]-hydrogenases from Desulfovi-
brio gigas [39–43] and the [FeFe]-hydrogenases from
Clostridium pasteurianum [44] and Desulfovibrio desulfuri-
cans [45] has shown that both enzyme types harbor deeply
buried active sites in which the site of H2 reactivity is a
single iron atom coordinated by diatomic ligands (carbon
Biological conversion of carbon dioxide and hydrogen Hawkins et al. 379
www.sciencedirect.com Current Opinion in Biotechnology 2013, 24:376–384

5. monoxide and cyanide). In the [NiFe]-enzymes, the iron
is also coordinated to a nickel atom, while in the [FeFe]-
hydrogenases it is bound by a second iron atom and linked
to a 4Fe-cluster. Both types of enzyme also contain
multiple [4Fe-4S] clusters, as well as hydrophobic gas
channels, that connect the catalytic sites to the molecular
surface [46]. These two enzymes differ with respect to
their distribution in nature, sensitivity to oxygen deacti-
vation, and in the assembly and maturation of their
catalytic sites [47]. For example, [NiFe]-hydrogenases
are widespread among bacteria and archaea, the [FeFe]-
enzymes are found in anaerobic bacteria and anaerobic
eukaryotes but not in the archaea, and the [Fe]-hydro-
genases are found only in certain methanogenic archaea.
The electrofuel production host R. eutropha H16 harbors
three [NiFe]-hydrogenases, all of which have been
characterized biochemically [48]. Two of them are used
to oxidize hydrogen, and thereby provide reducing
equivalents to the organism both for energy conservation
and biosynthesis, while the third type senses H2 in the
environment and serves a regulatory role. The cyto-
plasmic NAD(P)-reducing hydrogenase (SH) consists of
a heterodimeric H2-activating module (HoxHY) and a
heterodimeric NADH dehydrogenase module (HoxFU)
[49]. HoxH harbors the [NiFe] site, while HoxY contains
an iron–sulfur cluster. HoxF and HoxU contain multiple
iron–sulfur cluster and a flavin and these channel elec-
trons to NAD+
. SH also contains a fifth subunit (HoxI)
that is believed to be required for reducing NAD+
. The
NADH and NADPH generated by SH are primarily used
for biosynthetic processes, including autotrophic CO2
fixation. The other hydrogenase in R. eutropha used for
H2 activation is a heterodimeric membrane bound
[NiFe]-hydrogenase (MBH), which faces the periplasm
and is anchored to the membrane [50]. It oxidizes H2 and
feeds electrons to the quinone pool of the aerobic respir-
atory chain. MBH is made up of two subunits: HoxG is
the catalytic subunit while HoxK harbors three iron–
sulfur clusters. A di-heme b-type cytochrome HoxZ is
also part of the respiratory chain and it links MBH to the
quinone pool [48,50]. Hence, R. eutropha can efficiently
utilize H2 for carbon fixation both in terms of biosynthesis
via SH and in terms of ATP synthesis by aerobic respir-
ation via MBH.
P. furiosus is being engineered to fix CO2 via the 3HP/
4HB cycle and it contains three [NiFe]-hydrogenases, all
of which have been characterized biochemically [51].
Two of them are found in the cytoplasm (SHI, SHII)
and one is membrane bound (MBH). MBH is a respir-
atory complex encoded by 14 genes that oxidizes the
reduced ferredoxin generated from sugar fermentation
and evolves H2 and generates an ion gradient that is used
for ATP synthesis. SHI and SHII are thought to recycle
the H2 produced by MBH for biosynthetic purposes.
Each consists of four subunits that contain the [NiFe]
catalytic site, multiple iron–sulfur clusters, and flavin
adenine dinucleotide. SHI only uses NADP+
as an elec-
tron acceptor while SHII also uses NAD+
[52]. Hence,
even though P. furiosus is naturally a heterotroph, it does
have the capacity to utilize H2 and generate both
NADPH (via SHI and SHII) and NADH (via SHII) that
could be used to fix CO2 by a synthetic pathway. In the
case of the 3HP/4HB cycle, NADPH is the source of
reductant and, hence, SHI would be the key enzyme for
electrofuel generation. In this regard, the recently
reported ability to over-produce SHI by an order of
magnitude in an engineered P. furiosus strain [53] could
prove to be very useful for biofuel production.
In other organisms being considered for electrofuel pro-
duction, the situation is not so straightforward. For
example, there are five hydrogenases encoded in the
genome of Clostridium ljungdahlii [21
], but none have
been characterized biochemically. One is a [NiFe]-
enzyme that it is predicted to be membrane-bound, while
the other four are of the [FeFe] type, and all are predicted
to be cytoplasmic. Two of the [FeFe] hydrogenases
appear to be associated with other proteins that might
enable NAD+
reduction [21
]. The membrane-bound
[NiFe] hydrogenase is similar to the H2-oxidizing
MBH of R. eutropha discussed above, but the nature of
its physiological electron carrier is not known. Con-
sequently, it is not understood how C. ljungdahlii could
activate H2 and provide reducing power for CO2 fixation,
which is obviously required before the process could be
optimized through metabolic engineering.
Carbon dioxide capture
Any fuel or organic chemical produced from CO2 that
relies on biological carbon fixation depends on the con-
centration of CO2 in the environment. As such, mechan-
isms to concentrate CO2 have evolved to compensate for
the imbalance between the high demand of inorganic
carbon and low ambient CO2 concentration. It is well
known that some chemoautotrophs, various photoauto-
trophic microorganisms, green algae, and plants have
developed distinct mechanisms to increase the concen-
tration of CO2 in close proximity to ribulose-1,5-bispho-
sphate carboxylase/oxygenase (RubisCO), the CO2-fixing
enzyme of the CBB pathway [54,55]. This is necessary
because RubisCO has a low affinity for CO2 and does not
discriminate well between CO2 and the competing sub-
strate, O2. The O2-dependent oxygenase activity of
RubisCO is responsible for photorespiration, a wasteful
side reaction that leads to a net loss of carbon [56].
Carbonic anhydrase (CA), an essential component of CO2
concentrating mechanisms, catalyzes the reversible con-
version of CO2 and water to bicarbonate (HCO3
À
, the pKa
of carbonic acid is 6.4) [57,58]. CAs are both structurally
and functionally quite diverse, with five classes (a, b, g, d,
and z) widely distributed among plants, animals, fungi,
380 Energy biotechnology
Current Opinion in Biotechnology 2013, 24:376–384 www.sciencedirect.com

6. archaea, and bacteria, although the b-class are most
prevalent in CO2-fixing organisms [59,60
,61]. The cat-
alytic activity in CA relies on a metal cofactor, most
commonly zinc, which is coordinated by three histidine
residues (a, g, d) or two cysteines and a histidine (b and z).
The a, b, and g-classes all share a two-step zinc hydroxide
mechanism and are extremely fast enzymes (kcat/Km up to
108
MÀ1
sÀ1
).
Carbon concentrating mechanisms have only been
described for organisms that use the CBB cycle, however
exploitation of naturally occurring CO2-fixing pathways
for microbial electrofuels production may require engin-
eering of CA or attention to the molecular details for how
CO2 is fed to the carbon-fixing machinery in order to
optimize electrofuel production. The CO2-fixing
enzymes of 3HP, 3HP/4HB and DC/4HB cycles all use
bicarbonate rather than CO2 as substrate [32
], hence a
functional CA is likely a key requirement for an efficient
electrofuel production pathway. For example, the aceto-
genic C. acetobutylicum contains both b and g-types, while
the heterotrophic archaeon P. furiosus, which does not
naturally utilize CO2 as a carbon source but is the host for
an engineered 3HP/4HB pathway, appears to contain
only a g-type CA [58]. The extent to which the CA(s)
of the host might limit CO2-utilization by a heterologous
pathway is unclear at present. Moreover, as yet unchar-
acterized CO2-concentrating mechanisms might be pre-
sent in potential host organisms. In any case, the means
for concentrating and/or hydrating CO2 is obviously an
area that merits attention for electrofuel development.
Host development
The development of electrofuel hosts requires the avail-
ability of genetic tools, as well as a sound knowledge of
gene regulation and metabolism in the target host. Cur-
rent electrofuel projects are taking advantage of an estab-
lished body of knowledge for genetic manipulation in
well-studied organisms such as R. eutropha and Clostridium
spp., while others are working with newly developed
genetic systems (Table 2). There are two basic avenues
for electrofuel host development: engineer a natural
H2-utilizing autotroph to produce the desired electrofuel,
or engineer a suitable heterotroph to utilize CO2/H2 for
electrofuel production. In the latter case, choice of a
suitable host depends not only on the availability of a
genetic system but also tools for systems-level analysis
and the organism’s suitability for bioprocess production.
In the case of potentially suitable autotrophs, it may be
that these tools are not available or are still in the early
stages of development. Indeed, even though the
endogenous CO2-fixation machinery might be optimized,
diverting carbon flow to an electrofuel rather than to
native cellular metabolism may prove to be problematic.
The opposite is true for the heterotroph approach, where
preventing CO2-derived carbon from entering the host’s
native metabolism may be key to establishing an efficient
process. It is still too early to determine if the ‘auto-
trophic’ or the ‘heterotrophic’ strategy is better suited for
the generation of electrofuels.
R. eutropha serves as an example of the ‘autotrophic’
approach. A variety of tools and techniques exist for
genetic engineering of this H2-utilizing autotrophic
organism and there are both plasmid-based expression
systems [62] and chromosomal modifications via homolo-
gous recombination [63–66], as well as systems-level
analysis tools including microarrays [67]. While some
species of Clostridium are also natural H2-utilizing auto-
trophs, genetic tools for these organisms are still being
developed. So far they include plasmid-based and indu-
cible gene expression, antisense RNA knock-down, and
reporter systems [68,69], but techniques for chromosomal
modification have only recently emerged [70
]. These
efforts should enable clostridial hosts to be flexible elec-
trofuel producing platform organisms in the near future
[70
,71]. There is also a recent report of gene disruption
by cross-species complementation in the H2-oxidizing,
facultative autotroph, Metallosphaera sedula [72], although
the potential for metabolic engineering in this organism
will require continued development of its genetic system.
The second avenue for electrofuel development using
heterotrophs was recently demonstrated for the first time
Biological conversion of carbon dioxide and hydrogen Hawkins et al. 381
Table 2
Genetic tools for select electrofuels organisms. Current genetics tools for electrofuels organisms.
Organism Genome sequence Genetic tools
Ralstonia eutropha H16 Y Plasmid-based gene expression [62]
Gene knockout or deletion [63,65]
Chromosomal insertion [64]
Clostridium spp. Many Plasmid-based gene expression [68]
Anti-sense RNA silencing [68]
Expression reporter systems [68]
Chromosomal insertion [70
]
Pyrococcus furiosus Wild-type and COM1 Expression regulation [53]
Chromosomal insertion [27
,79]
Metallosphaera sedula Y Gene knockout or deletion [72]
www.sciencedirect.com Current Opinion in Biotechnology 2013, 24:376–384

7. in P. furiosus [27
]. Heterologous expression of the first
five genes from the 3HP/4HB pathway allowed P. furiosus
to utilize H2 and incorporate CO2 into 3HP, a crucial
intermediate in the carbon fixation pathway and a valu-
able industrial chemical building block. This was accom-
plished by using a highly competent strain that enables
chromosomal modification [73], the genome sequence of
which was recently reported [74]. The new genetic
system has also been successfully utilized for over-
expression of cytoplasmic hydrogenase I (SHI) [53],
thereby potentially allowing facile H2 oxidation and
NADPH production. Based on these promising prelimi-
nary results, efforts are underway for the construction of
the electrofuels production host utilizing the complete
M. sedula 3HP/4HB pathway for biological activation of
carbon dioxide into a variety of chemical and fuel mol-
ecules [3
].
Conclusion
Electrofuels are a promising paradigm that could improve
on the relatively poor efficiency of photosynthetically
produced biofuels and open up a program for scalable,
renewable liquid fuel production based on flexible energy
inputs. A handful of electrofuel organisms are in devel-
opment or have been recently reported, but opportunities
exist to expand the concept. The potential of harnessing
biological carbon fixation for production of key industrial
biomolecules holds great promise for displacing
petroleum-based feedstocks [75].
Acknowledgments
This work described here was supported in part by the US Department of
Energy Research through the ARPA-E Electrofuels Program (DE-
AR0000081 to RMK and MWA) and the Division of Chemical Sciences,
Geosciences and Biosciences, Office of Basic Energy Sciences (DE-FG05-
95ER20175 to MWA).
References and recommended reading
Papers of particular interest, published within the period of review,
have been highlighted as:
of special interest
of outstanding interest
1.
Conrado RJ, Haynes CA, Haendler BE, Toone EJ: Electrofuels: a
new paradigm for renewable fuels. In Advanced Biofuels and
Bioproducts. Edited by Lee JW. Springer; 2013:1037-1064.
Excellent conceptual overview of electrofuels paradigm by Department of
Energy Advanced Research Projects Agency-Energy (ARPA-E) with eco-
nomic analysis of electrofuel production.
2. Pearson RJ, Eisaman MD, Turner JWG, Edwards PP, Jiang Z,
Kuznetsov VL, Littau KA, di Marco L, Taylor SRG: Energy storage
via carbon-neutral fuels made from CO2, water, and renewable
energy. Proc IEEE 2012, 100:440-460.
3.
Hawkins A, Han Y, Lian H, Loder A, Menon A, Iwuchukwu I,
Keller M, Leuko T, Adams MWW, Kelly RM: Extremely
thermophilic routes to microbial electrofuels. ACS Catal 2011,
1:1043-1050.
Electrofuel concept review with a focus on thermophilic host production
systems.
4. Lovley DR, Nevin KP: Electrobiocommodities: powering
microbial production of fuels and commodity chemicals from
carbon dioxide with electricity. Curr Opin Biotechnol 2013,
24:385-390.
5.
Berg IA, Kockelkorn D, Ramos-Vera WH, Say RF, Zarzycki J,
Hu¨ gler M, Alber BE, Fuchs G: Autotrophic carbon fixation in
archaea. Nat Rev Microbiol 2010, 8:447-460.
Very thorough review of all six naturally occurring carbon fixation path-
ways.
6. Berg IA: Ecological aspects of the distribution of different
autotrophic CO2 fixation pathways. Appl Environ Microbiol
2011, 77:1925-1936.
7. Fuchs G: Alternative pathways of carbon dioxide fixation:
insights into the early evolution of life? Annu Rev Microbiol
2011, 65:631-658.
8. Hugler M, Sievert SM: Beyond the Calvin cycle: autotrophic
carbon fixation in the ocean. Annu Rev Mar Sci 2011, 3:261-289.
9.
Erb TJ: Carboxylases in natural and synthetic microbial
pathways. Appl Environ Microbiol 2011, 77:8466-8477.
Comprehensive review of carboxylases covering a wide range of bio-
chemical functions.
10. McKinlay J: Carbon dioxide fixation as a central redox cofactor
recycling mechanism in bacteria. Proc Natl Acad Sci U S A 2010,
107:11669-11675.
11.
Brigham CJ, Gai CS, Lu J, Speth DR, Worden RM, Sinskey AJ:
Engineering Ralstonia eutropha for production of isobutanol
from CO2, H2, and O2. In Advanced Biofuels and Bioproducts.
Edited by Lee JW. Springer; 2013:1065-1090.
Review of electrofuel prospects in R. eutropha using H2/CO2 to make
isobutanol.
12. Yang Y-H, Brigham CJ, Budde CF, Boccazzi P, Willis LB,
Hassan MA, Yusof ZAM, Rha C, Sinskey AJ: Optimization of
growth media components for polyhydroxyalkanoate (PHA)
production from organic acids by Ralstonia eutropha. Appl
Microbiol Biotechnol 2010, 87:2037-2045.
13. Brigham CJ, Kurosawa K, Rha C, Sinskey AJ: Bacterial carbon
storage to value added products. J Microb Biochem Technol S
2011, S3:1-13.
14. Atsumi S, Hanai T, Liao JC: Non-fermentative pathways for
synthesis of branched-chain higher alcohols as biofuels.
Nature 2008, 451:86-89.
15. Lan EI, Liao JC: Microbial synthesis of n-butanol, isobutanol,
and other higher alcohols from diverse resources. Bioresour
Technol 2012.
16. Higashide W, Li Y, Yang Y, Liao JC: Metabolic engineering of
Clostridium cellulolyticum for production of isobutanol from
cellulose. Appl Environ Microbiol 2011, 77:2727-2733.
17.
Li H, Opgenorth PH, Wernick DG, Rogers S, Wu T-Y, Higashide W,
Malati P, Huo Y-X, Cho KM, Liao JC: Integrated electromicrobial
conversion of CO2 to higher alcohols. Science 2012, 335:1596.
First literature report of electrofuel production in engineered R. eutropha
using electrochemically generated formate.
18.
Singer SW, Beller HR, Chhabra S, Chang CJ, Adler J: Microbial
electrocatalytic (MEC) biofuel production. In Advanced
Biofuels and Bioproducts. Edited by Lee JW. Springer; 2013:1091-
1099.
Review of electrofuel prospects in R. eutropha using electrochemically
generated H2.
19. Schiel-Bengelsdorf B, Du¨ rre P: Pathway engineering and
synthetic biology using acetogens. FEBS Lett 2012,
586:2191-2198.
20. Demler M, Weuster-Botz D: Reaction engineering analysis of
hydrogenotrophic production of acetic acid by
Acetobacterium woodii. Biotechnol Bioeng 2010, 108:470-474.
21.
Ko¨ pke M, Held C, Hujer S, Liesegang H, Wiezer A, Wollherr A,
Ehrenreich A, Liebl W, Gottschalk G, Du¨ rre P: Clostridium
ljungdahlii represents a microbial production platform based
on syngas. Proc Natl Acad Sci U S A 2010, 107:13087-13092.
First literature report of butanol production in engineered C. ljungdahlii
from synthesis gas.
22. Nevin KP, Hensley SA, Franks AE, Summers ZM, Ou J,
Woodard TL, Snoeyenbos-West OL, Lovley DR: Electrosynthesis
of organic compounds from carbon dioxide is catalyzed by a
382 Energy biotechnology
Current Opinion in Biotechnology 2013, 24:376–384 www.sciencedirect.com

8. diversity of acetogenic microorganisms. Appl Environ Microbiol
2011, 77:2882-2886.
23. Berg IA, Kockelkorn D, Buckel W, Fuchs G: A 3-
hydroxypropionate/4-hydroxybutyrate autotrophic carbon
dioxide assimilation pathway in Archaea. Science 2007,
318:1782-1786.
24. Estelmann S, Hugler M, Eisenreich W, Werner K, Berg IA, Ramos-
Vera WH, Say RF, Kockelkorn D, Gad’on N, Fuchs G: Labeling
and enzyme studies of the central carbon metabolism in
Metallosphaera sedula. J Bacteriol 2011, 193:1191-1200.
25. Han Y, Hawkins AS, Adams MWW, Kelly RM: Epimerase (Msed_
0639) and Mutase (Msed_0638, Msed_2055) convert (S)-
methylmalonyl-CoA to succinyl-CoA in the Metallosphaera
sedula 3-hydroxypropionate/4-hydroxybutyrate cycle. Appl
Environ Microbiol 2012, 78:6194-6202.
26. Hawkins AS, Han Y, Bennett RK, Adams MWW, Kelly RM: Role of
4-hydroxybutyrate-CoA synthetase in the CO2 fixation cycle in
thermoacidophilic Archaea. J Biol Chem 2012 http://dx.doi.org/
10.1074/jbc.M112.413195.
27.
Keller M, Schut GJ, Lipscomb GL, Menon A, Iwuchukwu I, Leuko T,
Thorgersen MP, Nixon WJ, Hawkins A, Kelly RM et al.: Exploiting
microbial hyperthermophilicity to produce an industrial
chemical using hydrogen and carbon dioxide. Proc Natl Acad
Sci U S A 2013. [in press].
The first literature report demonstrating heterologous expression and
utilization of carbon dioxide fixation genes in P. furiosus.
28. Yamamoto M, Arai H, Ishii M, Igarashi Y: Role of two 2-
oxoglutarate:ferredoxin oxidoreductases in Hydrogenobacter
thermophilus under aerobic and anaerobic conditions. FEMS
Microbiol Lett 2006, 263:189-193.
29. Thorgersen MP, Stirrett K, Scott RA, Adams MWW: Mechanism of
oxygen detoxification by the surprisingly oxygen-tolerant
hyperthermophilic archaeon, Pyrococcus furiosus. Proc Natl
Acad Sci U S A 2012, 109:18547-18552.
30.
Boyle NR, Morgan JA: Computation of metabolic fluxes and
efficiencies for biological carbon dioxide fixation. Metabol Eng
2011, 13:150-158.
Flux balance analysis that compares energetic costs of different common
metabolites according to carbon fixation pathway.
31. Fast AG, Papoutsakis ET: Stoichiometric and energetic
analyses of non-photosynthetic CO2-fixation pathways to
support synthetic biology strategies for production of
fuels and chemicals. Curr Opin Chem Eng 2012,
1:380-395.
32.
Bar-Even A, Noor E, Milo R: A survey of carbon fixation
pathways through a quantitative lens. J Exp Bot 2012, 63:2325-
2342.
Excellent quantitative analysis of carbon fixation pathway energetics.
33. Bennett BD, Kimball EH, Gao M, Osterhout R, Van Dien SJ,
Rabinowitz JD: Absolute metabolite concentrations and
implied enzyme active site occupancy in Escherichia coli. Nat
Chem Biol 2009, 5:593-599.
34.
Bar-Even A, Flamholz A, Noor E, Milo R: Thermodynamic
constraints shape the structure of carbon fixation pathways.
Biochim Biophys Acta 2012, 1817:1646-1659.
This excellent quantitative analysis reveals local thermodynamic char-
acteristics of each carbon fixation pathway and the types of biochemical
reactions they utilize.
35. Bar-Even A, Noor E, Lewis NE, Milo R: Design and analysis of
synthetic carbon fixation pathways. Proc Natl Acad Sci U S A
2010, 107:8889-8894.
36. Vignais PM, Billoud B: Occurrence, classification, and
biological function of hydrogenases: an overview. Chem Rev
2007, 107:4206-4272.
37. Vignais PM, Colbeau A: Molecular biology of microbial
hydrogenases. Curr Issues Mol Biol 2004, 6:159-188.
38. Fontecilla-Camps JC, Volbeda A, Cavazza C, Nicolet Y:
Structure/function relationships of [NiFe]- and [FeFe]-
hydrogenases. Chem Rev 2007, 107:4273-4303.
39. Pandelia M-E, Ogata H, Lubitz W: Intermediates in the catalytic
cycle of [NiFe] hydrogenase: functional spectroscopy of the
active, site. ChemPhysChem 2010, 11:1127-1140.
40. Volbeda A, Charon MH, Piras C, Hatchikian EC, Frey M, Fontecilla-
Camps JC: Crystal structure of the nickel-iron hydrogenase
from Desulfovibrio gigas. Nature 1995, 373:580-587.
41. Volbeda A, Martin L, Cavazza C, Matho M, Faber BW,
Roseboom W, Albracht SPJ, Garcin E, Rousset M, Fontecilla-
Camps JC: Structural differences between the ready and
unready oxidized states of [NiFe] hydrogenases. J Biol Inorg
Chem 2005, 10:239-249.
42. Ogata H, Kellers P, Lubitz W: The crystal structure of the [NiFe]
hydrogenase from the photosynthetic bacterium
Allochromatium vinosum: characterization of the oxidized
enzyme (Ni-A state). J Mol Biol 2010, 402:428-444.
43. Marques MC, Coelho R, De Lacey AL, Pereira IAC, Matias PM: The
three-dimensional structure of [NiFeSe] hydrogenase from
Desulfovibrio vulgaris Hildenborough: a hydrogenase without
a bridging ligand in the active site in its oxidised, ‘‘as-isolated’’
state. J Mol Biol 2010, 396:893-907.
44. Peters JW, Lanzilotta WN, Lemon BJ, Seefeldt LC: X-ray crystal
structure of the Fe-only hydrogenase (CpI) from Clostridium
pasteurianum to 1.8 angstrom resolution. Science 1998,
282:1853-1858.
45. Nicolet Y, Piras C, Legrand P, Hatchikian CE, Fontecilla-
Camps JC: Desulfovibrio desulfuricans iron hydrogenase: the
structure shows unusual coordination to an active site Fe
binuclear center. Structure 1999, 7:13-23.
46. Calusinska M, Happe T, Joris B, Wilmotte A: The surprising
diversity of clostridial hydrogenases: a comparative genomic
perspective. Microbiology 2010, 156:1575-1588.
47. Bo¨ ck A, King PW, Blokesch M, Posewitz MC: Maturation of
hydrogenases. In Advances in Microbial Physiology. Edited by
Poole RK. Academic Press; 2006:1-71.
48. Burgdorf T, Lenz O, Buhrke T, van der Linden E, Jones AK,
Albracht SPJ, Friedrich B: [NiFe]-Hydrogenases of Ralstonia
eutropha H16: modular enzymes for oxygen-tolerant
biological hydrogen oxidation. J Mol Microbiol Biotechnol 2005,
10:181-196.
49. Burgdorf T, van der Linden E, Bernhard M, Yin QY, Back JW,
Hartog AF, Muijsers AO, de Koster CG, Albracht SPJ, Friedrich B:
The soluble NAD+
-reducing [NiFe]-hydrogenase from
Ralstonia eutropha H16 consists of six subunits and can be
specifically activated by NADPH. J Bacteriol 2005,
187:3122-3132.
50. Saggu M, Zebger I, Ludwig M, Lenz O, Friedrich B, Hildebrandt P,
Lendzian F: Spectroscopic insights into the oxygen-tolerant
membrane-associated [NiFe] hydrogenase of Ralstonia
eutropha H16. J Biol Chem 2009, 284:16264-16276.
51. Jenney FE, Adams MWW: Hydrogenases of the model
hyperthermophiles. Ann N Y Acad Sci 2008, 1125:252-266.
52. van Haaster DJ, Silva PJ, Hagedoorn P-L, Jongajan JA,
Hagen WR: Reinvestigation of the steady-state kinetics and
physiological function of the soluble NiFe-hydrogenase I of
Pyrococcus furiosus. J Bacteriol 2007, 190:1584-1587.
53. Chandrayan SK, McTernan PM, Hopkins RC, Sun J, Jenney FE,
Adams MWW: Engineering hyperthermophilic Archaeon
Pyrococcus furiosus to overproduce its cytoplasmic [NiFe]-
hydrogenase. J Biol Chem 2012, 287:3257-3264.
54. Cannon GC, Heinhorst S, Kerfeld CA: Carboxysomal carbonic
anhydrases: structure and role in microbial CO2 fixation.
Biochim Biophys Acta 2010, 1804:382-392.
55. Badger MR, Price GD: CO2 concentrating mechanisms in
cyanobacteria: molecular components, their diversity and
evolution. J Exp Bot 2003, 54:609-622.
56. Tabita FR: Microbial ribulose 1,5-bisphosphate carboxylase/
oxygenase: a different perspective. Photosynth Res 1999, 60:1-28.
57. Tripp BC, Smith K, Ferry JG: Carbonic anhydrase: new insights
for an ancient enzyme. J Biol Chem 2001, 276:48615-48618.
Biological conversion of carbon dioxide and hydrogen Hawkins et al. 383
www.sciencedirect.com Current Opinion in Biotechnology 2013, 24:376–384

9. 58. Smith KS, Ferry JG: Prokaryotic carbonic anhydrases. FEMS
Microbiol Rev 2006, 24:335-366.
59. McDevitt ME, Lambert LA: Molecular evolution and selection
pressure in alpha-class carbonic anhydrase family members.
Biochim. Biophys. Acta 2011, 1814:1854-1861.
60.
Rowlett RS: Structure and catalytic mechanism of the b-
carbonic anhydrases. Biochim Biophys Acta 2010,
1804:362-373.
Great review on the biochemical features of the b-class carbonic anhy-
drases, which are most common in CO2-fixing organisms.
61. Ferry JG: The g class of carbonic anhydrases. Biochim Biophys
Acta 2010, 1804:374-381.
62. Sichwart S, Hetzler S, Bro¨ ker D, Steinbuchel A: Extension of the
substrate utilization range of Ralstonia eutropha strain H16 by
metabolic engineering to include mannose and glucose. Appl
Environ Microbiol 2011, 77:1325-1334.
63. Orita I, Iwazawa R, Nakamura S, Fukui T: Identification of
mutation points in Cupriavidus necator NCIMB 11599 and
genetic reconstitution of glucose-utilization ability in wild
strain H16 for polyhydroxyalkanoate production. J Biosci
Bioeng 2012, 113:63-69.
64. Kawashima Y, Cheng W, Mifune J, Orita I, Nakamura S, Fukui T:
Characterization and functional analyses of R-specific
enoyl coenzyme A hydratases in polyhydroxyalkanoate-
producing Ralstonia eutropha. Appl Environ Microbiol 2012,
78:493-502.
65. Lindenkamp N, Volodina E, Steinbuchel A: Genetically modified
strains of Ralstonia eutropha H16 with b-ketothiolase gene
deletions for production of copolyesters with defined 3-
hydroxyvaleric acid contents. Appl Environ Microbiol 2012,
78:5375-5383.
66. Brigham CJ, Budde CF, Holder JW, Zeng Q, Mahan AE, Rha C,
Sinskey AJ: Elucidation of b-oxidation pathways in Ralstonia
eutropha H16 by examination of global gene expression. J
Bacteriol 2010, 192:5454-5464.
67. Brigham CJ, Speth DR, Rha C, Sinskey AJ: Whole-genome
microarray and gene deletion studies reveal regulation of the
polyhydroxyalkanoate production cycle by the stringent
response in Ralstonia eutropha H16. Appl Environ Microbiol
2012, 78:8033-8044.
68. Tracy BP, Jones SW, Fast AG, Indurthi DC, Papoutsakis ET:
Clostridia: the importance of their exceptional substrate and
metabolite diversity for biofuel and biorefinery applications.
Curr Opin Biotechnol 2012, 23:364-381.
69. Lee J, Jang Y-S, Choi SJ, Im JA, Song H, Cho JH, Seung DY,
Papoutsakis ET, Bennett GN, Lee SY: Metabolic engineering of
Clostridium acetobutylicum ATCC 824 for isopropanol–
butanol–ethanol fermentation. Appl Environ Microbiol 2012,
78:1416-1423.
70.
Al-Hinai MA, Fast AG, Papoutsakis ET: Novel system for efficient
isolation of Clostridium double-crossover allelic exchange
mutants enabling markerless chromosomal gene deletions
and DNA integration. Appl Environ Microbiol 2012,
78:8112-8121.
Recent breakthrough in Clostridium genetics that opens the door for
precise metabolic engineering.
71. Heap JT, Kuehne SA, Ehsaan M, Cartman ST, Cooksley CM,
Scott JC, Minton NP: The ClosTron: mutagenesis in Clostridium
refined and streamlined. J Microbiol Methods 2010, 80:49-55.
72. Maezato Y, Johnson T, McCarthy S, Dana K, Blum P: Metal
resistance and lithoautotrophy in the extreme
thermoacidophile Metalosphaera sedula. J Bacteriol 2012,
194:6856-6863.
73. Lipscomb GL, Stirrett K, Schut GJ, Yang F, Jenney FEJ, Scott RA,
Adams MWW, Westpheling J: Natural competence in the
hyperthermophilic archaeon Pyrococcus furiosus facilitates
genetic manipulation: construction of markerless deletions of
genes encoding the two cytoplasmic hydrogenases. Appl
Environ Microbiol 2011, 77:2232-2238.
74. Bridger SL, Lancaster WA, Poole FL, Schut GJ, Adams MWW:
Genome sequencing of a genetically tractable Pyrococcus
furiosus strain reveals a highly dynamic genome. J Bacteriol
2012, 194:4097-4106.
75. Werpy T, Peterson G, Aden A, Bozell J, Holladay J, White J,
Manheim A: Top Value Added Chemicals from Biomass: Volume
I — Results of Screening for Potential Candidates from Sugars and
Synthesis Gas. U.S. Department of Energy-Energy Efficiency and
Renewable Energy; 2004.
76. Zhu X-G, Long SP, Ort DR: What is the maximum efficiency with
which photosynthesis can convert solar energy into biomass?
Curr Opin Biotechnol 2008, 19:153-159.
77. Parida B, Iniyan S, Goic R: A review of solar photovoltaic
technologies. Renew Sust Energ Rev 2011, 15:1625-1636.
78. Holladay JD, Hu J, King DL, Wang Y: An overview of hydrogen
production technologies. Catal Today 2009, 139:244-260.
79. Basen M, Sun J, Adams MWW: Engineering a hyperthermophilic
archaeon for temperature-dependent product formation.
mBio 2012, 3:1-8.
384 Energy biotechnology
Current Opinion in Biotechnology 2013, 24:376–384 www.sciencedirect.com