COBIOT-1169; NO.

OF PAGES 9

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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 electrofuelproducing 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:XXXXXX

This review comes from a themed issue on Energy biotechnology
Edited by Eric J Toone and Johannes H de winde

Available online XXX

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 largescale. In particular, these include low efficiency of solar
energy conversion (Figure 1) and competition for agricultural resources. Recent initiatives in the U.S. and elsewhere 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
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synthetic biologists, metabolic engineers, and microbiologists to equip native CO2-fixing species or autotrophs with
pathways for targeted fuel production, or confer autotrophy
on heterotrophic host organisms, or both. The range of
possible sources currently being explored for low-potential, 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 production from hydrogen and CO2 is shown
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 extensively 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 microorganisms that are currently being considered that use hydrogen (Table 1).
The most ubiquitous CO2-fixation pathway is the Calvin
BensonBassham (CBB) cycle found in plants, algae,
cyanobacteria, purple bacteria, and also in some proteobacteria, such as Ralstonia eutropha. R. eutropha is a metabolically diverse, facultatively autotrophic bacterium that
can grow on sugars, fatty acids, amino acids, triacylglycerides 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 2ketoisovalerate pathway for branched chain amino acid
synthesis [11] from H2 and CO2. This strategy has
already been successfully used in E. coli to produce
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COBIOT-1169; NO. OF PAGES 9

2 Energy biotechnology

Figure 1

(a)

CH2OH
O OH

1.8-2.4%b

0.18-0.2%c

OH
a

Wet-milling, EH

6%

4.5-

CBP

OH
OH

PS

15-2

0% d

10-13%e,f

PV

WEHP

H2/CO2

TBD

(b)
50-70%f
WEHP
70-85%f
SMR

H2/CO2

TBD

50-70%f
WEHP
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. aZhu et al. [76], bAssuming grain starch represents 40% of total corn biomass, cConrado
et al. [1], dParida et al. [77], eAssuming 65% overall electrolysis efficiency, fHolladay et al. [78].

Figure 2
+

NADP

n-butanol
NADPH
CO2
Fixation

Acetyl-CoA
Pyruvate

CO

HCO

farnesene
long chain alkenes
isobutanol

(1) Generating Reducing Power

(2) Incorporation of Inorganic Carbon

(3) Microbial Synthesis

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.
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Biological conversion of carbon dioxide and hydrogen Hawkins et al.

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)

Ralstonia eutropha H16

Betaproteobacteria

Ralstonia eutropha H16

Pyrococcus furiosus

Betaproteobacteria
Archaeon

Electrochemically
generated H2
H2
H2

Clostridium ljungdahlii

Acetogenic bacteria

H2

isobutanol and other higher alcohols from sugars [14,15],

as well as in Clostridium cellulolyticum to produce isobutanol from cellulose [16]. R. eutropha can also use electrochemically 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 WoodLjungdahl (WL) pathway. The most
energetically efficient pathway for CO2 fixation is the
WL 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 2,3butanediol 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-hydroxypropionate (3HP), dicarboxylates (DC) and/or 4-hydroxybutyrate (4HB). One version is found in members of the
thermoacidophilic archaeal order Sulfolobales, which
includes species in the genera Sulfolobus and Metallosphaera [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 metabolism via succinyl-CoA rather than acetyl-CoA
[9,10,14,15,24]. The enzymatic details of this pathway
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CO2 fixation cycle

CBB cycle
CBB cycle
3HP/4HB cycle from
M. sedula
WL pathway

Status

References

PHB, butanol, farnesene,

long-chain alkenes
Isobutanol
n-butanol

Target products

[18]

D
D

[11]
[3]

n-butanol, ethanol

[21]

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 engineered 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 temperaturedependent 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 CO2fixing 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 WL 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-oxidizing 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 separate [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 advantageous in bioprocessing, especially under conditions
where maintaining strict anoxia is costly [11,29].
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4 Energy biotechnology

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 different 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 WL
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 reactions 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 thermodynamically feasible. One common explanation for
this is based on the different electron carriers used. For
example, ferredoxin (Eo < 400 mV) has a lower
reduction potential than NAD(P)H (Eo = 320 mV),
but the extra energetic contribution of oxidizing two
ferredoxin molecules compared to one NADPH molecule
under standard conditions (20 kJ mol 1) does not equal
the energy released by ATP hydrolysis (over 50 kJ mol 1)
[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 pattern in the reaction profile of carbon fixation enzymes
[34]. Inspection of the reduction potentials of halfreactions 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
Current Opinion in Biotechnology 2013, 24:19

in each carbon fixation pathway are coupled directly or

indirectly to carboxylation and carboxyl reduction reactions. Some pathways manage to reduce the ATP requirements of carbon fixation by coupling unfavorable
reactions to exergonic reactions other than ATP hydrolysis. In the case of the WL 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 prospect 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 approximately 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 C4glyoxylate cycles. These pathways all produce glyoxylate, 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
23 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 interconversion of molecular hydrogen and protons in the presence of
a suitable electron carrier. Based on structural and biochemical analysis, hydrogenases can be categorized by
the metal atoms in their active sites: [NiFe]-hydrogenases, [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 Desulfovibrio gigas [3943] and the [FeFe]-hydrogenases from
Clostridium pasteurianum [44] and Desulfovibrio desulfuricans [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
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Biological conversion of carbon dioxide and hydrogen Hawkins et al.

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 deactivation, 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]-hydrogenases 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 cytoplasmic 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 ironsulfur cluster. HoxF and HoxU contain multiple
ironsulfur cluster and a flavin and these channel electrons 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 respiratory 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 respiration 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 respiratory 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]
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catalytic site, multiple ironsulfur clusters, and flavin

adenine dinucleotide. SHI only uses NADP+ as an electron 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 production, 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. Consequently, 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 concentration of CO2 in the environment. As such, mechanisms 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 photoautotrophic microorganisms, green algae, and plants have
developed distinct mechanisms to increase the concentration of CO2 in close proximity to ribulose-1,5-bisphosphate 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 substrate, 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 conversion of CO2 and water to bicarbonate (HCO3 , 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,
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6 Energy biotechnology

archaea, and bacteria, although the b-class are most

prevalent in CO2-fixing organisms [59,60,61]. The catalytic 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 M 1 s 1).
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 engineering 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 acetogenic 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 uncharacterized CO2-concentrating mechanisms might be present 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 availability of genetic tools, as well as a sound knowledge of
gene regulation and metabolism in the target host. Current electrofuel projects are taking advantage of an established 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 organisms 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 hosts
native metabolism may be key to establishing an efficient
process. It is still too early to determine if the autotrophic 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 homologous recombination [6366], as well as systems-level
analysis tools including microarrays [67]. While some
species of Clostridium are also natural H2-utilizing autotrophs, genetic tools for these organisms are still being
developed. So far they include plasmid-based and inducible 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 electrofuel 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

Table 2
Genetic tools for select electrofuels organisms. Current genetics tools for electrofuels organisms.
Organism

Genome sequence

Ralstonia eutropha H16

Clostridium spp.

Many

Pyrococcus furiosus

Wild-type and COM1

Metallosphaera sedula

Current Opinion in Biotechnology 2013, 24:19

Genetic tools
Plasmid-based gene expression [62]
Gene knockout or deletion [63,65]
Chromosomal insertion [64]
Plasmid-based gene expression [68]
Anti-sense RNA silencing [68]
Expression reporter systems [68]
Chromosomal insertion [70]
Expression regulation [53]
Chromosomal insertion [27,79]
Gene knockout or deletion [72]

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Biological conversion of carbon dioxide and hydrogen Hawkins et al.

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 valuable industrial chemical building block. This was accomplished 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 overexpression of cytoplasmic hydrogenase I (SHI) [53],
thereby potentially allowing facile H2 oxidation and
NADPH production. Based on these promising preliminary 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 molecules [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 development 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 (DEAR0000081 to RMK and MWA) and the Division of Chemical Sciences,
Geosciences and Biosciences, Office of Basic Energy Sciences (DE-FG0595ER20175 to MWA).

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