Biological conversion of carbon dioxide to

photosynthetic fuels and electrofuels

Han Li
ab
and James C. Liao
*
abc
To achieve sustainable growth of human society, fossil fuels must eventually be replaced with renewable
resources. Ultimately, the energy and carbon in fuels and chemicals synthesized must come from the sun
and CO
2
directly. Biological systems hold the promise to catalyze the synthesis of such fuels or chemicals.
This article discusses recent advances in developing biofuel production processes from CO
2
, which include
photosynthetic processes using algae and cyanobacteria and the non-photosynthetic electrofuel
processes using Ralstonia eutropha and other lithoautotrophic microorganisms. Each of these processes
involves strengths and weaknesses. While none of these processes have achieved industrial success, the
challenges involved may point the direction for further improvement within the limit of theoretical
possibility. Finally, all biological processes produce cell mass rich in protein. Regenerating ammonium by
deamination of hydrolyzed proteins may close the loop of the global nitrogen cycle, which is also one
of the major challenges in large scale biological processes.
Broader context
An ideal and sustainable fuel and chemical production process should derive energy from sunlight and carbon skeleton from CO
2
directly. Photosynthetic
microorganisms have the capability to accomplish these tasks by coupling the so-called light reaction of photosynthesis (converting solar energy to
biochemical energy) to the dark reaction and subsequent carbon metabolism (converting CO
2
to fuel). Alternatively, the light reaction can be replaced with
man-made devices such as photovoltaic cells. Biologically accessible energy converted from photons or photovoltaic electricity can then drive the carbon xation
reactions to produce a desirable compound using metabolically engineered pathways. To close the nitrogen cycle, the protein in the cell mass generated can be
deaminated to regenerate ammonia while producing more carbon-based fuels or chemicals.
Introduction
Fossil energy sources such as oil, coal, and natural gas supply
about 82% of US's total energy need.
1
While the total reserve of
fossil resources on earth is nite, our energy consumption
grows at a stunning rate of about 30% every decade.
2
This
situation is certainly not sustainable in the long run. The energy
problem is further complicated by short-term geopolitical and
economic instabilities. Moreover, CO
2
produced from fossil fuel
combustion has been implicated to contribute to climate
changes. These reasons argue for an eventual replacement of
fossil energy sources. In particular, fossil-derived liquid fuels
constitute more than 90% of the energy used in the trans-
portation sector, which cannot be replaced unless major
changes in infrastructure are in place.
To achieve sustainability, the energy must ultimately come
from the sun, and the carbon skeletons of liquid fuels must be
derived from CO
2
. In general, solar energy harvesting and CO
2
reduction can be accomplished using either man-made devices
or biological systems (Fig. 1). Each has its pros and cons. Solar
energy harvesting using man-made devices has achieved
reasonably high eciency (1045%), but the energy is output in
the form of electricity, whose storage remains problematic for
transportation applications. Also, CO
2
reduction to liquid fuels
by man-made processes is heretofore inecient and non-
specic. On the other hand, biological systems have utilized
photosynthesis to capture solar energy and reduce CO
2
to
biomass for millions of years. The typical photosynthesis e-
ciency from solar to biomass energy is less than 1% for plants,
although the opportunity for improvement is enormous thanks
to the advances in genomic and molecular biology tools. In
particular, biological reduction of CO
2
to make long-chain
reduced carbon compounds is eective and specic for CO
2
re-
utilization. With metabolic engineering and synthetic biology
tools, biological systems can be tailored to make compounds of
interest with specic structures and conformations. This capa-
bility cannot be easily achieved using non-biological methods.
a
Department of Chemical and Biomolecular Engineering, University of California Los
Angeles, Los Angeles, USA. E-mail: liaoj@ucla.edu; Fax: +1 310 206-4107; Tel: +1
310 825-1656
b
The Molecular Biology Institute, University of California Los Angeles, Los Angeles, USA
c
Institute for Genomics and Proteomics, University of California Los Angeles, Los
Angeles, USA
Cite this: Energy Environ. Sci., 2013, 6,
2892
Received 30th May 2013
Accepted 31st July 2013
DOI: 10.1039/c3ee41847b
www.rsc.org/ees
2892 | Energy Environ. Sci., 2013, 6, 28922899 This journal is The Royal Society of Chemistry 2013
Energy &
Environmental Science
MINIREVIEW
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Typically, biological fuel production processes depend on
plant biomass. In particular, gasoline and diesel substitutes
have been produced using engineered microbial catalysts from
biomass-derived substrates, including simple sugars,
39
cellu-
lose,
10,11
and minimally treated biomass.
12
However, the har-
vesting, processing, and decomposing of recalcitrant biomass
are still challenging.
13
The capability of biological systems to catalyze fuel produc-
tion from biomass can be extended to CO
2
. Thus, instead of
plants, CO
2
-xing microorganisms can be metabolically engi-
neered to produce liquid fuels directly, bypassing the lignocel-
lulose processing issues. In this scenario, CO
2
xation does not
strictly depend on light. The CO
2
-xing dark reactions in
autotrophic microorganisms can be powered by energy and
reducing equivalence derived from a variety of sources in
addition to solar energy. However, the energy needed for CO
2
xation must ultimately come from the sun. The conversion of
solar energy to drive biological CO
2
xation and fuel production
can again be accomplished using two approaches (Fig. 1): bio-
logical or non-biological. In the former case, the cell utilizes the
complete system of photosynthesis, including both the light
and dark reactions, and directly produces liquid fuel. In the
latter approach, a man-made device is used to harvest sunlight
in the form of electricity, which then powers the biological
production of fuels from CO
2
. Such processes have been dub-
bed electrofuel production.
Neither approach competes with food crops for farmland
and bypasses the recalcitrance problem of lignocellulose, while
fully capitalizing on the biological capability to synthesize
liquid fuels with high specicity and eciency. We discuss
below the recent progress and remaining challenges in these
strategies.
Biofuel production from photosynthetic
microorganisms
The rst scenario of photosynthetic production of fuel without
lignocellulosic biomass is to utilize algae. The extraordinary
capacity of some algal species to accumulate lipids has made them
attractive candidates for biofuel production (Fig. 2). It has been
proposed that algal biofuel can replace the petroleum usage of the
whole United States by only using less than 4% of the land area of
the country, which compares favorably with other biofuel crops
such as corn and oil palm.
14
Previously, the majority of the work
was focused on isolation and selection of algae species with
supreme lipid accumulation levels, fast growth rate, simple
nutrient requirement, and high light-utilization eciencies.
15
Recently, the development of next-generation sequencing and
molecular biology technologies has made the targeted engineering
of algal metabolism possible. For example, under conditions
which favor the triacylglycerol accumulation in algae such as
nitrogen and sulfur deprivation conditions, the genes that showed
dierential expression have been identied using high throughput
RNA-sequencing technologies,
16,17
which provided insight into the
cellular regulation of the lipid metabolism. Such advanced tech-
nologies are expected to improve algal oil production signicantly.
To produce fuels and chemicals directly from CO
2
without
going through lipid synthesis, cyanobacteria are the organisms of
choice. Cyanobacteria are photosynthetic prokaryotic microor-
ganisms and are more tractable for genetic engineering. For
example, synthetic pathways have been introduced into two of
the model organisms Synechococcus elongatus PCC 7942 and
Synechocystis sp. PCC 6803 for the production of ethanol,
18
iso-
butyraldehyde,
19
isobutanol,
19
1-butanol,
20,21
isoprene,
22
ethylene,
23
2-methyl-1-butanol,
24
2,3-butanediol,
25
and 1,2-pro-
panediol
26
(Fig. 2). In addition, cyanobacteria produce an array of
alkanes with diverse carbon numbers and structures
27,28
(Fig. 2),
some of which are only found in cyanobacteria. Recently, the
genes responsible for alkane production in cyanobacteria have
been identied and characterized.
5
Although simpler than algae,
metabolic engineering of cyanobacteria takes considerably more
time and eort compared with other commonly studied and
industrially utilized prokaryotes. The diculties lie in multiple
copies of the genome that present problems in genetic
knockout,
29,30
the limited number of well characterized and
controllable promoters and other genetic tools,
31
and the rela-
tively poorly characterized metabolic regulation compared with
the traditional industrial hosts.
Although suggested to be scalable, these biofuel production
processes using photosynthetic microorganisms still need to
overcome substantial challenges to be economically viable. First,
the eciency of biological photosystems is constrained by the
nature of the biological molecules used, which can only utilize
radiation within a limited spectrum. As a consequence, about 50%
of solar energy cannot be used.
32
Second, the biological photosys-
tems have not been optimized for the maximal energy conversion
when light is abundant. Therefore the eciency of solar energy
capture is also limited by the saturation eect and other complex
cellular regulations.
32,33
Third, large-scale culturing of photosyn-
thetic organisms represents a new bio-production paradigm that
Fig. 1 General schemes of solar energy harvesting and carbon xation by bio-
logical and articial approaches. Man-made devices are relatively ecient for
solar energy harvesting and reducing power generation; while biological meta-
bolic pathways are more versatile and specic for synthesis of carbon-based fuels
and chemicals from carbon dioxide. A hybrid process that combines the articial
light reaction and the biological dark reaction has been recently proposed
and demonstrated.
This journal is The Royal Society of Chemistry 2013 Energy Environ. Sci., 2013, 6, 28922899 | 2893
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requires large two-dimensional (2D) light exposing surface areas,
since sucient light exposure is only available within a layer of
around 20 centimeters from the surface. Compared to the
conventional three-dimensional (3D) microbial fermentation
processes, which have been used in the industrial scale for
centuries, the 2D process still requires substantial breakthroughs
to be economically competitive. For example, the open-pond
culture faces the diculties in nutrient delivery, product collec-
tion, culture maintenance, and water loss.
34
Alternatively, closed
photo-bioreactors may be needed to maximize sunlight utilization
and cell growth,
14,35,36
particularly for genetically modied organ-
isms. However, the cost is inevitably increased. In addition, the
product toxicity issue in photosynthetic chemical and fuel
production needs to be addressed. Higher alcohols may disrupt
the structure of the cell membrane, which contains the essential
light harvesting apparatus of photosynthesis. For example, it has
been reported that cyanobacterium S. elongatus PCC 7942 showed
signicant growth retardation with 750 mg L
1
isobutanol
19
or
600 mg L
1
2-methyl-1-butanol.
24
In situ removal of the product
might be one solution,
19
which may require closed photo-biore-
actor systems in a large scale. Alternatively, strain evolution and
genetic engineering may yield production strains with higher
tolerance. In-depth understanding of the cell physiology in
photosynthetic microorganisms is needed.
Electrofuel: separation of light and dark
reactions
The expense of direct photosynthetic production of fuels largely
lies in the cost of 2D photo-bioreactors, which are needed to
distribute light throughout the culture. On the other hand, the
traditional 3D microbial bioreactors that can hold a large
amount of cultures in the bulk of the bioreactor are relatively
inexpensive, but cannot distribute light to the bulk. One way to
circumvent the need for photo-bioreactors is to separate the
light and dark reactions of the photosynthesis process, so
that the light reactions can be substituted by man-made
photovoltaic solar panels or wind turbines. Both devices
generate intermittent electricity, which causes a major problem
in storage. If the intermittent electricity can be utilized to drive
the dark reactions in a bulk bioreactor, the electricity storage
problem can be solved and the need for a 2D photo-bioreactor
can be avoided. In addition, this approach could have better
sun-to-fuel eciency. For example, plants growing at the
current average growth rate of 1 kg biomass dry weight per m
2
per year in the United States capture only about 0.28% of the
incident solar energy,
37
whereas man-made solar cells collect
energy from sunlight and generate electricity with relatively
high eciencies ranging from 10 to 40%.
38,39
The high sunlight
harvesting eciencies will greatly reduce the land usage.
37
Furthermore, it expands the boundaries of biofuels by exploring
the vast repertoire of lithoautotrophic microorganisms which
can x CO
2
in the dark and have diverse metabolic and physi-
ological features. For example, while the photosynthetic plants,
cyanobacteria, and algae used for traditional biofuel production
utilize the CalveBensonBassham (CBB) cycle exclusively for
CO
2
xation, a number of other CO
2
xation pathways, such as
the WoodLjungdahl pathway, exist in lithoautotrophic micro-
organisms which have higher energy eciencies
40
(Fig. 2).
Direct electron transfer to CO
2
-xing microorganisms
Transfer of electrons to the microbes can occur either directly
from an electrode or indirectly through an electron mediator
Fig. 2 Metabolic pathways for biofuel and chemical synthesis using autotrophic microorganisms. CBB cycle, CalvinBensonBasshamcycle; TCAcycle, tricarboxylic acid
cycle; ACP, acyl carrier protein.
2894 | Energy Environ. Sci., 2013, 6, 28922899 This journal is The Royal Society of Chemistry 2013
Energy & Environmental Science Minireview
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(Fig. 3). Early studies have suggested that some species such as
Geobacter metallireducens
41
are able to accept electrons directly
from electrodes as the respiration electron donor. It has also
been suggested that electricity can directly drive methane
production from CO
2
by a microbial biolm containing
predominantly a methanogen, Methanobacterium palustre.
42
Recently, a broad range of microorganisms including Spor-
omusa species, Clostridium species, and Moorella thermoacetica
have been demonstrated to be able to accept electrons directly
from the electrode and produce native fermentation products
such as acetate and 2-oxobutyrate from CO
2
(ref. 43 and 44)
(Fig. 2). These processes achieved high current eciencies with
low overpotential. Moreover, one of the organisms that is shown
to be capable of accepting electrons directly from electrodes,
Clostridium ljungdahlii,
45
has recently been engineered to
produce 1-butanol, a gasoline substitute, from fructose. The
results indicated the possibility that the direct electron transfer
approach may be used to make liquid fuels using CO
2
and
electricity.
However, several biological and engineering diculties have
to be considered. First, fundamental metabolic engineering
barriers need to be overcome for the production of non-native
liquid fuels. These anaerobic acetogenic microbes do not
perform respiration and rely largely on fermentative pathways
to generate ATP. If the carbon ux is diverted away from the
native fermentation product formation, the overall fuel yield
may be drastically reduced due to energy deciency.
40
To solve
this problem, basic energy conversion mechanism and meta-
bolic regulations in these organisms need to be understood.
Alternatively, acetate produced by acetogens from CO
2
can be
fed to an aerobic organism in a separate reactor to produce long
carbon chain products.
46
Second, direct electron transfer
involves a 2D biolm-based production process,
47
which enjoys
the favorable features of long-term stability and robustness in
lab scales but has not been used in industrial scales.
48,49
For the
microbes to accept electrons, they need to be constrained to the
biolms on the electrodes. As such, a large electrode surface
area is needed to support large scale production, which repre-
sents higher cost and requires specially designed bioreactors.
Delivery of electrons to microbes via carriers
Alternatively, electrons can be delivered to the microbes via
chemicals (Fig. 3). To be sustainable in a large scale, these
carriers need to be derived from electrochemical reactions with
minimal cost and without adverse environmental eects.
Hydrogen and formic acid are two top choices for this purpose
(Fig. 3). Hydrogen has a low solubility in water, which signi-
cantly limits its mass transfer rate from the gas phase to the
microbes in the liquid phase. In addition, the safety issues
associated with hydrogen utilization will increase the cost in
large scale manufacturing. The direct electron transfer from the
electrode to the microbes mentioned above may overcome the
mass transfer issue, but replace it with other challenges in scale
up. Alternatively, soluble electron carriers could be used such as
formic acid. Formic acid is highly soluble in aqueous solution
with no safety issues. It can also be produced electrochemically
using water and CO
2
.
50
Furthermore, the formate utilization
pathway is present in a broad range of microorganisms. Other
inexpensive, non-toxic electron carriers are also possible, but
large-scale environmental impact needs to be considered.
Hydrogen generated from water splitting can be used as the
energy and electron source for lithoautotrophic organisms.
Current electrolytic hydrogen generation processes have high
energy eciencies of more than 50%.
39,51
By coupling with solar
photovoltaics or wind energy, the estimated price of renewable
hydrogen (3.6 to 7.6 $ per kg) is closer to that of natural gas
derived hydrogen (1 to 2 $ per kg).
52
Recently, photo-electro-
chemical cells using earth-abundant materials have been
actively studied as another method to split water using solar
energy.
5255
The photo-electrochemical cells mimic the natural
photosynthetic light reactions in that they both convert solar
energy to chemical energy in H
2
equivalents. The remaining
challenges are (1) capturing the energy in H
2
and converting to
biologically usable reducing equivalents; (2) reducing CO
2
to
universal metabolic building blocks such as pyruvate and
acetyl-CoA; and (3) knitting the carboncarbon bonds using the
building blocks to produce fuels with desired structures.
56
A wide range of lithoautotrophic microorganisms can use
hydrogen as the energy source, among which Ralstonia eutropha
has been well studied as the model organism.
57
R. eutropha is
facultative chemolithoautotrophic and has the unique oxygen-
tolerant [NiFe]-hydrogenases, which confer ease of handling
Fig. 3 Direct and indirect methods to transfer electrons to the microorganisms. (a)
Hydrogen and formate as the mediators to deliver electrons to engineered Ralstonia
eutropha cells for higher alcohol production from carbon dioxide. (b) Ammonia as
the electron donor for the autotrophic growth of Nitrosomonas europaea cells. (c)
Reduced neutral red (NRH) delivers electrons to support growth and fumarate
reduction in Actinobacillus succinogenes. (d) Acidithiobacillus ferrooxidans can utilize
Fe
2+
as the energy source to power carbon xation and support growth. (e) A broad
range of microorganisms including Sporomusa species, Clostridium species, and
Moorella thermoacetica accept electrons directly from the electrode and produce
native fermentation products such as acetate from CO
2
. (f) A microbial biolm
containing predominantly a methanogen, Methanobacterium palustre, directly
obtains electrons fromthe cathode to drive methane production fromCO
2
. Acetate
was also present to support the growth of the biolm.
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and make the organism a favorable host for metabolic engi-
neering and large scale production. Recently, biosynthetic
pathways for branched-chain higher alcohols (isobutanol and
3-methyl-1-butanol) have been introduced to R. eutropha
58,59
(Fig. 2). The engineered strain produced 1 g L
1
higher alco-
hols from CO
2
using electrochemically produced hydrogen as
the sole energy source.
58
These results demonstrated the feasi-
bility of electrofuel production using H
2
as the intermediate,
and the biocatalysts of potentially higher eciencies are yet to
be explored. For example, the thermophilic hydrogen-utilizing
bacterium Hydrogenobacter thermophilus has been shown to use
the reductive TCA cycle for CO
2
xation,
60,61
which has higher
energy eciency than the CBB cycle in R. eutropha. It has also
been suggested that the highly ecient 3-hydroxypropionate/
4-hydroxybutyrate (3-HP/4-HB) CO
2
xation cycle in thermo-
philic archaea might be introduced into a genetically tractable,
hyperthermophilic, and hydrogen-utilizing host such as
Pyrococcus furiosus.
62,63
As discussed above, the low solubility and the safety
concerns of hydrogen represent a major hurdle for its utiliza-
tion in a large scale bacterial culture system. To overcome this
problem, formate can be used as an electron carrier instead of
hydrogen. In addition to being produced by electrochemical
reduction of CO
2
,
50
it is also a major byproduct in chemical
conversion of biomass.
64
Similar to hydrogen, multiple lith-
oautotrophic organisms are known to utilize formate as the
electron donor by using formate dehydrogenases that produce
NADH from formate while releasing CO
2
as the substrate for the
CBB cycle.
58
The engineered R. eutropha described above
58
was
also able to produce 1.2 g L
1
higher alcohols using formate as
the sole carbon and energy source.
To demonstrate the direct conversion of electricity to liquid
fuel, an integrated electro-microbial bioreactor was constructed
using formate as an electron transfer intermediate.
58
In this
system, both electrochemical production of formate and
biochemical conversion of formate to fuel are conducted in the
same reactor, thus avoiding the separation cost of formate and
minimizing the back reaction of the electrochemical system. In
this system, the formic acid is formed by the electrochemical
reduction of CO
2
in the aqueous solution using indium as the
cathodic catalyst. The formic acid produced is utilized by the
engineered Ralstonia cells to generate CO
2
and NADH. Thus,
formate serves as a carrier to deliver both CO
2
and the reducing
equivalent to the cell. CO
2
is then converted to higher alcohols,
isobutanol and 3-methyl-1-butanol, through the CBB cycle and
the engineered fuel production pathway.
3
One complication of
this integrated electro-microbial production system is that the
reactive compounds may be generated from the anode reaction
in the microbial growth medium. In particular, the generation
of nitric oxide and superoxide was detected and suggested to
trigger cellular response as demonstrated using a transcrip-
tional reporter system.
58
This problem is reminiscent of the
challenges in biological photosystems, where reactive species
are commonly generated. Instead of relying on the sophisti-
cated biological anti-stress systems, the problem of free radicals
generated in the electro-microbial system was mitigated by
shielding the anode from the bulk of the reactor. Alternatively,
medium optimization and other anode coating methods could
be developed for further improvement of eciency.
Other alternative electron carriers have also been reported
(Fig. 3). Growth of the lithoautotroph Nitrosomonas europaea
using CO
2
and electricity-generated ammonia is one example.
47
In this case, N. europaea cells utilized the reducing energy in
ammonia and secreted the oxidized end product nitrite, which
was continuously recycled to a separated electrochemical
module and reduced back to ammonia with around 100%
current eciencies. The challenge of this system is the low
growth rate and the evaporation of ammonia from the system,
causing drop in eciency. Another chemolithoautotroph Acid-
ithiobacillus ferrooxidans can utilize Fe
2+
as the energy source to
power carbon xation,
6567
which makes it another attractive
host for electrofuel production. Some articial redox carriers
such as neutral red can also be used to introduce reducing
energy to biological systems to drive metabolism,
68
which
suggests the possibilities that novel articial electron carriers
with dierent chemical properties could be designed to deliver
electrons to desired microorganisms. However, the cost for
their large scale production and environmental impacts need to
be addressed.
Electrofuels as a means for electricity storage
The current method of electricity storage via batteries suers
from the low energy density, which generally ranges between 0.1
and 0.7 MJ kg
1
(or 0.52.0 MJ L
1
).
69
In contrast, the energy
density of gasoline is around 45 MJ kg
1
. Given the limited on-
board space in the vehicles, the low energy density of batteries
greatly hampered their usage in the transportation sector.
Although major innovations in lithium-ion battery technology
have been made recently,
7073
ve times greater energy densities
are required for the future all-electric vehicles to have a 300400
mile driving range.
73
To match the performance of internal
combustion engines in the global scale, batteries with orders of
magnitude higher energy densities may be necessary. Alterna-
tively, electrolytic water splitting can store electrical energy in
chemical bonds in H
2
molecules with eciencies higher than
50%. However, the volumetric energy density of H
2
is low
(5.6 MJ L
1
at 700 bar and 8.5 MJ L
1
as liquid hydrogen) and H
2
utilization in the transportation sector remains dicult. The
development of electrofuels provides a promising approach for
storing intermittent electricity, such as solar and wind power-
generated electricity, in the form of liquid fuels that can be used
for transportation directly.
Nitrogen recycling for biofuel production
systems
As discussed, liquid fuels can be directly produced using CO
2
by
photosynthetic or lithoautotrophic microorganisms. The
production of such fuels and their subsequent combustion
forms a closed carbon cycle and thus reduces the net carbon
emission. However, many nutrients required in the biological
fuel production schemes are utilized in a one-pass, non-recy-
clable manner, among which the reduced nitrogen
2896 | Energy Environ. Sci., 2013, 6, 28922899 This journal is The Royal Society of Chemistry 2013
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(ammonium) is required in the greatest amount
56,7476
for the
synthesis of proteins and nucleotides. Some cyanobacteria
species can produce ammonium from nitrogen in the air,
7779
which may represent an opportunity to solve the problem.
However, most autotrophic microorganisms mentioned above
do not x nitrogen. Thus, conventional agriculture as well as
future biological production of fuels must rely on the articial
nitrogen reduction process, known as the HaberBosch process,
to provide ammonium as the nitrogen source. This process is
energy intensive and environmentally unfriendly. And the
nitrogenous nutrients used in biological production are
currently not recycled.
To close the nitrogen cycle, ammonium needs to be freed
from the biomass residues of microbial fermentation and
reused to build new biocatalysts (Fig. 4). Moreover, it would be
ideal if the deaminated carbon skeletons in protein-rich
biomass residues could be converted to fuels or chemicals. The
traditional ways for biomass residue treatment include anaer-
obic digestion,
80,81
which employs a series of reactions carried
out by acidogenic bacteria and methanogenic archaea in the
absence of oxygen. The process can convert biomass to CO
2
and
methane, while releasing other mineral nutrients. The inter-
mediate product hydrogen and the nal product methane can
be used as biofuels.
80
Recently, a new approach has been
demonstrated.
74
Microbial cells were engineered to produce
higher alcohols from waste protein with high yield.
74
Nitrogen
metabolic pathways in E.coli were redesigned to strip amino
groups from amino acids, and higher alcohol production
pathways were introduced to convert the carbon skeletons of
amino acids into fuels.
To realize the potential of nitrogen recycling from the
biomass waste, other studies have been focusing on optimizing
the treatment process of biomass to release amino acids and
other constituents.
82,83
Alternatively, microorganisms have also
been engineered to display proteases on the surface and can
utilize proteins directly.
84
The use of protein-rich biomass residuals achieves several
advantages, including closing the nitrogen cycle, and increasing
the overall energy and carbon yield of biological processes. In
addition, it allows the use of algae biomass and animal wastes
as resources to derive ammonia as a nutrient and carbon
compounds as fuel. When used in the algal processes, it avoids
the need for a long nutrient starvation period for lipid accu-
mulation since accumulated biomass will not be wasted.
Similar benets are realizable in any biological processes for
fuel production.
Conclusion
Ultimately, biofuel production should use only CO
2
, H
2
O, and
sunlight as starting materials and energy input. Toward this
goal, various photosynthetic and lithoautotrophic microor-
ganisms have been engineered for biofuel production, which
potentially represents the most direct and ecient route from
CO
2
to fuels. In addition, a specialized biological system for
recycling reduced nitrogen from biomass waste has also been
constructed with the prospect of minimizing nutrient dissi-
pation from the biofuel production scheme. Although the
initial successes are promising, substantial scientic and
engineering barriers remain for these processes to be scalable
and economically viable. Meanwhile, the vastly diverse pop-
ulation of autotrophic microorganisms in the environment still
awaits exploration as the hosts for CO
2
conversion. Such bio-
logical resources and the rapidly advancing next-generation
sequencing and biomolecular tools will surely facilitate prog-
ress toward eventual replacement of fossil fuels.
References
1 Energy Information Administration, Monthly Energy Review
March 2012, http://www.eia.doe.gov/emeu/mer/pdf/pages/
sec1_7.pdf.
2 Energy Information Administration, International energy
outlook 2011, http://www.eia.gov/forecasts/ieo/.
3 S. Atsumi, T. Hanai and J. C. Liao, Nature, 2008, 451, 8689.
4 K. Zhang, M. R. Sawaya, D. S. Eisenberg and J. C. Liao, Proc.
Natl. Acad. Sci. U. S. A., 2008, 105, 2065320658.
5 A. Schirmer, M. A. Rude, X. Li, E. Popova and S. B. del
Cardayre, Science, 2010, 329, 559562.
6 B. B. Bond-Watts, R. J. Bellerose and M. C. Chang, Nat. Chem.
Biol., 2011, 7, 222227.
7 C. R. Shen, E. I. Lan, Y. Dekishima, A. Baez, K. M. Cho and
J. C. Liao, Appl. Environ. Microbiol., 2011, 77, 29052915.
8 Y. Dekishima, E. I. Lan, C. R. Shen, K. M. Cho and J. C. Liao,
J. Am. Chem. Soc., 2011, 133, 1139911401.
9 C. Dellomonaco, J. M. Clomburg, E. N. Miller and
R. Gonzalez, Nature, 2011, 476, 355359.
10 W. Higashide, Y. Li, Y. Yang and J. C. Liao, Appl. Environ.
Microbiol., 2011, 77, 27272733.
11 G. Bokinsky, P. P. Peralta-Yahya, A. George, B. M. Holmes,
E. J. Steen, J. Dietrich, T. Soon Lee, D. Tullman-Ercek,
C. A. Voigt, B. A. Simmons and J. D. Keasling, Proc. Natl.
Acad. Sci. U. S. A., 2011, 108, 1994919954.
12 A. J. Wargacki, E. Leonard, M. N. Win, D. D. Regitsky,
C. N. Santos, P. B. Kim, S. R. Cooper, R. M. Raisner,
A. Herman, A. B. Sivitz, A. Lakshmanaswamy,
Fig. 4 Nitrogen recycling for biofuel production systems. To achieve nitrogen
neutral biofuel production, ammonia and carbon portions in the protein waste are
separated. The free ammonia is fed back to the microbial process as a nutrient. And
the carbon skeletons of the amino acids are converted to the biofuel product.
Overall, thanks to the nitrogen recycling process, the input of the biofuel production
system are only sunlight, carbon dioxide, and water. And the output is biofuels.
This journal is The Royal Society of Chemistry 2013 Energy Environ. Sci., 2013, 6, 28922899 | 2897
Minireview Energy & Environmental Science
P
u
b
l
i
s
h
e
d

o
n

0
1

A
u
g
u
s
t

2
0
1
3
.

D
o
w
n
l
o
a
d
e
d

b
y

A
c
a
d
e
m
i
a

S
i
n
i
c
a

-

T
a
i
p
e
i

o
n

1
4
/
0
8
/
2
0
1
4

1
1
:
0
6
:
0
4
.

View Article Online
Y. Kashiyama, D. Baker and Y. Yoshikuni, Science, 2012, 335,
308313.
13 D. Graham-Rowe, Nature, 2011, 474, S6S8.
14 D. R. Georgianna and S. P. Mayeld, Nature, 2012, 488, 329
335.
15 L. Rodol, G. Chini Zittelli, N. Bassi, G. Padovani, N. Biondi,
G. Bonini and M. R. Tredici, Biotechnol. Bioeng., 2009, 102,
100112.
16 R. Miller, G. Wu, R. R. Deshpande, A. Vieler, K. Gartner,
X. Li, E. R. Moellering, S. Zauner, A. J. Cornish, B. Liu,
B. Bullard, B. B. Sears, M. H. Kuo, E. L. Hegg,
Y. Shachar-Hill, S. H. Shiu and C. Benning, Plant
Physiol., 2010, 154, 17371752.
17 D. Gonzalez-Ballester, D. Casero, S. Cokus, M. Pellegrini,
S. S. Merchant and A. R. Grossman, Plant Cell, 2010, 22,
20582084.
18 J. Dexter and P. Fu, Energy Environ. Sci., 2009, 2, 857864.
19 S. Atsumi, W. Higashide and J. C. Liao, Nat. Biotechnol.,
2009, 27, 11771180.
20 E. I. Lan and J. C. Liao, Metab. Eng., 2011, 13, 353363.
21 E. I. Lan and J. C. Liao, Proc. Natl. Acad. Sci. U. S. A., 2012,
109, 60186023.
22 P. Lindberg, S. Park and A. Melis, Metab. Eng., 2010, 12, 70
79.
23 J. Ungerer, L. Tao, M. Davis, M. Ghirardi, P.-C. Maness and
J. Yu, Energy Environ. Sci., 2012, 5, 89989006.
24 C. R. Shen and J. C. Liao, Energy Environ. Sci., 2012, 5, 9574
9583.
25 J. W. Oliver, I. M. Machado, H. Yoneda and S. Atsumi, Proc.
Natl. Acad. Sci. U. S. A., 2013, 110, 12491254.
26 H. Li and J. C. Liao, Microb. Cell Fact., 2013, 12, 4.
27 V. M. Dembitskii, I. Dor, I. Shkrob and M. Aki, Bioorg. Khim.,
2001, 27, 130140.
28 P. Bhadauriya, R. Gupta, S. Singh and P. Bisen, World J.
Microbiol. Biotechnol., 2008, 24, 139141.
29 M. Matsuoka, K. Takahama and T. Ogawa, Microbiology,
2001, 147, 20772087.
30 M. Griese, C. Lange and J. Soppa, FEMS Microbiol. Lett., 2011,
323, 124131.
31 D. Geerts, A. Bovy, G. de Vrieze, M. Borrias and P. Weisbeek,
Microbiology, 1995, 141(Pt 4), 831841.
32 X. G. Zhu, S. P. Long and D. R. Ort, Curr. Opin. Biotechnol.,
2008, 19, 153159.
33 D. C. Ducat, J. C. Way and P. A. Silver, Trends Biotechnol.,
2011, 29, 95103.
34 N. Savage, Nature, 2011, 474, S15S16.
35 E. Molina, J. Fernandez, F. G. Acien and Y. Chisti, J.
Biotechnol., 2001, 92, 113131.
36 C. Y. Chen, K. L. Yeh, R. Aisyah, D. J. Lee and J. S. Chang,
Bioresour. Technol., 2011, 102, 7181.
37 R. Agrawal, N. R. Singh, F. H. Ribeiro and W. N. Delgass,
Proc. Natl. Acad. Sci. U. S. A., 2007, 104, 48284833.
38 R. R. King, D. C. Law, K. M. Edmondson, C. M. Fetzer,
G. S. Kinsey, H. Yoon, R. A. Sherif and N. H. Karam, Appl.
Phys. Lett., 2007, 90, 183516.
39 R. Agrawal and N. R. Singh, Annu. Rev. Chem. Biomol. Eng.,
2010, 1, 343364.
40 A. G. Fast and E. T. Papoutsakis, Curr. Opin. Chem. Eng.,
2012, 1, 380395.
41 K. B. Gregory, D. R. Bond and D. R. Lovley, Environ.
Microbiol., 2004, 6, 596604.
42 S. Cheng, D. Xing, D. F. Call and B. E. Logan, Environ. Sci.
Technol., 2009, 43, 39533958.
43 K. P. Nevin, T. L. Woodard, A. E. Franks, Z. M. Summers and
D. R. Lovley, mBio, 2010, 1(2), e00103e00110.
44 K. P. Nevin, S. A. Hensley, A. E. Franks, Z. M. Summers, J. Ou,
T. L. Woodard, O. L. Snoeyenbos-West and D. R. Lovley, Appl.
Environ. Microbiol., 2011, 77, 28822886.
45 M. Kopke, C. Held, S. Hujer, H. Liesegang, A. Wiezer,
A. Wollherr, A. Ehrenreich, W. Liebl, G. Gottschalk and
P. Durre, Proc. Natl. Acad. Sci. U. S. A., 2010, 107, 13087
13092.
46 G. Stephanopoulos, Bioprocess and microbe engineering for
total carbon utilization in biofuel production, US Pat. US
20110177564A1, 2011.
47 W. O. Khunjar, A. Sahin, A. C. West, K. Chandran and
S. Banta, PLoS One, 2012, 7, e44846.
48 R. Gross, K. Buehler and A. Schmid, Biotechnol. Bioeng.,
2013, 110, 424436.
49 B. Rosche, X. Z. Li, B. Hauer, A. Schmid and K. Buehler,
Trends Biotechnol., 2009, 27, 636643.
50 S. Ikeda, T. Takagi and K. Ito, Bull. Chem. Soc. Jpn., 1987, 60,
2517.
51 Committee on Assessment of Resource Needs for Fuel Cell
and Hydrogen Technologies and National Research
Council, Transitions to Alternative Transportation
TechnologiesA Focus on Hydrogen, The National Academies
Press, 2008.
52 J. Newman, P. G. Hoertz, C. A. Bonino and J. A. Trainham,
J. Electrochem. Soc., 2012, 159, A1722A1729.
53 S. Y. Reece, J. A. Hamel, K. Sung, T. D. Jarvi, A. J. Esswein,
J. J. Pijpers and D. G. Nocera, Science, 2011, 334, 645648.
54 Y. Hou, B. L. Abrams, P. C. Vesborg, M. E. Bjorketun,
K. Herbst, L. Bech, A. M. Setti, C. D. Damsgaard,
T. Pedersen, O. Hansen, J. Rossmeisl, S. Dahl,
J. K. Norskov and I. Chorkendor, Nat. Mater., 2011, 10,
434438.
55 H. Zhu, N. Song, H. Lv, C. L. Hill and T. Lian, J. Am. Chem.
Soc., 2012, 134, 1170111708.
56 R. Conrado, C. Haynes, B. Haendler and E. Toone, in
Advanced Biofuels and Bioproducts, ed. J. W. Lee, Springer,
New York, 2013, ch. 38, pp. 10371064.
57 A. Pohlmann, W. F. Fricke, F. Reinecke, B. Kusian,
H. Liesegang, R. Cramm, T. Eitinger, C. Ewering,
M. Potter, E. Schwartz, A. Strittmatter, I. Voss,
G. Gottschalk, A. Steinbuchel, B. Friedrich and B. Bowien,
Nat. Biotechnol., 2006, 24, 12571262.
58 H. Li, P. H. Opgenorth, D. G. Wernick, S. Rogers, T. Y. Wu,
W. Higashide, P. Malati, Y. X. Huo, K. M. Cho and
J. C. Liao, Science, 2012, 335, 1596.
59 J. Lu, C. J. Brigham, C. S. Gai and A. J. Sinskey, Appl.
Microbiol. Biotechnol., 2012, 96, 283297.
60 T. Kawasumi, Y. Igarashi, T. Kodama and Y. Minoda, Int. J.
Syst. Bacteriol., 1984, 34, 510.
2898 | Energy Environ. Sci., 2013, 6, 28922899 This journal is The Royal Society of Chemistry 2013
Energy & Environmental Science Minireview
P
u
b
l
i
s
h
e
d

o
n

0
1

A
u
g
u
s
t

2
0
1
3
.

D
o
w
n
l
o
a
d
e
d

b
y

A
c
a
d
e
m
i
a

S
i
n
i
c
a

-

T
a
i
p
e
i

o
n

1
4
/
0
8
/
2
0
1
4

1
1
:
0
6
:
0
4
.

View Article Online
61 H. Arai, H. Kanbe, M. Ishii and Y. Igarashi, J. Bacteriol., 2010,
192, 26512652.
62 A. S. Hawkins, Y. Han, H. Lian, A. J. Loder, A. L. Menon,
I. J. Iwuchukwu, M. Keller, T. T. Leuko, M. W. W. Adams
and R. M. Kelly, ACS Catal., 2011, 1, 10431050.
63 M. W. Keller, G. J. Schut, G. L. Lipscomb, A. L. Menon,
I. J. Iwuchukwu, T. T. Leuko, M. P. Thorgersen,
W. J. Nixon, A. S. Hawkins, R. M. Kelly and M. W. Adams,
Proc. Natl. Acad. Sci. U. S. A., 2013, 110, 58405845.
64 R. Xing, W. Qi and G. W. Huber, Energy Environ. Sci., 2011, 4,
21932205.
65 R. C. Blake, G. T. Howard and S. McGinness, Appl. Environ.
Microbiol., 1994, 60, 27042710.
66 P. I. Harvey and F. K. Crundwell, Appl. Environ. Microbiol.,
1997, 63, 25862592.
67 J. Valdes, I. Pedroso, R. Quatrini, R. J. Dodson, H. Tettelin,
R. Blake, 2nd, J. A. Eisen and D. S. Holmes, BMC Genomics,
2008, 9, 597.
68 D. H. Park, M. Laivenieks, M. V. Guettler, M. K. Jain and
J. G. Zeikus, Appl. Environ. Microbiol., 1999, 65, 29122917.
69 J. M. Tarascon and M. Armand, Nature, 2001, 414, 359367.
70 I. Kovalenko, B. Zdyrko, A. Magasinski, B. Hertzberg,
Z. Milicev, R. Burtovyy, I. Luzinov and G. Yushin, Science,
2011, 334, 7579.
71 Z. Peng, S. A. Freunberger, Y. Chen and P. G. Bruce, Science,
2012, 337(6094), 563566.
72 P. G. Bruce, S. A. Freunberger, L. J. Hardwick and
J.-M. Tarascon, Nat. Mater., 2012, 11, 1929.
73 M. M. Thackeray, C. Wolverton and E. D. Isaacs, Energy
Environ. Sci., 2012, 5, 78547863.
74 Y.-X. Huo, K. M. Cho, J. G. L. Rivera, E. Monte, C. R. Shen,
Y. Yan and J. C. Liao, Nat. Biotechnol., 2011, 29, 346351.
75 Y. X. Huo, D. G. Wernick and J. C. Liao, Curr. Opin.
Biotechnol., 2012, 23, 406413.
76 D. G. Wernick and J. C. Liao, Appl. Microbiol. Biotechnol.,
2013, 97, 13971406.
77 I. Berman-Frank, P. Lundgren, Y. B. Chen, H. Kupper,
Z. Kolber, B. Bergman and P. Falkowski, Science, 2001, 294,
15341537.
78 A. S. Steunou, D. Bhaya, M. M. Bateson, M. C. Melendrez,
D. M. Ward, E. Brecht, J. W. Peters, M. Kuhl and
A. R. Grossman, Proc. Natl. Acad. Sci. U. S. A., 2006, 103,
23982403.
79 B. L. Flaherty, F. Van Nieuwerburgh, S. R. Head and
J. W. Golden, BMC Genomics, 2011, 12, 332.
80 G. Antonopoulou, H. N. Gavala, I. V. Skiadas,
K. Angelopoulos and G. Lyberatos, Bioresour. Technol.,
2008, 99, 110119.
81 V. N. Gunaseelan, Biomass Bioenergy, 1997, 13, 83114.
82 J. M. Romero Garcia, F. G. Acien Fernandez and
J. M. Fernandez Sevilla, Bioresour. Technol., 2012, 112, 164
170.
83 R. E. Teixeira, Green Chem., 2012, 14, 419427.
84 K. Y. Hara, S. Kim, H. Yoshida, K. Kiriyama, T. Kondo,
N. Okai, C. Ogino, H. Fukuda and A. Kondo, Appl.
Microbiol. Biotechnol., 2012, 93, 14951502.
This journal is The Royal Society of Chemistry 2013 Energy Environ. Sci., 2013, 6, 28922899 | 2899
Minireview Energy & Environmental Science
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