Review

Embracing Biological Solutions

to the Sustainable Energy Challenge
Oluwakemi Adesina,1,5 Isao A. Anzai,1,5 José L. Avalos,2,3,4,* and Buz Barstow1,*

Biological solutions hold unique advantages to address challenges in sustainable The Bigger Picture
energy. Living organisms have evolved for billions of years to solve problems in The world today is in a state of
catalysis, material synthesis, carbon fixation, and energy capture and storage, enormous transition. In the
including not only photosynthesis but also older metabolisms that rely on metal coming few decades, billions of
oxidation and reduction. These capabilities offer solutions to problems in sus- people are likely to leave poverty.
tainable energy, including the safe use of nuclear power, the construction and re- This unprecedented development
cycling of batteries, the extraction and processing of rare earth elements, and will be one of the greatest
the carbon-neutral or even carbon-negative synthesis of hydrocarbon fuels. Bio- opportunities to improve global
logical self-repair, self-assembly, and self-replication offer the ability to deploy quality of life and public health
these capabilities on a global scale, and evolution can be harnessed to accelerate ever presented, but it will only be
engineering. In this review, we discuss the opportunities for applied biology to realized if this development is
contribute to the sustainable energy landscape, the challenges faced, and cut- done right. The challenge of
ting-edge bioengineering that draws inspiration from fundamental research sustainable energy is not just to
into biophysics, metabolism, catalysis, and systems biology. provide energy without polluting
the atmosphere with fossil carbon
INTRODUCTION but also to do so on a global scale
The widespread use of biology has the potential to revolutionize the sustainable and at a cost that everyone can
energy landscape. It is fair to say that the use of applied biology has transformed afford. Thanks to capabilities
medicine over the last 70 years, from the first mass production of penicillin to recom- ranging from room-temperature
binant protein drugs such as erythropoietin, insulin, and human growth hormone. and pressure catalysis to self-
Looking to the future, synthetic biology has the potential to revolutionize the pro- assembly, biology offers first-draft
duction of vaccines, drug delivery, treatment of cancer, and regenerative medicine.1 solutions to problems in
We believe that with increased support and awareness, applied biology could make sustainable energy such as the
a similar mark on sustainable energy and, by doing so, protect and improve human safe use of nuclear energy, the
health and development by mitigating climate change;2 revolutionizing access to capture and storage of solar
energy; improving air and water quality; producing, processing, and distributing power, and even the construction
food; and preserving the natural environment. of advanced materials. The goal of
applied and synthetic biology in
The widespread use of intermittent sources of renewable energy such as wind and so- energy is to provide engineering
lar power; energy storage;3 nuclear power;4 energy-saving advanced materials such tools and address fundamental
as carbon composites;5 and biofuels6 have all been identified as key aspects of a questions needed to transform
future sustainable energy infrastructure. However, the cost of energy storage remains these templates into practical
high, and its durability is low; the storage of spent nuclear fuel remains technically and solutions.
politically contentious, and the further expansion of biofuels raises the specter of
competition among land for agriculture, land for wilderness, and land for energy
crops.7 All of these technological, economic, and political challenges will be ampli-
fied by the increasing energy needs of the developed and developing worlds.

Potential for Biology in the Sustainable Energy Infrastructure

A significant difference between medicinal and energy products is in their required
scale. Although only a few grams of penicillin or a few micrograms of erythropoietin
are required for therapeutic effect, a car carrying one person will consume kilograms

20 Chem 2, 20–51, January 12, 2017 ª 2017 Elsevier Inc.

Figure 1. A Map of This Article
Biological capabilities relevant to sustainable energy (left) and sustainable energy applications (right). Alongside each potential application, we have
noted the most useful biological capability for that application, followed by additional capabilities.

of gasoline every day. Even though erythropoietin produced with a very low conver-
sion efficiency from glucose is very successful in the market, given the high price of
pharmaceuticals, the same is not true for biofuels. We believe that this dramatically
different efficiency requirement means that the problems faced in the application of
biology to energy are fundamentally different from those faced in the application of
biology to health.

However, biology has several capabilities that provide it with the potential to make
substantive contributions in sustainable energy (Figure 1L). Biology offers catalytic
capabilities in energy capture and storage,8 fuel synthesis,6 metal reduction,9 metal
oxidation,10 and material synthesis.11 Biology also offers the capability to self-repli-
cate, self-assemble from Earth-abundant elements, and self-repair, which provide
the potential to develop technologies that are unparalleled in their low cost and de-
ployability8 such as agriculture.

Although the use of biology in many areas of sustainable energy might look fanciful,
it is useful to imagine the state of cancer therapy less than half a century ago. At the
time, a diagnosis of cancer was more often than not a death sentence. However,
thanks to sustained commitment to both fundamental and clinical research into
the biology of cancer, and the diagnostic and therapeutic advances derived from
them, many cancers are now manageable or even curable.
1Department of Chemistry, Frick Chemistry
This article details the capabilities that biology brings to sustainable energy and Laboratory, Princeton University, Washington
seven promising, but still developing, applications for biology to make contributions Road, Princeton, NJ 08544, USA
2Department of Chemical and Biological
to the sustainable energy infrastructure (Figure 1R). These applications are not only
Engineering, Hoyt Laboratory, Princeton
in biofuels but also in enabling non-biological sustainable energy technologies. We University, William Street, Princeton,
have ordered these applications by the prominence of the role that biology plays in NJ 08544, USA
them from supporting an abiotic technology to applications that are fundamentally 3The Andlinger Center for Energy and the
biological. In each application section, we discuss the importance of the challenge, Environment, Princeton University, 86 Olden
Street, Princeton, NJ 08544, USA
the applied and fundamental biological obstacles, particularly biochemical ones, 4Department of Molecular Biology, 119 Lewis
that need to be overcome to construct a practical solution to the problem. Thomas Laboratory, Washington Road,
Princeton, NJ 08544, USA
5Co-first
Capabilities 1 and 2: Bio-catalysis and Self-Assembly author
The challenge of sustainable energy is not simply one of providing energy without *Correspondence: javalos@princeton.edu (J.L.A.),
polluting the atmosphere with fossil carbon, but doing this on a global scale and buz@princeton.edu (B.B.)
at a price that is affordable worldwide. Two of the most basic features of biology, http://dx.doi.org/10.1016/j.chempr.2016.12.009

Chem 2, 20–51, January 12, 2017 21

bio-catalysis and self-assembly, make biology uniquely suited to the problem of sus-
tainable energy. We have outlined these capabilities and others that are derived
from them in a Venn diagram in Figure 1L. Sustainable applications of biology
that rely on these capabilities are listed in Figure 1R. Together, these diagrams
form a map of this article (Figure 1).

Capabilities 3 and 4: Self-Repair and Self-Replication; and Energy Harvesting, CO2

Fixation, and Chemosynthesis
There is no better example of the huge potential for biology to contribute to the
sustainable energy infrastructure than the enormous power channeled by photosyn-
thesis across the globe,12 largely without human intervention. Photosynthesis
demonstrates that renewable energy can be captured and stored by inexpensive
self-assembling catalysts on an enormous scale. Across the globe’s land and oceans,
photosynthesis stores z4,000 exajoules (EJ) (4 3 1021 J) of solar energy as biomass
over the course of a year. This corresponds to an average instantaneous energy stor-
age rate of z130 terawatts (TW).12 By contrast, world energy consumption in 2012
stood at only z550 EJ year
1 (an instantaneous rate of z18 TW).13 Because sunlight
is both intermittent and diffuse, the storage, harvesting, and ease of transport of
captured solar energy are just as important as the initial capture. Naturally occurring
photosynthetic organisms have the remarkable ability to store solar energy and
atmospheric CO2 in a variety of energy-dense compounds, including starch,
sucrose, cellulose, and oils.

Even as world energy use increases, the amount of energy channeled by photosyn-
thesis will remain larger for some time to come. Total world energy use is projected
to increase to z800 EJ year
1 (z30 TW) by 2040.13 Should current average rates of
increase continue until 2100, world energy consumption could stand at z 1,400 EJ
year
1 (z45 TW).13 Only if we imagine that 10.9 billion people14 (the highest projec-
tion of the human population in 2100) were to use energy at the rate that the average
American does today (z10 kW15) would world annual energy usage approach or
exceed the z4,000 EJ year
1 channeled by natural photosynthesis. However, future
energy-efficiency standards and technologies are likely to reduce this number.
Furthermore, not all forms of sustainable energy that we consume now or in the
future need to be derived from photosynthesis (e.g., wind, solar, nuclear, hydroelec-
tric, and geothermal power), which relieves the pressure to expect photosynthesis to
provide all of our energy demands, despite its huge potential.

Another advantage of biological systems is their remarkable ability for self-repair.

Whereas a tree trunk is far less resistant to breakage from severe insults than the
steel frames used to support solar photovoltaics, biological systems offer high
degrees of tolerance to degradation by day to day wear and tear, precisely the
sort of low-grade insult that limits the lifespan and hence penetration of modern
renewable technologies. For instance, Sequoia trees have functional lifespans in
excess of 3,000 years.16 By contrast, solar photovoltaics have functional lifespans
that will likely be measured in decades.17 The self-repair of biological systems could
one day offer the potential to dramatically increase the lifespan of renewable energy
investments. Much as with conventional energy sources such as nuclear and fossil
fuel-fired power plants, extensions in lifespan increase the value that can be
extracted from renewable energy investments, hence greatly raising their competi-
tiveness with conventional energy installations.

Intuitively, we might expect that biological capture and storage of solar energy as
biomass is both cheaper and more straightforward than an abiotic alternative

22 Chem 2, 20–51, January 12, 2017

 A B

Figure 2. World and US Energy Consumption

(A) World energy consumption in 2010. 23
(B) US energy consumption from July 2015 to June 2016. 15

because it relies on agriculture, one of the oldest and most mature technologies.
Simple cost estimates back this intuition. Studies indicate that the cost of producing
biomass and delivering it to the farm gate ranges from $40 per dry ton of loblolly
pine to $70 per dry ton of switchgrass.18 Assuming a lower heating value
of z19 kJ g
1,19 the cost of biological energy capture and storage is between
$2 per gigajoule (GJ) for loblolly pine and $4 GJ
1 for switchgrass. On the basis
of current cost estimates, the average extraction cost of Saudi crude is approxi-
mately $6 GJ
1,20 whereas the production cost of North American shale oil is
approximately $14 GJ
1.20 Blankenship et al.21 noted that the proper point of com-
parison for biological to abiotic solar energy conversion is with photovoltaic-driven
H2 production. Recent work by Rodriguez et al.22 estimates the lowest possible cost
of photovoltaic-driven production of H2 (including solar concentration, photovol-
taic, and electrolyzer costs but not including costs such as system maintenance
and land), and its compression and distribution range is approximately $2 kg
1 or
$6 GJ
1. Simply put, today the cost of capturing a GJ of energy from the Sun in
the US Midwest by agriculture is potentially lower than it is to extract a GJ from
the Arabian desert or the Marcellus Shale.

The ease and low cost of deployment and self-repair of biology are two of the rea-
sons that liquid fuels (biofuels) made from recently biologically fixed carbon (usually
within the last growing season, in contrast to millions of years ago) are the most suc-
cessful solar energy storage and capture schemes yet invented. Figure 2 shows a
breakdown of the sources of energy consumed in the US and around the world.
Biomass-derived energy provides 4.9% of all US energy consumption.15 This is

Chem 2, 20–51, January 12, 2017 23

approximately ten times the energy supplied by solar and approximately equal to all
other sustainable energy sources combined aside from nuclear.15 Globally, biomass
provided 10% of all energy (much of it for heating) in 2010. This is larger than all sus-
tainable energy sources combined, including nuclear.23 Furthermore, the longevity
and deployability of biology opens the possibility of radically democratizing energy
generation, allowing rural communities in both the developing and developed
worlds a supply of clean, cheap, and tradable energy that could revitalize rural econ-
omies around the world.24

The challenge to applied biology is to leverage the low cost of energy capture by
finding ways to effectively transform fixed carbon into useable fuels with the same
ease as refining crude oil into gasoline. Fortunately, biology offers an amazing
collection of enzymes that are able to work in concert to transform fixed carbon in
biomass to a variety of infrastructure-compatible fuels and fuel precursors. As bio-
catalysts can operate at room temperature and pressure and in relatively small-scale
installations, these conversions have the potential to be highly efficient in compari-
son with thermochemical processes such as Fischer-Tropsch and in principle will pro-
duce no net CO2 over their lifetime.

The relatively small-scale installations enabled by biological catalysts are a signifi-

cant advantage in the production of biofuels. Biological catalysts can be incorpo-
rated into bacteria and yeast that convert plant biomass into fuels, encoded into
photosynthetic microorganisms such as algae and cyanobacteria to allow direct con-
version of the primary products of carbon fixation into fuels, or perhaps even engi-
neered into plants. This potential for small-scale conversion removes the need for
centralized conversion plants, dramatically easing the logistics of conversion and
better matching it with the diffuse capture of solar energy.

Capability 5: Material Synthesis

Natural biology (sequences, translated molecules, and organisms evolved in the
wild, as opposed to those designed, assembled, or evolved in the laboratory) syn-
thesizes a truly amazing range of materials for hunting and foraging; defense and
protection; and structural support. For example, Caerostris darwini major ampullate
spider silk requires seven times more energy per unit volume to break than Kevlar
and 14 times more than carbon fiber.11 Latex rubber, derived from the Hevea brasi-
liensis tree, offers a unique combination of high compliance, resilience, elasticity,
abrasion resistance, malleability, impact resistance, and heat dispersion that is un-
matched by synthetic rubbers.25 Mineralizing microorganisms can construct nano-
structured and hierarchical porous structures much like those found in the electrodes
of batteries but are able to do so under benign ambient conditions26 and have even
been used in the development of concrete that self-repairs.27

Capability 6: Control of Metal Redox State

Biology also offers unparalleled control over the redox state of metals. Electroactive
microbes use specialized extracellular electron transfer (EET) protein complexes to
move electrons to, from, or between their metabolism and a remarkable variety of
external substrates, ranging from metal ions to solid surfaces, including electrodes
(Figure 3).

Electroactive microbes can be broadly divided into two classes: metal reducers
and metal oxidizers. EET gives metal-reducing microbes such as Shewanella onei-
densis MR-1 the ability to breathe onto far more substances than just O2, including
Fe, Mn, U, Tc, Cr, Np, Pu, iodate, selenite, tellurite, and vanadate.28 This gives

24 Chem 2, 20–51, January 12, 2017

 A B

Figure 3. Extracellular Electron Transfer Machinery

(A) A generalized model of the S. oneidensis electron outflow machinery: A, MtrA; B, MtrB; C, MtrC; D, OmcA; I, the complex I NADH quinone
oxidoreductase; and Q, CymA.
(B) A generalized model of the proposed electron uptake machinery used by the neutrophilic iron-oxidizing microbe Sideroxydans lithotrophicus:
A, MtoA; B, C, D, and Q are thought to be proteins homologous to MtrB, MtrC, OmCA, and CymA, respectively; and ? denotes parts of the electron
uptake machinery responsible for reduction of NAD(P) + that are still unknown. 36

metal-reducing microbes potential applications in bio-remediation, including the

cleanup of radionuclide contaminated sites, where metal reduction can dramatically
reduce the mobility of some contaminating metals in the environment.29,30 In addi-
tion, the ability of these microbes to reduce solid surfaces such as electrodes makes
them a key component in microbial fuel cells that can extract electrical energy from
waste and sediments.31

In the archetypal electroactive microbe, Shewanella oneidensis MR-1, EET is

enabled by an extensive network of c-type cytochrome electron transfer proteins,
including the mtr EET operon, which encodes a set of specialized outer membrane
cytochromes that connects the electron transport chain (protein I in Figure 3A) in the
inner membrane of the cell to the external environment9 (proteins A–D and Q in Fig-
ure 3A). This mechanism is responsible for carrying most of the electron flux from
metabolism in the cytoplasm of the cell to external electron acceptors beyond the
outer membrane28 (Figure 3A). Many other metal-reducing electroactive microbes
share similar systems.

A growing body of evidence suggests that a variant of the EET system that is tuned
for electron uptake into the inner membrane of the cell (proteins A–D and Q in Fig-
ure 3B) and metabolism (the protein marked ? in Figure 3B) is used by carbon-fixing
neutrophilic metal-oxidizing microbes.32 We have drawn a proposed schematic of

Chem 2, 20–51, January 12, 2017 25

the electron uptake machinery in a neutrophilic iron-oxidizing microbe such as Side-
roxydans lithotrophicus ES-1 in Figure 3B.

Many electroactive microbes (Figures 3B, 9C, and 9D) are able to transfer electrons
over long distances between the cell surface and external substrates at high rates by
using conductive pili (sometimes called bacterial nanowires).33 Some electroactive
microbes can use this type of solid-matrix conduction to transfer electrons over
centimeter-length scales.34

Ancestors of the metal-oxidizing microbes are thought to have powered the Earth’s
biosphere before the advent of oxygenic photosynthesis.35 Early in the Earth’s his-
tory, there was significant flux of reduced iron into the oceans from hydrothermal
activity.35 Iron-oxidizing electroactive microbes were able to take advantage of
this reduced Fe as an electron and energy source and use it to power autotrophic
carbon-fixing metabolism.35 Metal-reducing electroactive organisms, much like
today’s Shewanella oneidensis MR-1, were able to use the now oxidized Fe as a
terminal electron acceptor for anaerobic respiration, allowing it to be recycled by
subduction, driving an enormous global Fe cycle and depositing the Banded Iron
Formations, some of the earliest known rocks.35,36 Canfied et al.35 estimate that
the primary production of an Fe-cycling-based ecosystem could have been as
high as 6 Gt year
1 of organic carbon, only approximately eight times lower than
the primary production of today’s photosynthetically driven ecosystem. This sug-
gests that the total power flux through such an Fe-cycling system could have been
as high as 10 TW, enough to support a thriving ecosystem.

The ability of contemporary electroactive metal-oxidizing organisms to grow

autotrophically by carbon fixation32 suggests that these organisms possess a mech-
anism to transfer electrons that have reached the inner membrane (at z
100 mV
versus standard hydrogen electrode [SHE], low energy) to reduce NAD(P)H
(at z
320 mV versus SHE, higher energy).36 However, the exact mechanism of
this ‘‘uphill pathway’’ (the protein marked ? in Figure 3B) that acts analogously to a
voltage transformer remains unknown.36

Iron-oxidizing microbes are already used on industrial scales today: Acidithiobacillus

ferrooxidans is a key component of the copper bio-leaching operation at the Escon-
dida Mine in the Atacama Desert in Chile. A. ferrooxidans oxidizes Fe(II) to produce
Fe(III), which then oxidizes Cu(I) to Cu(II), reforming Fe(II) and leaching Cu(II)37 (Fig-
ure 4A). This bio-leaching heap, visible from orbit, is 5 km long and 2 km wide and
has a volume of approximately one trillion liters, making it arguably the world’s
largest bioreactor.37

APPLICATION A: ENABLING THE SAFER USE OF NUCLEAR POWER

Problem in Sustainable Energy
Nuclear fission is an extremely large potential source of carbon-free base load power
for the sustainable energy infrastructure.4 However, the widespread deployment of
nuclear power is limited by concerns over the proliferation of enriched radioactive
material; long-term security of the nuclear fuel supply; and environmental contami-
nation risks. These contamination risks come from accidents during power plant
operation and nuclear material transport; reactor decommissioning; and uncer-
tainties over the stability of long-term storage repositories for spent nuclear fuel
and nuclear waste. The consequences of these risks are magnified because the
redox state of many of the components of nuclear fuel, spent nuclear fuel, and

26 Chem 2, 20–51, January 12, 2017

 A B

C D

Figure 4. Microbial Metal Mobilization and Demobilization Mechanisms

(A) Metal-oxidizing organisms such as A. ferrooxidans are able to solubilize metals including copper and uranium either by indirect oxidation via iron or
by direct oxidation. Although bio-oxidation is most commonly associated with the mobilization of metals in bio-remediation, it can also be used to de-
mobilize metals such as Fe(II) under neutral conditions.
(B) Phosphate-solubilizing fungi such as Aspergillus niger ATCC 1015 are thought to be able to precipitate metals from ore by secreting organic acids
that can dissolve minerals such as those found in REE-containing ores.
(C) Bio-reduction by organisms such as S. oneidensis and G. sulfurreducens can reduce soluble U(VI) to mononuclear U(IV), which can immediately
precipitate or form insoluble uraninite crystals.
(D) Phosphates generated by microbes including Citrobacter sp. can precipitate U(VI) as uranyl phosphate, and Rahnella sp. can mineralize uranium as
autunite.
(E) Carboxyl and phosphate groups on the surface of organisms, including Roseobacter sp. AzwK-3b and S. oneidensis, can bind middle REEs and heavy
REEs, respectively, facilitating the separation of REEs mixed in solution.

high level waste renders them water soluble and hence highly mobile in the environ-
ment.29 The long-running controversy over the security and safety of groundwater at
the nuclear waste storage repository at Yucca Mountain was emblematic of this
problem.

Chem 2, 20–51, January 12, 2017 27

Opportunity for Biological Solution
Although there are a number of physicochemical remediation methods for the
cleanup of uranium mining sites, decommissioned nuclear sites, waste-processing
facilities, and short- and long-term waste storage facilities and accident sites,
microbes offer the potential for low cost, low environmental impact, and long-
term remediation.30 Microbes offer several mechanisms29 for the de-mobilization
of metals, three of which are discussed here: bio-reduction; bio-mineralization;
and bio-sorption (Figures 4C–4E). In addition, we discuss two mechanisms that
are typically used for metal mobilization: bio-oxidation37 and bio-demineraliza-
tion38 (Figures 4A and 4B). These mechanisms are used not just in bio-remediation
but might also find use in the recovery of energy-critical elements (application 2);
the synthesis of battery electrodes (application 3); and hybrid photosynthesis
(application 7).

In addition to environmentally common metals such as Fe and Mn, electroactive

microbes such as Shewanella oneidensis MR-1, Geobacter metallireducens, and
Geobacter sulfurreducens are able to bio-reduce (Figure 4C) and hence precipitate
radionuclides, such as U(VI),39 found as contaminants at nuclear-associated sites
through their EET machinery (Figure 3A). Of the three bio-remediation mechanisms
discussed here, this has received the most attention and is considered the most
promising.29

Metals can also be precipitated through bio-mineralization by microbially generated li-

gands such as sulfide or phosphate, or as carbonates or hydroxides in response to local-
ized alkaline conditions at the cell surface (Figure 4D). Bio-mineralization of uranium as
uranyl phosphate has been demonstrated with Citrobacter sp., and species including
Rahnella sp., Bacillus sp., and Aeromonas sp. can precipitate uranium as autunite.30
Sulfide precipitation by sulfate-reducing bacteria such as Desulfovibrio sp. and
Desulfotomaculum sp. has recently garnered interest.30

Additionally, metals can be removed from solution either through sorption onto the
surface of a cell or through electrostatic attraction to negatively charged groups or
binding to carboxyl, amine, hydroxyl, phosphate, and sulfhydryl groups40,41 (Fig-
ure 4E). However, at the time of writing bio-sorption is not widely considered to
be suitable for bio-remediation of nuclear-associated sites.29,30

In situ bio-remediation using bio-reduction has been successfully demonstrated in

pilot-scale field trials at several US Department of Energy (DOE) sites, and bio-
mineralization has been investigated for potential use at the DOE Oak Ridge site.29

Microbes can also be used to mobilize U, offering an environmentally friendly route

for its extraction from ore (Figure 4B). Pal et al.42 recently demonstrated the use
of A. ferrooxidans for the direct bio-oxidation and bio-leaching of uranium, and
Rashidi et al.43 demonstrated the use of A. ferrooxidans with Fe cycling for uranium
bio-leaching.

Challenges for Biological Solution

Genetic engineering of microorganisms used in bio-remediation of nuclear waste
could further expand the role that biology can play in a wider adoption of nuclear
energy. Although each type of cleanup and each individual cleanup site poses its
own unique set of challenges, we see at least four challenges for enhancement
of bio-remediation with advanced genetic engineering: stabilizing remediated
contaminants; reducing the solubilization of Pu(IV); enhancing the tolerance of

28 Chem 2, 20–51, January 12, 2017

bio-remediation organisms to co-contaminating metals and radiation; and manag-
ing the competition for reduction between radionuclides and nitrate.

Long-Term Stability of Remediated Contaminants

Bio-reduction by electroactive microbes has been shown to effectively reduce U(VI),
the predominant and highly water soluble oxidation state of U in nuclear fuel, to insol-
uble mononuclear U(IV) and uraninite crystals.29,30 A number of studies have shown at
least medium-term stability of uraninite but indicate the potential for re-oxidation
and re-solubilization of mononuclear U(IV).29 At the present time, it is unclear whether
the final form of the biologically precipitated uranium (uraninite or mononuclear
U(IV)) can be controlled genetically. If this is indeed the case, this could prove to
be an important avenue for the improvement of bio-remediation technology.

The long-term stability of precipitated radionuclides is also helped by a long-term

supply of electron donor to allow bio-reducing microbes to continue operating
long after an initial cleanup. Acetate is often the preferred electron donor for bio-
reduction,30 but this requires periodic replenishment. Broadening the base of
electron and carbon donors in genetically engineered or newly found bio-reduction
organisms to utilize organics commonly found in soil could significantly improve
long-term remediation.

Solubilization of Pu(IV)
Although U(VI) is highly mobile in the environment, Pu(IV), the predominant contam-
inating form of Pu from nuclear fuel and waste, is mercifully immobile. However,
Pu(IV) can be reduced by naturally occurring microorganisms to Pu(III), which is
highly mobile and hence significantly more dangerous.29 The development of
engineered microbes incapable of this reduction, or that are able to oxidize Pu(III)
(Figure 4E) and that displace such naturally occurring microbes would go a long
way to ameliorating this risk.

Microbial Tolerance to Uranium, Co-contaminating Metals, and Radiation

Counterintuitively, the most common hazard at most nuclear-associated sites is not
radiation30 but the chemical toxicity of radionuclides and other co-contaminating
transition metals such as copper and cadmium, and lead.30 Dissolved uranium typi-
cally has been shown to inhibit microbial growth at concentrations above 100 mM.30
However, uranium concentrations of 250 mM or more are often found at contami-
nated sites.30

Although the radiation-tolerant microbe Deinococcus radiodurans is able to tolerate

orders of magnitude higher radiation doses than those anticipated at many nuclear
sites30 and uranium concentrations as high as 2.7 mM,30 exposure to only 1.8 mM of
the co-contaminant cadmium led to 70% inhibition of its growth.30 In addition, as
D. radiodurans is gram positive, and hence lacks an outer membrane, it will be an
extremely challenging feat to add the S. oneidensis EET machinery to it, which relies
heavily on the outer membrane.28

Thus, it is thought that metal-tolerant rather than radiation-tolerant strains are far
more useful for most remediation scenarios.30 Many bacteria present in uranium-
contaminated environments offer both adequate metal and radiation tolerance.30
However, many of these microbes are currently non-cultivable,30 making the discov-
ery of their genetic basis for these traits challenging. Understanding the enhanced
radiation- and metal-tolerance of environmental isolates from nuclear-contaminated
sites and combining this with the EET capabilities of organisms such as S. oneidensis

Chem 2, 20–51, January 12, 2017 29

and Geobacter species presents a considerable challenge and opportunity for sys-
tems and synthetic biology to improve bio-remediation.

Nitrate Competition
Nitrates are often found as a co-contaminant at many nuclear sites. The lower redox
potential of nitrates relative to U(VI) means that bio-reduction organisms preferen-
tially reduce nitrates at the expense of uranium precipitation.29,30 However, although
nitrate is a poor oxidant of U(IV), in combination with iron it can re-oxidize and hence
re-solubilize bio-reduced uranium.29 Development of organisms able to switch off
nitrate reduction at high U(VI) concentrations, but restore it after U(VI) reduction is
complete presents a significant opportunity for improvement of bio-remediation.

APPLICATION B: MINING AND SEPARATION OF RARE EARTH

ELEMENTS
Problem in Sustainable Energy
The rare earth elements (REEs; the lanthanides and scandium and yttrium) are widely
recognized to be vital ingredients for current and future sustainable energy technol-
ogies.44 Yttrium barium copper oxide is used in high-temperature superconductors
and energy-efficient light bulbs. Dysprosium, neodymium, praseodymium, and
samarium are essential constituents of the high-field magnets used by automotive
electric motors and wind-turbine generators. Scandium is used in high-strength light-
weight alloys. Europium and terbium are used as phosphors in solid-state lighting.
Finally, lanthanum, cerium, praseodymium, and neodymium are used in the anodes
of nickel metal hydride batteries. Rare earths, despite their name, are abundant in the
Earth’s crust, but their extraction, separation, and recycling are uniquely challenging
because of the lack of concentrated reserves and their chemical similarity.

Mining REEs
Unlike many metals, REEs are widely dispersed throughout the Earth’s crust.
Approximately 95% of known reserves of REEs are found in the minerals bastnäsite
(REE-FCO3), monazite (light REE-PO4), and xenotime (heavy REE-PO4).45 However,
typically REEs are not concentrated in veins and hence need to be extracted from
extremely large volumes of ore. For example, in addition to REE-PO4, monazite usu-
ally contains the thorium minerals cheralite (ThCa(PO4)2) and huttonite (ThSiO4), and
uranium.45

Established techniques for REE extraction rely on high temperatures and harsh
chemical treatments such as high-temperature (R140 C) chemical leaching with
(1) either concentrated sulfuric acid or NaOH followed by acid treatment or (2) heat-
ing with CaCl2 and CaCO3 at temperatures exceeding R975 C followed by leaching
with 3% HCl.46 These conventional techniques are both energy intensive and pro-
duce large volumes of toxic uranium and thorium waste.46

Separation of REEs
In addition to the difficulties of extraction, the chemical similarity of the lanthanide
REEs, their trivalency, and similar ionic radii make both physical and chemical sepa-
ration from one another difficult.47 This poses a challenge not only for refining but
also for recycling these scarce and critical metals from metal-bearing solid and liquid
wastes.48

Opportunities and Challenges for Biological Solution

Two recent breakthroughs have highlighted the potential for the use of applied
biology in rare earth mining and separation.

30 Chem 2, 20–51, January 12, 2017

Mining REEs
Brisson et al.38 recently demonstrated biological REE extraction from monazite ore.
In contrast to uranium and copper bio-extraction, which relies on bio-oxidation, this
work took advantage of bio-demineralization by using the phosphate-solubilizing
fungi Aspergillus niger ATCC 1015, Aspergillus terreus ML3-1, and Paecilomyces
spp. strain WE3-F (Figure 4E). This single-pass process achieved 3%–5% recovery
of REE from monazite ore with low leaching of thorium waste. Although this is not
yet competitive with the CaCl2/CaCO3 process (89% REE recovery and low thorium
leaching) and NaOH process (98% REE recovery but notable thorium leaching),38
bio-leaching has the potential to significantly reduce the energy footprint of REE
extraction because it can proceed at room temperature.

Multiple mechanisms for microbial phosphate solubilization have been proposed. The
predominant mechanism is thought to be the production of organic acids,49 which
reduce the pH, causing an increase in the solubility of phosphate minerals. Significant
advances in the understanding of bio-leaching mechanisms to isolate and identify
bio-leaching compounds could allow the optimization of this process to achieve an
economically viable alternative to conventional REE extraction processes.38

Separation of REEs
The bacterial cell surface is an ideal surface for the separation of REEs from solution.
Middle REEs can preferentially adsorb to the carboxyl groups present on the surface
of cells.40 Similarly, the heaviest REEs, which have a pKa of z2.0 can preferentially
bind to phosphate groups, also with a pKa of z2.0, present on the bacterial cell
surface.41

Bonificio and Clarke47 demonstrated REE separation by using bio-sorption (Fig-

ure 4E) of mixed REEs by microbes immobilized on an assay filter followed by
selective desorption with decreasing pH. Bonificio and Clarke were also able to
demonstrate variation of the REE separation factor by using four different
microbes: Roseobacter sp. AzwK-3b, S. oneidensis, Sphingobacterium sp., and
Halomonas sp.47 Given the rich variety of bacterial surface chemistries, they specu-
late that other bacteria might exhibit significantly greater differentiation in binding
different lanthanides.

Although this process is currently only at laboratory scale and has not been opti-
mized, it is possible to concentrate a solution of equal concentrations of each lantha-
nide to nearly 50% of the three heaviest lanthanides (Tm, Lu, and Yb) in just two
passes, surpassing existing industrial practice, while being considerably more envi-
ronmentally benign.47 With a greater understanding of the surface chemistry and
specific functional groups involved in lanthanide binding, and the genetics and sys-
tems biology of bio-sorption, it might be possible to engineer a microbe capable of
highly selective REE separation.

APPLICATION C: ENABLING SUSTAINABLE MANUFACTURING OF

BATTERIES
Problem in Sustainable Energy
Energy storage on a large scale is almost sure to be a key feature of the sustainable
energy infrastructure.3 By time shifting the availability of renewables and reducing
congestion at point of load, energy storage systems from a few kWhr to multiple
MWhr could have significant impacts not only on CO2 production but also on the re-
silience and distribution integrity of the grid.50 Batteries are an attractive solution to
this problem because they combine energy storage capabilities with high power

Chem 2, 20–51, January 12, 2017 31

density and cyclability, but at least three challenges stand in the way of their wide-
spread adoption: lifespan, carbon footprint, and constraints on the global supply
of key ingredients.

Lifespan
A significant challenge in the scale-up of battery energy storage is the cost per unit of
energy stored compounded by a limited cycle and calendar life.51 This problem is
most acutely felt in transportation: the short lifespan and hence rapid depreciation
in value of batteries is a significant contributing factor to the high total cost per
mile of electrical vehicles.52 Without a significant breakthrough in lifetime and
cost, electrochemical energy storage is likely to be limited to niche markets and
applications.

Carbon Footprint
Adding to the problem of limited lifespan, the manufacturing processes available for
the production of battery components typically require high temperatures and large
expenditures of energy. The use of nanostructured electrode materials is thought to
be one of the most attractive routes to make dramatic improvements in the capacity,
rate capability, cycling life, and safety of batteries.53 However, the creation of
electrodes with such tailored morphologies has often required the use of high-tem-
perature synthesis.54 It is estimated that the high-temperature (700 C) ceramic pro-
duction process for a single 1 kWhr lithium-ion battery requires more than 400 kWhr
of energy and results in the emission of 75 kg of CO2.54

Lithium Supply Constraints

The increasing global demand for lithium, and its uneven distribution across the
world, has raised fears of a lithium supply constriction.54 This has inspired re-
searchers to look for ways to recycle lithium and for new battery chemistries that
use either alternative monovalent (Na+ and K+) or divalent (Mg2+ and Ca2+) cat-
ions.54 Of these approaches, Na offers the most widespread availability, although
its higher molecular weight means that a reduction in specific energy density should
be expected.

Opportunities for Biological Solution

Manufacture
The recently developed ionothermal synthesis process has enabled researchers to
reduce the energy requirements of manufacturing battery electrodes with tailored
morphologies by dropping the synthesis temperature of phosphate and silicates
to approximately 200 C, a considerable improvement over the high-temperature
ceramic process.26 Recent pioneering work on the biological synthesis of electrode
materials at room temperature offers the potential to further reduce the energy cost
and carbon footprint of batteries while also greatly increasing the scalability of bat-
tery production.

Viruses offer the potential for templating of nanostructured electrodes at ambient

temperatures. Pomerantseva et al.55 have demonstrated the use of the tobacco
mosaic virus as a template for nanostructured V2O5 cathodes. Furthermore, Lee
et al.56 have demonstrated the use of engineered viruses as templates for the con-
struction of high-rate FePO4/single-walled carbon nanotube electrodes at room
temperature. Although this approach does not yet allow the formation of lithium-
based electrodes, essential for a practical lithium-ion cell, and cannot be used on
a large scale, it effectively demonstrates the use of genetic programming in elec-
trode material templating.

32 Chem 2, 20–51, January 12, 2017

Metal-oxidizing microbes have been used to generate components for lithium bat-
teries. Miot et al.57 used the anaerobic Fe(II)-oxidizing bacterium Acidovorax sp.
strain BoFeN1 to produce a-FeOOH at room temperature; abiotically converted
this to a-Fe2O3; and then demonstrated its use as an electrode material in lithium
batteries. These proof-of-principle experiments suggest that biological principles
and even whole microorganisms could become integral parts of the design and
assembly of battery components in the years to come.

Resource Availability
Recent work has demonstrated the abiotic construction of Na-containing organic
electrodes.58 As biological systems rely heavily on Na-, K-, and Mg-containing
organic complexes, these results suggest the possibility of biologically synthesizing
electrodes for non-lithium batteries. Batteries with organic, biomass-sourced elec-
trodes could potentially be recycled by biodegradation to liberate the CO2 used
in electrode construction for further biomass production.54 In addition, as
mentioned above, biology can also offer environmentally friendly solutions such
as bio-leaching for the extraction of REEs and other metals such as Ni and Co, which
could reduce the carbon footprint of battery electrode manufacturing.54

Recycling
Materials extraction is a key driver of the energy cost of battery manufacturing, mak-
ing recycling increasingly important.54 The chemical complexity of batteries makes
their recycling a multi-step process. For instance, hydrometallurgy involves acid-
base leaching, solvent extraction, chemical precipitation, and electrochemical
processes, each of which incurs both carbon and energy costs, along with local envi-
ronmental effects. Here, too, as in the mining of REEs and uranium, biological pro-
cesses such as bio-leaching (Figures 4A and 4C) can provide an environmentally
benign low-energy alternative to conventional recycling methods.54 A key challenge
will be determining the genetic make-up of microbes best suited to battery
recycling.

APPLICATION D: ENABLING THE SUSTAINABLE MANUFACTURING OF

LIGHTWEIGHT MATERIALS
Problem in Sustainable Energy
Lightweight composite materials that contain fibers and a polymer matrix have the
potential to revolutionize the energy footprint of transportation, radically reducing
the weight and aerodynamic drag of cars, aircraft, and drones, and even improving
safety.5 However, the costs of manufacture and imbued energy59 in these compos-
ites are serious drawbacks that threaten to limit the ultimate scale of their adoption.

Opportunity for Biological Solution

In contrast to synthetic carbon fibers, naturally sourced fibers such as kenaf, flax,
jute, hemp, and sisal have very low imbued energy, along with low density, accept-
able specific strength properties, and ease of separation.60 Biologically sourced
polymers such as cellulosic plastics, polylactides (PLA), starch plastics, and soy-
based plastics are now moving into mainstream use,61 and microbial-synthesized
polyhydroxyalkanoates (PHAs) and polyhydroxybutyrates (PHBs) have attracted
attention.61

The use of bio-plastics in automobiles can be traced as far back as the 1940s,60 and
there has been renewed interest in recent years in the use of natural fibers to lighten
the weight of automobile components and make them biodegradable.61 Biologi-
cally based composites with crop-derived fibers and plastics have been used in

Chem 2, 20–51, January 12, 2017 33

the construction of several internal components of cars for more than a decade.61
However, there are several challenges for the use of composites of natural fibers
in exterior structural body panels, including inconsistent physical properties that
vary with growing conditions; poor compatibility with polymeric matrices; moisture
sensitivity; and low microbial resistance.61

Genetic engineering has the potential to address some limitations of natural fibers in
composite applications. Spider silks have remarkable material properties, often
exceeding those of synthetic fibers, and matching those of even the most advanced
materials such as carbon nanotube fibers.11 Transgenic organisms have been engi-
neered to produce recombinant silk proteins,11 opening the possibility of micro-
bially producing engineered silks in bulk that could be tailored for structural
applications.

Furthermore, microbial biofilms such as the Escherichia coli curli film are composed
of amyloid nanofibers that self-assemble from the small protein subunits, which also
have remarkable materials properties.62 Nguyen et al.62 recently demonstrated an
engineered variant of the curli biofilm that is able to perform functions as diverse
as nanoparticle bio-templating, substrate adhesion, and the covalent immobiliza-
tion of catalytic proteins. The amyloid fibers that compose this engineered
film are extremely robust, being able to withstand boiling in detergents and
extended incubation in solvents.62 These studies suggest that similar biofilms are
potentially suitable for mechanically demanding applications under harsh chemical
environments.

APPLICATION E: CONVERTING BIOMASS TO BIOFUELS

Problem in Sustainable Energy
Despite many of the impressive features of electrochemical energy storage systems
such as batteries, the unique advantages of liquid fuels as energy storage molecules
are difficult to overstate. It seems most likely that liquid fuels will play an essential
role in the energy infrastructure for a long time to come because of their unrivaled
high energy density, particularly in transportation.

Compared with battery-powered vehicles, liquid-fuel-powered vehicles can have

extremely long ranges. The maximum range of flight is determined by the ratio of
the energy stored by the aircraft at takeoff to its aerodynamic drag.63 The energy
storage capacity is a function of the energy density of the fuel (C); the fraction of
mass of the aircraft dedicated to fuel at takeoff (f), and the energy recovery efficiency
of the engine (ε). The aerodynamic drag is determined by the drag coefficient cd; the
frontal area, fA, and the acceleration due to gravity, g. MacKay estimated the range
of flight of an aircraft as

εfC
dmax = 1=2
(Equation 1)
gðcd fa Þ
and validated this formula by accurately estimating the maximum ranges of aircraft
and birds.63

The lift-to-drag ratio (cdfA)
1/2 of a 747 aircraft is approximately 17, and fuel accounts
for z50% of its mass at takeoff (f). The energy density (C) of jet fuel is 46 3 106 J kg
1
and the energy recovery efficiency of a turbo-fan engine is z1/3. With this informa-
tion, Equation 1 estimates the maximum flight range of a 747 as z13,300 km
(compared with a record maximum flight range of 16,000 km with a tail-wind assist
from the jet stream63).

34 Chem 2, 20–51, January 12, 2017

By contrast, whereas the energy recovery efficiency of an electric motor is close to
80%, the energy density of a lithium-ion battery is almost two orders of magnitude
smaller than that of a hydrocarbon (0.36–0.875 3 106 J kg
1). Assuming that 50% of
the mass of the aircraft is dedicated to batteries, the flight range of lithium-ion-
battery-powered aircraft is limited to only z100–250 km.

Hydrocarbon fuels are well embedded into several industries, particularly aviation.
Hydrocarbon fuels have considerable safety advantages and best practice in their
use is well established. Moreover, they are supported by regulation and a multi-tril-
lion dollar worldwide distribution infrastructure.

Although we highlight the shortcomings of present-day battery technology, this

should not be taken as negativity. We see many good reasons to be optimistic about
developments in battery science and technology, and many more are likely to be
cited by experts in the field. However, we raise this issue to point out that electrifi-
cation of the transportation infrastructure is not a panacea at present, and we should
not assume that the challenges facing biofuels are any less solvable than those facing
batteries and transport electrification.

Currently, biofuels offer one of the few, if not the only, routes to carbon-neutral
transportation fuels and certainly the only route to carbon-negative fuels through
the BECCS (Bio-Energy with Carbon Capture and Storage) process.64 The recent
advances in cellulosic ethanol production strengthens support for projections that
suggest that biofuels could be expanded significantly through the use of forest
waste and high-yield crops grown on marginal land.65

However, although the ultra-low costs of carbon and energy capture by biomass are
highly attractive, challenges in transforming this captured carbon into useful fuels
remain. Most significantly, the market penetration of present-day biofuels (ethanol)
is limited because of low compatibility with current engine technologies, setting a
low ceiling on the amount of fossil gasoline that can be displaced by carbon-neutral
alternatives. Although the DOE has approved ethanol gasoline blends of 10%, 15%,
and even 85%, (E10, E15, and E85, respectively) only flexible-fuel vehicles can use
E85. Furthermore, E15 is not recommended for use with vehicles built before the
2001 model year. As a result, the vast majority of ethanol used in the US is in a
10% blend with gasoline (E10). Even when E15 achieves full penetration in the US
market, the low energy density of ethanol (z2/3 that of gasoline) means that only
10% of the energy currently obtained from fossil fuel can be displaced. Further
complicating matters, the gasoline storage and distribution infrastructure is incom-
patible with blends higher than E15. As this infrastructure represents trillions of dol-
lars of decades-long investments, it is extremely unlikely that blends above E15 will
gain market traction in the foreseeable future. Although the US Renewable Fuel
Standard requires that 36 billion gallons of renewable fuel be blended into gasoline
per year by 2022, the current distribution infrastructure only allows the safe use of
z23 billion gallons of ethanol per year. Thus, without improving the compatibility
of the biofuels we produce, we are headed toward massive overproduction of
ethanol for which we simply lack the infrastructure to utilize as a biofuel.

Opportunities for Biological Solution

In the near term, there are a number of areas where applied biology can contribute
to the production of biofuels from cellulosic biomass. Firstly, current large-scale pro-
cesses to convert cellulose to ethanol rely on thermochemical pretreatment66 and
enzymatic hydrolysis with fungal cellulases, which render these processes very

Chem 2, 20–51, January 12, 2017 35

36 Chem 2, 20–51, January 12, 2017
Figure 5. Reverse b-Oxidation/ABE and Fatty Acid Synthesis Pathways
The reverse b-oxidation/acetone-butanol-ethanol (ABE) and fatty acid synthesis pathways are used to create a variety of fuel products, including
isopropanol, 1-butanol, 4-methyl-1-pentanol, 1-hexanol, n-pentane, 1-pentanol, fatty alcohol, long- and short-chain alkanes, and fatty acid ethyl esters
(FAEEs). Pathway data derived from Cheon et al. 67 with additional pathways from Sheppard et al. 68 and Tseng et al. 69 Fuel products in green and
magenta boxes are gasoline or biodiesel replacements, respectively. Orange indicates an intermediate derived from ketoacid pathways.

expensive. We anticipate that biological solutions, such as more active bacterial cel-
lulases such as those from Clostridium thermocellum, will enhance cellulose conver-
sion.70 In addition, it will be necessary to find biological and chemical approaches to
convert lignin (which is currently used as boiler fuel) to more valuable compounds to
increase the economic solvency of biorefineries.71 In addition, genetic engineering
and directed evolution of biofuel-producing microorganisms to increase their toler-
ance to products as well as toxic impurities in cellulosic hydrolysates will have the po-
tential to increase biofuel titers and productivities.

Recent advances in synthetic biology and metabolic engineering have allowed engi-
neered microbes to convert sugars to a variety of advanced biofuels that are much
more compatible with the current transportation infrastructure. Engineered meta-
bolic pathways have been demonstrated to produce butanol,72 oleaginous fuels
such as biodiesel,73 branched-chain alcohols,74 medium-chain fatty acids,75 and
alkanes.76 A selection of these pathways for the production of gasoline-like mole-
cules, biodiesels, and aviation fuels are shown in Figures 5, 6, and 7. It is also
possible to engineer enzymes and metabolic pathways to develop microbial strains
that produce unnatural products with potentially better fuel properties than some of
the products that natural biology provides68,69,77 (Figures 5, 6, and 7).

The enzymatic catalysts that constitute these engineered pathways can be incorpo-
rated into either bacteria or yeast that convert plant biomass into fuels. In the longer
term, we see considerable potential for the development of crop strains that, while
retaining much of the resilience to environmental insults that naturally occurring
plants possess, will be designed to be considerably easier to break down inside a
reactor, perhaps with specific synthetic enzymes produced by the plants themselves
that do not occur naturally and are activated at the command of the biofuel
manufacturer.

APPLICATION F: ENHANCING THE EFFICIENCY OF PHOTOSYNTHESIS

Problems in Sustainable Energy
The growing use of biofuels has provoked concern over the displacement of
wilderness ecosystems, displacement of agriculture, and disruption of agricultural
communities.7 Although there is no solid evidence that biofuels have yet to mean-
ingfully influence long-term global food prices, even during the commodities
boom of 2006–2008,78 the possibility that greatly expanded biofuel production
could affect global food supply exists. On reviewing the potential contributions
for biomass to world primary energy, Slade et al.79 noted that changes to diet; agri-
cultural patterns; increased deforestation; and increases in crop productivity would
most likely be required for sourcing more than 100 EJ year
1 from biomass given the
current photosynthetic efficiencies (18% of the current world primary energy use of
550 EJ year
1).13

Although many sectors of the global economy could be electrified, transportation

stands out as being heavily dependent on carbon-based energy carriers. Current
world transportation energy requirements stand at z 110 EJ year
1 in 2012 and
are predicted to rise to z163 EJ year
1 by 2040.13 If current and projected trends

Chem 2, 20–51, January 12, 2017 37

Figure 6. Isoprenoid Synthesis Pathways
Pathway data derived from Cheon et al. 67 with additional pathways from Peralta-Yahya et al. 6 and Yang et al. 80 Products in green boxes are gasoline
substitutes, and those in blue boxes are suitable jet-fuel replacements. Products in red boxes are other valuable products derived from these pathways.

38 Chem 2, 20–51, January 12, 2017

Figure 7. Ketoacid Pathway for Production of Hydrocarbon Fuels
Pathway data derived from Cheon et al. 67 with additional enzymatic steps from Zhang et al. 77 Fuel molecules highlighted in green are gasoline
substitutes. Orange indicates a ketoacid-pathway-derived intermediate that is used in other pathways. Asterisks represent mutated enzymes.

Chem 2, 20–51, January 12, 2017 39

in transportation energy use13 were to continue in linear fashion until 2100, the
global transportation energy demand would stand at z 270 EJ year
1. Although
it is difficult to make such long-range predictions, and there are good reasons to
think that there may be a natural ceiling to global transportation needs that will
be reached earlier, innovations in transportation technology and rising incomes
around the world could also usher in a new era of global-scale mobility that requires
much more energy than is used today. Given that the potential exists for future in-
creases in biofuel demand that will place serious stresses on the biosphere, agricul-
ture, and human civilization, we believe that efforts to improve the efficiency of
photosynthesis should be taken seriously.

Opportunities for Biological Solution

What Causes the Inefficiency of Photosynthesis?
Zhu et al.81 conducted a survey of the energy loss mechanisms in photosynthesis and
calculated upper bounds for the full-spectrum conversion efficiency of C3 and C4
photosynthesis of 4.6% and 6%. These upper bounds are noticeably higher than
even the best overall photosynthetic efficiencies seen in the field: z2.4% for C3
plants, including sugar beets, barley potatoes, and apples; z 3.4% for C4 plants
such as Miscanthus giganteus; and z3% for algae grown in bubbled photo-bioreac-
tors.82 However, when averaged across the globe, photosynthesis is only estimated
to capture between 0.25%8 and 1%21 of the incident solar flux.

The overall efficiency of photosynthesis appears to be kinetically limited by

RuBisCO, the central carboxylating enzyme in C3 and C4 photosynthesis.83 Although
the rate of photosynthesis increases linearly with illumination at low light levels, this
rate plateaus at between 10% of the maximum solar flux for shade plants and 20% for
sun plants such as sugar cane.8 We built a simple model of microbial photosynthesis
that compares how much carbon fixation can be performed by the carboxylating
enzymes packed into the volume of the cell to the amount of light that can be inter-
cepted by the cell’s cross-section.82 The model indicates that the supply of photons
can quickly exceed the ability of the cell to productively use them for carbon fixa-
tion.82 For convenience, we have restated the model below.

The photon interception cross-section of a cell can be estimated by calculating its

radius of gyration. Assuming a cylindrical cell like E. coli with a diameter d of z1.3 mm
and a length l of z3.9 mm,84 the radius of gyration is
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
Rg = ðd=4Þ2 + ðl=12Þ2 = 0:43 mm; (Equation 2)

and the effective light interception cross-section is

Ai = pRg2 = 0:57 mm2 : (Equation 3)

The number of photons that can be intercepted by this cross-section per second can
be calculated by considering the solar power per square meter (P1), the fraction of
the energy in the solar spectrum that can be used for photosynthesis (z49%82), and
the average energy per mole of photosynthetically active photons (Eg z 205 kJ81).
Thus, the delivery rate of photosynthetically active photons to the cell is

n_g = 0:487P1 NA Ai Eg : (Equation 4)

The photon demand of the cell can be calculated by estimating the amount of
RuBisCO hosted by the cell and the catalytic rate of this enzyme. Assuming that
the cell’s dry weight (md) is z433 fg (BioNumbers: 10389285) and 52.4% of this

40 Chem 2, 20–51, January 12, 2017

Figure 8. Simple Model for the Saturation of Photosynthesis
The plot shows the number of photosynthetically active photons incident on a cell with a light
interception cross-section of z0.6 mm 2 as a function of incident solar power. 82 The dashed
horizontal red line shows an upper estimate of photon demand for carbon fixation under ambient
CO2 concentrations. The photon supply begins to exceed demand at z18% of the peak solar
power at ground level (z185 W m
2 ).

dry weight is protein (fp) (BioNumbers: 101955), the total dry weight of protein in the
cell is

mp = md fp = 433 3 0:52 = 227 fg: (Equation 5)

Leaves maximize carbon fixation by expressing extremely large amounts of

RuBisCO; in fact, this enzyme makes up z50% of leaf soluble protein (fR).81 With a
molecular weight of 65 kDa per catalytic unit (mR) (BioNumbers: 105007), the esti-
mated number of RuBisCO catalytic units in the cell is

nR = mp fR NA mR = 227 fg 3 0:5 3 NA =65 kDa = 1 3 106 : (Equation 6)

Assuming a photon demand (ngC) of 8 (for C3 photosynthesis) to 12 (C4) photons per

carbon fixation, the total photon demand rate and a catalytic rate (rR) range from 3
(BioNumbers: 108735) to 1286 CO2 s
1, the photon demand rate of all RuBisCO cat-
alytic units is

n_gR = ngC rR nR ; (Equation 7)

24nR %n_gR %144nR ; (Equation 8)
0:25 3 108 s
1 %n_gR %1:51 3 108 s
1 : (Equation 9)
The photon delivery rate (Equation 4) is plotted for a range of solar intensities, from
0 to 1,030 W m
2 (the peak solar power at ground level), alongside the maximum
photon demand (Equation 7) in Figure 8. This model suggests that the Calvin-Benson
cycle can usefully utilize only between z3% and 18% of the maximum solar flux, close
to the point at which photosynthesis typically saturates.8 Rather than allowing this
light to pass through, plants typically dissipate this excess light as heat by denying
it to competitors by shading and limiting the overall solar energy capture ability of
a plot of land or a volume of liquid in a photo-bioreactor87 (Figure 9A). Gains in photo-
synthetic rate by increases in the amount of RuBisCO per leaf seem largely to have
been exhausted given that this protein can constitute up to z50%81,88 of leaf soluble
protein, and it is doubtful that more could be added without health consequences to
the leaf or the use of undesirable quantities of fertilizer.81

Chem 2, 20–51, January 12, 2017 41

 A B C D

Figure 9. Photosynthetic and Electrosynthetic Organisms

(A) Conventional photosynthetic microbes.
(B) Photosynthetic microbes with truncated antenna complexes.
(C) Solid-matrix EET-capable electroactive microbes that are able to absorb electricity directly into carbon-fixing metabolism.
(D) H2-oxidizing microbes that use a hydrogenase enzyme to oxidize H2 to provide a source of intracellular reducing equivalents for carbon fixation and fuel
synthesis. Whereas photosynthetic microbes can only accept solar energy, electroactive microbes can accept renewable electricity from many sources.

Challenges
Is Higher Carbon Uptake Possible?
Although the overall efficiency of photosynthesis appears to be limited by the low
catalytic rate of RuBisCO, it is worth asking whether any enzyme could do the job
faster. Theoretical calculations of the maximum rate of air capture of CO2 in abiotic
systems suggest that there is considerable room for improvement. Johnston et al.89
calculated that perfect chemical absorbers could achieve carbon uptake rates as
high as 7 3 1015 molecules cm
2 s
1. Using a simpler method, Brenner et al.8 calcu-
lated a similar maximum carbon uptake rate from the atmosphere of 5 3 1015 mol-
ecules cm
2 s
1. The annually averaged net carbon uptake rate (when accounting for
carbon loss due to plant respiration) from the atmosphere is z2 3 1014 molecules
cm
2 s
1 by wetlands;89 z1 3 1014 molecules cm
2 s
1 by forests, cultivated
land, and tropical woodland and savanna.89 By contrast, the maximum instanta-
neous net carbon uptake rate by corn leaves at 300 ppm CO2 and high illumination
is z2 3 1015 molecules cm
2 s
1,89 which is comparable with the maximum
observed instantaneous carbon fixation rate of z2 3 1015 molecules cm
2 s
1.8
This suggests that there is at least a factor of 2.5–3.5 for improvement in maximum
instantaneous carbon uptake rate and a factor of 25–35 in annually averaged rate,
especially if sites with highly engineered plants were thoughtfully spaced across
the globe to avoid the creation of CO2 depletion zones that would limit the rate
of diffusional transport at downwind sites.89

Could Higher Carbon Uptake Be Achieved?

Despite decades of effort to produce a faster RuBisCO, this enzyme may already be
optimized.90 Although there are high-catalytic-rate variants of RuBisCO,86 this

42 Chem 2, 20–51, January 12, 2017

increased speed invariably comes with the loss of ability to discriminate O2 and
CO2.90 This increases the rate of fixation of O2 in place of CO2 and results in energy
loss due to photorespiration.

Fortunately, RuBisCO is not the only carboxylating enzyme in nature: at least six
alternative carbon-fixation cycles do not rely on RuBisCO.83 In addition, Bar-Even
et al.83 proposed two computationally designed carbon-fixation cycles that are
composed of naturally occurring and engineered enzymes in combinations that
are not found in nature. These cycles have potentially higher carbon-fixation rates
and lower energy demands than naturally occurring cycles.83 Furthermore, Siegel
et al.91 recently demonstrated a de novo computationally designed enzyme that
enabled the construction of a new carbon-fixation pathway that is predicted to
use carbon more efficiently than any naturally occurring one-carbon assimilation
pathway. A recent pioneering demonstration of the reconstitution of the Calvin-Ben-
son cycle in E. coli by Antonovsky et al.92 lays the foundation for an apples to apples
comparison of the performance of the naturally occurring and synthetic carbon-fix-
ation cycles and pathways in a common host organism.

Could Light Utilization Be Improved?

A further source of inefficiency in photosynthesis is the limited use of the solar spec-
trum and the competition for photons between the overlapping absorption spectra
of photosystems I and II.21 Recently discovered chlorophylls from the cyanobacteria
Chlorogloepsis fritschii PCC 9212 and Synechococcus sp. PCC 7335 with absorption
spectra that peak in the far red could allow use of more of the solar spectrum.93

Melis87 and colleagues have succeeded in improving photosynthetic conversion and

productivity of algae by up to 3-fold by shortening the chlorophyll antenna size of
the photosystems into algae. This intervention minimizes excess light absorption
and allows more algae and hence more carbon-fixing enzyme to participate in
photosynthesis, increasing the overall rate of photosynthesis of a culture. We
compare an unmodified photosynthetic microbe with full-size antennae with a modi-
fied one with truncated antennae in Figures 9A and 9B.

If organisms that achieve such high levels of photosynthetic efficiency can be engi-
neered, they will be significantly different from any naturally occurring organism. Key
to their public acceptance will be effective measures to prevent their escape into the
unspoiled wildernesses, prevention of pollution, and depletion of the natural gene
pool. Rovner et al.94 have developed organisms with recoded genomes that are
dependent on externally supplied synthetic amino acids, laying the foundation for
effective measures to prevent the escape of genetically modified organisms.

APPLICATION G: STORING RENEWABLE ELECTRICITY AS BIOFUELS

In the near future, we can expect considerable reductions in the cost of solar elec-
tricity thanks to the Moore’s Law pace of photovoltaic cost reduction (called Swan-
son’s Law95). The cost of photovoltaics (on a dollar per MW of peak power basis) has
fallen with an exponent of z
0.1 year
1 since 1976 and more recently with an expo-
nent of z
0.3 year
1. Recent power purchase agreements for solar photovoltaic
power have seen its cost drop to below $30 MWhr
1, or $8.33 GJ
1, for the first
time.96 Meanwhile, electrical conversion efficiencies are already as high as z26%
for single-junction and z46% for multi-junction devices97 and continue to rise. If
these trends continue, it is imaginable that the cost of producing a GJ of solar elec-
tricity could soon be lower than the cost of storing a GJ as biomass. If this cost

Chem 2, 20–51, January 12, 2017 43

reduction is realized, it could be leveraged by carbon-fixing metabolisms driven
by either the oxidation of H2 or electron uptake through solid-matrix EET that
could allow the flexibility of metabolism to be combined with the efficiency of pho-
tovoltaics in a hybrid photosynthetic process called electrosynthesis98 (Figures 9C
and 9D).

The use of direct flow of electrical current or transfer of a soluble mediator such as H2
has the potential to greatly improve the distribution of charge and energy to carbon-
fixing cells and ameliorate the excess-light-consumption problem that limits the
overall efficiency of photosynthesis even more effectively than truncated photo-
system antennae (Figure 9B). Additionally, because most renewable electricity
sources are inherently electrical, this process has the potential to not only increase
the efficiency of storage of solar energy over that of photosynthesis but also
provide a method of storing renewable electricity from all sources as biofuels (Fig-
ures 9C and 9D).

Both the solid-matrix EET (Figure 9C) and H2 oxidation (Figure 9D) methods for
transferring electrical energy to metabolism have their own unique set of advantages
and disadvantages. Figure 9 shows a schematic of an unmodified photosynthetic
microbe alongside a modified photosynthetic microbe with truncated photosystem
antennae, and EET- and H2-oxidation-driven electrosynthetic organisms. This field
offers the opportunity to interface cutting-edge abiotic engineering, including
nanofabrication, with cutting-edge genetic engineering.

Hydrogen-Mediated Electrosynthesis
To date, H2 oxidation has enjoyed the most success in engineered electrosynthesis.
Liu et al.99 used H2 produced by a low-cost anode (the bionic leaf) to power CO2 fix-
ation and the production of the bioplastic PHB and alcohols by the H2-oxidizing bac-
terium Ralstonia eutropha.

Four outstanding features of H2-mediated EET offer the potential for high solar-to-
liquid-fuel conversion efficiencies. First, it is aided at the outset by the already high
efficiencies of commercial electrolyzers, which are capable of storing electrical en-
ergy as H2 with efficiencies approaching 80%21 and with room for further improve-
ment in terms of efficiency and cost. Second, because of its extremely low viscosity,
H2 can be transported over long distances with minimal energy loss.82 This feature is
particularly attractive because it allows the possibility for H2 generation and carbon
fixation to be spatially separated and independently optimized. Third, H2-oxidizing
microbes directly couple the oxidation of H2 to the reduction of NAD(P)+. This min-
imizes potential mismatches within H2-oxidizing organisms. The redox potential of
H2/2H+ + 2e
is
0.42 V relative to the SHE, whereas for NAD+ + H++ 2e
/
NADH, it is
0.32 V, representing only z8% of the energy of a 1.2 eV photon.
Finally, we estimated that the protein requirements of H2 oxidation are between
only 0.04% and 0.7% of the total dry protein mass of the cell82 to fully supply the
needs of RuBisCO.

However, a common concern regarding H2-mediated electron transport is the

electron-delivery-rate limitations imposed by the low solubility of H2 in water
(0.00016 g/100 g H2O for H2 versus 0.169 g/100 g H2O for CO2 at room temperature
and pressure). One of the authors built simple models of hydrogen- and EET-medi-
ated electrosynthesis to demonstrate the effects of the transfer mechanism on
system geometry.82 For a reactor with a headspace with a total pressure of 1 atmo-
sphere containing 80% H2, 10% O2, and 10% CO2 (these H2 and O2 concentrations

44 Chem 2, 20–51, January 12, 2017

are very high but are perhaps industrially acceptable), a reactor surface area of
106 m2 is required for full utilization of the solar energy captured by a 1 m2 solar
panel.82 This is not intrinsically a problem in itself, but creative engineering will
most likely be required to maintain energy conversion efficiency, minimize H2 loss,
maintain an acceptable level of safety, and mitigate the effects of proton consump-
tion due to fuel synthesis on solution pH.82 An ingenious solution to this problem is
the hollow-fiber gas reactor,100 in which R. eutropha biofilms are immobilized on the
surface of hollow fibers. A high H2 atmosphere is maintained on the exterior of the
fiber, and a high O2 atmosphere is maintained on the inside. This permits packaging
of high surface areas within a reasonable volume while affording the culture access to
both gases, but it minimizes direct mixing and thus circumvents some of the safety
concerns of operating a high-O2 and high-H2 atmosphere.

Extracellular Electron-Transfer-Mediated Electrosynthesis

Solid-matrix EET is speculated to have the most promise for electrosynthesis given
the high rates of electron transfer observed in microbial fuel cells.98 We built a simple
model82 of electron uptake by solid-matrix EET and calculated that the electrode
area needed for full use of the sunlight incident on a 1 m2 solar panel would need
to be only 10 m2 if a 1-cm-thick conductive biofilm that achieved the maximum
current densities achieved in nature could be made.82 If such a conductive biofilm
could be made reproducibly, these could greatly relieve the need for highly
extended surface areas inside an electrosynthesis reactor highlighted by the analysis
of H2-mediated electron transport. A tantalizing area of research is the construction
of nanostructured surfaces to control biofilm formation.101

Determining whether these estimates of current density are achievable with low-
energy losses will require a deeper physical understanding of the mechanisms of
solid-matrix EET. It has been proposed that pili proteins act like organic semicon-
ductors, or metals,102 or that they involve redox-gradient-driven hopping.103 A res-
olution to this debate would greatly aid attempts to estimate maximum transfer rates
and energy losses in solid-matrix EET.

Additionally, as the mechanism used by metal-oxidizing microbes to transfer

electrons from the quinone pool at low energy (z
100 mV versus SHE) to the
NAD(P)H pool at high energy (z
320 mV versus SHE) remains undiscovered, we
cannot say whether this ‘‘uphill pathway’’ incurs an energy-efficiency penalty.36 Eluci-
dating this mechanism would greatly aid understanding of the upper limits of effi-
ciency of this type of electrosynthesis.

REALIZING AMBITIONS IN BIOLOGICAL ENGINEERING: SYNTHETIC

AND SYSTEMS BIOLOGY TOOLS
Realizing these ambitious engineering goals for applied biology in sustainable energy
will require extensive genetic engineering. Synthetic organisms devoted to bioenergy
will likely look very different from naturally occurring organisms. Fortunately, in the last
decade, remarkable advances have been made in gene synthesis, genome editing,
directed evolution, bio-sensing, gene sequencing, and genome analysis tools.

Gene and Genome Editing

Recent advances in lambda-red and CRISPR/Cas9 for prokaryotic and eukaryotic en-
gineering have led to dramatic advances in the ease of gene addition, deletion,
modification, and replacement. These tools have ushered in a step change in the
ability to make extensive gene modifications and insert large sections of foreign
DNA into organisms.1

Chem 2, 20–51, January 12, 2017 45

Protein Engineering
Recent years have seen dramatic increases in our ability to engineer individual pro-
teins. Directed evolution is a powerful strategy for engineering proteins without the
need for a complete understanding of the relationship between amino acid
sequence and protein structure and function, which still eludes us. This approach
is based on our ability to generate enormous diversity in the primary sequences of
proteins by sequential random mutations, followed by artificial selection of variants
that acquire an improvement in the trait of interest.104 Protein engineering by
directed evolution is a perfect example of how a scientist can apply the principles
of Darwinian evolution to develop a synthetic selection of unnatural biological func-
tions. A limitation of this approach is the need to develop efficient assays from which
to select mutants with new capabilities, which are often difficult or impossible to
devise. Another challenge is the need to carry out laborious selections for each
mutagenic iteration, which is usually the rate-limiting step in protein engineering
by this approach.

De novo protein design is a powerful approach to engineering proteins with

completely new folds and functions that have never been tapped by natural evolu-
tion.105 The computational methods in this approach are assisted by our current
understanding of the physical principles ruling protein folding, as well as empirical
observations of the relationships between amino acid sequence and three-dimen-
sional protein structure. This technology has enabled the de novo design of enzymes
needed for a de novo carbon assimilation pathway,91 as well as proteins for self-
assembling biomaterials.105

Biosensors
Microbial engineering for the production of biofuels, chemicals, and other green
chemicals of interest is limited by the inability to develop high-throughput technol-
ogies to accelerate strain development. New advances in genetically encoded
biosensors will help overcome this limitation by monitoring in real time the concen-
trations of metabolites or products of interest,106 and reporting them with easy to
read and quantifying signals, such as fluorescence, luminescence, or colorimetry.
Such biosensors enable high-throughput screens and sorting technologies, such
as fluorescence-activated cell sorting, to expedite microbial strain development.
A great challenge here is in developing active sites that bind to desired target chem-
icals and mechanisms to transduce this binding into fluorescent, colorimetric, or
luminescent signals.

Whole-Genome Engineering
The type of functionality that we seek will likely require extensive modification across
the genome. New technologies such as multiplex automated genome engineering
(MAGE)107 have made simultaneous modification of multiple sections of a microbial
genome in parallel possible.

Genome engineering has made it possible to recode organisms to change stop

codons across the genome and add in unnatural amino acids to engineer
new capabilities in engineered enzymes by de novo design,105 prevent viral
infection (a common problem in organisms used on a massive scale), or prevent
escape of engineered organisms (a reasonable concern in organisms used in the
open). Recoded organisms1 open the possibility of constructing synthetic organ-
isms that have limited chance of survival in the wild, greatly easing public
acceptance.

46 Chem 2, 20–51, January 12, 2017

Current pressing challenges are the development of tools for synthetic biology and
directed evolution of organisms beyond the traditional model organisms such as
E. coli and S. cerevisiae. Such tools would enable whole-genome engineering of
non-model organisms such as autotrophic organisms that bring unique capabilities
to sustainable energy.

Genome Analysis
With all of the recent advances in the ability to modify and even completely write
new genomes, we are still left with the question of what to write. Despite the
amazing progress in sequencing over the past decade, the full genetic basis for
many of the complex biological behaviors that we would like to exploit remains
unknown. In fact, it is fair to say that the percentage of genes of unknown
function in the average genome sequenced today is the same (30%–40%) as it
was more than a decade ago.108 The reductionist approach to studying funda-
mental biological phenomena alone is unlikely to resolve this situation. Instead,
it will be necessary to take a systems biology approach to study the different
aspects of cell physiology, such as cell division, cell signaling, and metabolism,
in order to further improve our ability to harness and engineer desirable biological
traits.

Fortunately, over the past 7 years, there have been considerable advances
in genomic characterization tools for systems biology such as Tn-seq108 for
characterization of pooled transposon insertion mutant collections. Several of the
authors developed the Knockout Sudoku method to develop whole-genome
knockout collections for esoteric organisms at dramatically low cost and high
speed.109 This technology enabled the construction of the first whole-genome
knockout collection for an electroactive microbe, S. oneidensis, built with the specific
intent of discovering mechanisms of electron uptake by metal-oxidizing microbes.

CONCLUSIONS
Today, biofuels embody the most prominent use of biology in sustainable energy by
achieving the lowest cost and most widely deployed method for capturing and stor-
ing solar energy. However, the further expansion of traditional biofuels technology
has provoked significant concerns over their environmental impact, causing many to
question whether biology has any future in sustainable energy.

Rather than viewing these concerns as reasons to reject biofuels, we believe that the
challenges facing biofuels are no more intractable than the challenges faced by non-
biological energy technologies. The advances in genetic engineering and our
increasing understanding of basic biology reassure us that these challenges are solv-
able. Moreover, the astounding capabilities of biology to control cell replication and
self-assembly, the redox state of metals, carbon fixation, and materials and chemical
synthesis give us confidence that in the future, biology will not only be used in the
production of biofuels. We envision applications for biology both in supporting
and enabling traditionally abiotic technologies such as nuclear power, battery en-
ergy storage, and the production and recycling of energy-critical materials and
advanced materials for making lighter vehicles. Furthermore, we foresee the appli-
cation of biology in new technologies where it will play a central role such as
enhanced and hybrid photosynthesis.

We envision two paradigms in the development of applied biology technologies:

one that relies on the accrual of incremental innovations on top of naturally occurring

Chem 2, 20–51, January 12, 2017 47

organisms but that, when compounded, offers the potential for transformative ef-
fects in energy. The second relies on larger innovations driven by the application
of answers to basic research questions.

The potential of the incremental development paradigm can be illustrated by

analogy with developments in computation over the last 25 years. Computers
of 25 years ago share much of the same DNA (metaphorically) as a computer
of today: both use electronics that operate on transistors synthesized by CMOS
technology, have a von Neumann architecture, could (or do) run a variant of the
Unix operating system, use a graphical user interface based on high-speed
rendering; and can exchange information through Transmission Control Proto-
col/Internet Protocol (TCP/IP). However, parallel technical developments in
batteries, lightweight high-resolution displays, encryption, and wireless chipsets
have led to a dramatically different experience of the computer from a special-
ized device to one that is shockingly ubiquitous, and has a dramatic power for
the user.

Although organisms used in the synthesis of biofuels in the future will share much of
the same DNA (literally) as organisms used today, compounded incremental
advances in the enzymes used for fuel synthesis and genes for enhanced tolerance
to fuel and environmental conditions could produce an organism that is radically
different from the original wild counterpart, one with a transformative effect on
the market for liquid fuels. We anticipate that a similar development paradigm could
be applied to organisms used in bio-remediation, the production of advanced ma-
terials, and mining of energy-critical elements.

The application of biology to sustainable energy will also require finding solutions to
problems at the intersection of applied and basic research. Much as the invention of
the airplane required advances in the basic science of aerodynamics, sustainable en-
ergy applications involving biology will require continued basic research into the
fundamental principles of biology, ranging from evolutionary mechanisms and
light-independent autotrophic metabolisms to carbon-fixation pathways and funda-
mental properties of biological materials.

AUTHOR CONTRIBUTIONS
O.A., I.A.A., J.L.A., and B.B. wrote, revised, and approved the manuscript.

ACKNOWLEDGMENTS
This work was supported by Princeton University startup funds and a Career Award at
the Scientific Interface from the Burroughs Wellcome Fund to B.B. and by the Alfred
P. Sloan Foundation to J.L.A.

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