Download Journal of Biotechnology Alternative routes to biofuels: Light

Journal of Biotechnology 142 (2009) 87–90
Contents lists available at ScienceDirect
Journal of Biotechnology
journal homepage: www.elsevier.com/locate/jbiotec
Alternative routes to biofuels: Light-driven biofuel formation
from CO2 and water based on the ‘photanol’ approach
K.J. Hellingwerf ∗ , M.J. Teixeira de Mattos
Molecular Microbial Physiology Group, Swammerdam Institute for Life Sciences and Netherlands Institute for Systems Biology, University of Amsterdam,
Nieuwe Achtergracht 166, NL-1018 Amsterdam, The Netherlands
a r t i c l e
i n f o
Article history:
Received 4 November 2008
Received in revised form 29 January 2009
Accepted 3 February 2009
Keywords:
Biofuel production
Cyanobacteria
Photosynthesis
Fermentation
Ethanol
Butanol
a b s t r a c t
For a sustainable energy future, the development of efficient biofuel production systems is an important
prerequisite. Here we describe an approach in which basic reactions from phototrophy are combined in single organisms with key metabolic routes from chemotrophic organisms, with C3 sugars as
Glyceraldehyde-3-phosphate as the central linking intermediate. Because various metabolic routes that
lead to the formation of a range of short-chain alcohols can be used in this approach, we refer to it as the
photanol approach. Various strategies can be explored to optimize this biofuel production strategy.
© 2009 Elsevier B.V. All rights reserved.
1. Introduction
Photosynthesis, be it in natural or in artificial form, is a key
element in a sustainable, global, supply of energy. In this process, a reaction center, often assisted by light-harvesting antennae,
converts the free energy of visible irradiation emitted by the sun
into the redox free energy of electrons (Fig. 1). In artificial photosynthesis (photovoltaics) the redox energy is directly converted
into electricity and therefore artificial photosynthesis is optimally
suited for electricity supply, particularly because of the minimal
number of free energy conversion steps involved.
For fuel supply, however, the electrons with their more negative
redox potential, have to be stored stably in chemical compounds
(like H2 , CH3 OH, etc.) that can be transported at low cost to the
required site of combustion. In spite of a considerable amount of
work to achieve this, artificial photosynthesis has not yet provided
stable catalysts to convert the electrons, derived from water, into
stable fuel products. Rather than chemical catalysts, one can also
set up an in vitro system, using the molecular components of natural photosynthesis, i.e. Photosystems I and II (Slater, 1976). However,
particularly Photosystem II is very sensitive to auto-inactivation
(Aro et al., 1993), and the in vitro systems lack auto-regenerative
capacity. For these reasons we estimate that natural photosynthe-
∗ Corresponding author. Tel: +31 20 5257055.
E-mail address: [email protected] (K.J. Hellingwerf).
0168-1656/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.jbiotec.2009.02.002
sis is much better suited for large-scale sustainable fuel production
than artificial systems.
In natural photosynthesis, based on chlorophototrophy (Bryant
and Frigaard, 2006), light emitted by the sun, is captured by the
antennae of phototrophic organisms on the surface of the earth
and transferred via resonance energy transfer to dedicated reaction centers, the Photosystems I and II. There, the free energy is
stored by a series of electron transfer events, catalyzed by these
two photosystems and a cytochrome b6 /f complex, that convert the
free energy of light into the biochemical energy of NADPH. Simultaneously, the electron transfer steps generate a proton gradient
that can lead to the formation of ATP, in an amount matching the
requirements for conversion of both these nucleotides, together
with CO2 , into phosphorylated carbohydrates, via the Calvin cycle
(Calvin, 1989). The main catalytic components for natural photosynthesis are housed in the thylakoid membrane. Both its intrinsic
electron-transfer pathway and its underlying structure are highly
conserved in evolution and are used in micro-organisms and in the
chloroplasts of plants and algae.
2. Current biofuel production strategies
Similar to CO2 , also protons can be used as the final electron
acceptor in natural photosynthesis (Kruse et al., 2005; Prince and
Kheshgi, 2005), thus producing overall (a mixture of) oxygen and
hydrogen. In the following, however, we will concentrate on the
former electron acceptor. Since the new millennium several alter-
88
K.J. Hellingwerf, M.J. Teixeira de Mattos / Journal of Biotechnology 142 (2009) 87–90
Fig. 1. Schematic representation of the photosynthesis process. R: reactant; P: product. EET: excitation energy transfer.
native strategies have been developed for large-scale conversion of
CO2 into biofuels:
(i) The first of these implies a two-step procedure: First formation
of (plant-derived) biomass, followed by a second processing
step for conversion to products like methane and ethanol.
Methane formation from biomass waste has been developed
as a technology already several decades ago. The large-scale
production of ethanol has been pioneered in Brazil, from sugarcane (Goldemberg, 2007), and more recently also in the US from
various crops, in particular from corn (Hill et al., 2006). This latter approach, however, has become controversial for various
reasons (Marris, 2006). First of all, the use of food crops for
fuel production has led to sharp increases in food prices. Second, environmental concerns have been expressed regarding
the environmental impact of the application of these processes
at the very large scale, e.g. because of local nutrient imbalances
and extent of net CO2 sequestration (Searchinger et al., 2008).
(ii) Above discussions about the net energy yield and environmental impact of large-scale ethanol production has led to
the development of several alternatives, often referred to
as ‘second-generation biofuels’ (Chisti, 2008; Vasudevan and
Briggs, 2008). For example, an economically viable alternative has been found in the harvesting of plants (or their seeds,
e.g. rapeseed). As many seeds are rich in tri-glycerides, transesterification with methanol at high pH (i.e. alkali catalyzed)
leads to the straightforward formation of methyl-esters of longchain fatty acids, i.e. biodiesel, which can be directly used as a
gasoline additive (Vasudevan and Briggs, 2008). Yet additional
sources of triglycerides are available: Many algae form considerable amounts of these lipids, which upon harvesting and
extraction can be processed in the same way as plant-derived
oils (Chisti, 2007). For many species the neutral lipid content
is in the order of ∼30% by weight, but for selected species this
content may go up to 80% (e.g. Schizochytrium sp., Botryococcus braunii, etc.). It should be noted, however, that for some of
these organisms the neutral lipid fraction is dominated by polyisoprenoids (Ratledge, 2004) rather than triglycerides, such as
the botryococcenes (Metzger and Largeau, 2005). Nevertheless,
these latter may be converted into useful fuel through ‘cracking’.
Because of the inherent disadvantages of ‘first-generation’ biofuels, several proposals have been made for improvement. For the
first stage of the process these include the improvement of the efficiency of photosynthesis (Mussgnug et al., 2007) and the use of
phototrophic microorganisms because of their inherently higher
biomass yield per unit surface area (Janssen et al., 2003; Dismukes
et al., 2008), the use of more than only the polysaccharide fraction
of the plant cells, including the lignin fraction (Weng et al., 2008),
the application of genetic engineering to alter plant composition
and to facilitate monomer release from the biopolymers (Bouton,
2007; Sticklen, 2008). For the second stage of the process it has
been proposed to e.g. alter the solvent specificity of the solventproducing organism, like to butanol (Durre, 2007) or a mixture of
branched-chain alcohols (Atsumi et al., 2008), to increase its solvent tolerance (Stephanopoulos, 2007), or even to substitute it for
an entirely chemical process (Agrawal et al., 2007).
Independent of these proposed improvements, suggestions for
further altering and optimizing biofuel production systems have
been made. An intriguing one of these is to use an engineered
cyanobacterium (i.e. Synechococcus leopoliensis) equipped with the
cloned bacterial cellulose synthase genes from Gluconobacter xylinus (Nobles and Brown, in press). This recombinant cyanobacterium
produces extra-cellular deposits of non-crystalline cellulose; particularly its non-crystalline nature makes this polymer an ideal
feedstock for biofuel production of various alcohols. Relevant in
this respect is that cellulose production in this configuration is not
limited by the intracellular storage capacity of the producer cell,
a limitation that does apply to e.g. glycogen and poly-alkanoate
formation. Significantly, homologous expression enzymes that catalyze the formation of extracellular cellulose has also been observed
in natural isolates of cyanobacteria, like in the filamentous Crinalium epipsammum (Dewinder et al., 1990).
3. The ‘photanol approach’: combining phototrophy and
chemotrophy
Besides many attractive features, the first- and secondgeneration biofuel production systems described above do also
have a number of less desirable aspects. The first-generation
approach suffers from the disadvantage that in the first step a very
complex product is formed that requires significant efforts before a
major part of it can be converted to useful fuel. Second generation
systems have the inherent limitation of the cellular lipid storage
capacity (although botryococcenes are described to be ‘deposited’
outside of the producing cells) and require a complex biosynthetic
machinery, with a limited capacity, and a nitrogen-limited growth.
We therefore think that it is relevant to develop further alternatives.
In the realm of (micro)biology, molecular physiology has
revealed two basic modes of life: The phototrophic and the
chemotrophic mode (Madigan and Parker, 2003). A subclass of
organisms that uses the former mode, the chlorophotoautotrophic
organisms, convert CO2 and minerals into new cells, using the
energy of (sun)light. In their intermediary metabolism, the NADPH
and ATP generated at the thylakoid membrane drive a cyclic
metabolic pathway, called the Calvin cycle, in which CO2 is converted into C3 sugars like Glyceraldehyde-3-phosphate. These latter
intermediates are key intermediates that feed most of the remaining anabolic pathways (Madigan and Parker, 2003).
In chemotrophic organisms C3 sugars play a key role in
catabolism: A wide range of sugars is first degraded to the C3 level,
after which they can be converted to CO2 , provided that sufficient
oxygen is available, or to a range of more reduced products. The latter is particularly important when anaerobic conditions prevail (for
instance in the ABE fermentation in clostridia (Awang et al., 1988;
Ezeji et al., 2007), the basis of the classical ‘solvatogenesis process’),
but takes place also under (micro)aerobic conditions, e.g. as a form
of ‘overflow metabolism’ (Deboer et al., 1990). A well-known example of the latter is the ethanol formation in Crabtree-positive yeasts
like Saccharomyces cerevisiae (Vandijken et al., 1993).
The key role of C3 sugars in both modes of metabolism brings up
the question whether or not it would be possible to combine them
in one organism (see also Fig. 2). Using the methods of synthetic
K.J. Hellingwerf, M.J. Teixeira de Mattos / Journal of Biotechnology 142 (2009) 87–90
89
Fig. 2. Schematic representation of the proposed ‘Photanol process’: By combining key activities from the phototrophic mode of life and from chemotrophy, a solvent cassette
system can be designed for the production of a range of organic solvents.
biology (Hasty et al., 2002), this should lead to a type of metabolism
that one might classify as ‘photofermentative’. It would circumvent
the need to first convert CO2 into the complex mixture of biopolymers of which the average cell is composed (protein, nucleic acids,
cell walls, neutral and phospholipids, etc.) and to apply a series
of subsequent processing steps to convert this complex mixture
into a specific biofuel. Accordingly, the overall efficiency of the
biofuel production process may be significantly increased. In the
theoretical limit the number of incident photons per area could be
quantitatively converted into e.g. ethanol, which implies a theoretical limit of ∼105 l/ha/yr.
For the production of ethanol from CO2 in the cyanobacterium
Synechococcus sp. strain PCC 7942, Deng and Coleman (1999) (see
also Fu, 2009) that this approach is feasible by heterologous expression of two enzymes only. The former investigators selected the
coding sequences of pyruvate decarboxylase (pdc) and alcohol
dehydrogenase II (adh) from the bacterium Zymomonas mobilis and
showed that in the resulting recombinant strain ethanol accumulates up to 5 mM in a period of four weeks.
Extending on this idea, through application of molecular genetic
engineering, a set of strains can be constructed that will each lead
to any of a series of desired products, including various alcohols
and organic acids, well-know from microbial fermentation processes. We refer to this approach of biofuel production as the
‘Photanol approach’. Furthermore, by a properly timed blocking of
anabolism of the host organism, e.g. through a lack of minerals in the
growth medium, it should be possible to redirect all carbon derived
from CO2 to a pre-selected fermentative pathway. The genetic
engineering and synthetic biology required for this approach is
most straightforwardly done in the cyanobacterium Synechocystis
PCC6803 because of the natural transformability (Shestakov and
Khyen, 1970; Dzelzkalns and Bogorad, 1986) of this species and the
many genomics analyses to which it has been subjected (e.g. Hihara
et al., 2001; Shastri and Morgan, 2005).
4. Challenges
To make the Photanol approach an economically viable strategy, several scientific challenges still lie ahead. One of these is
the downstream processing of the products formed. Nevertheless, for many aspects of this process use can be made of the
knowledge base derived from the ABE fermentation (Awang et
al., 1988). From such considerations it is clear already that ideally
the growth temperature of the production organism should be as
close as possible to the boiling point of the product formed; furthermore, the product should have very low solubility in water,
or both. Nevertheless, this may lead to problems of product toxicity, a phenomenon that possibly can be counteracted through
the (engineered) involvement of broad-specificity efflux pumps.
In the light of these considerations it is relevant to note that
genes encoding an ethylene synthase have been cloned successfully in the cyanobacterium Synechococcus elongatus PCC 7942
(Takahama et al., 2003). Significant rates of ethylene production
were reported.
Because of the oxygenic character of photosynthesis in
cyanobacteria, one of the important criteria in the selection of all
coding sequences to be heterologously expressed will be the oxygen
sensitivity of the encoded enzymes.
References
Agrawal, R., Singh, N.R., Ribeiro, F.H., Delgass, W.N., 2007. Sustainable fuel for the
transportation sector. Proceedings of the National Academy of Sciences of the
United States of America 104, 4828–4833.
Aro, E.M., Virgin, I., Andersson, B., 1993. Photoinhibition of photosystem. II. Inactivation, protein damage and turnover. Biochim. Biophys. Acta 1143, 113–134.
Atsumi, S., Hanai, T., Liao, J.C., 2008. Non-fermentative pathways for synthesis of
branched-chain higher alcohols as biofuels. Nature 451, 86-U13.
Awang, G.M., Jones, G.A., Ingledew, W.M., 1988. The acetone-butanol-ethanol fermentation. Crit. Rev. Microbiol. 15 (Suppl. 1), S33–S67.
Bouton, J.H., 2007. Molecular breeding of switchgrass for use as a biofuel crop. Curr.
Opin. Genet. Dev. 17, 553–558.
Bryant, D.A., Frigaard, N.U., 2006. Prokaryotic photosynthesis and phototrophy illuminated. Trends Microbiol. 14, 488–496.
Calvin, M., 1989. 40 years of photosynthesis and related activities. Photosynth. Res.
21, 3–16.
Chisti, Y., 2007. Biodiesel from microalgae. Biotechnol. Adv. 25, 294–306.
Chisti, Y., 2008. Biodiesel from microalgae beats bioethanol. Trends Biotechnol. 26,
126–131.
Deboer, J.P., Demattos, M.J.T., Neijssel, O.M., 1990. d(−)lactic acid production by
suspended and aggregated continuous cultures of Bacillus-Laevolacticus. Appl.
Microbiol. Biotechnol. 34, 149–153.
Deng, M.D., Coleman, J.R., 1999. Ethanol synthesis by genetic engineering in
cyanobacteria. Appl. Environ. Microbiol. 65, 523–528.
Dewinder, B., Stal, L.J., Mur, L.R., 1990. Crinalium-epipsammum Sp-Nov—a filamentous cyanobacterium with trichomes composed of elliptic cells and containing
poly-beta-(1,4) glucan (cellulose). J. Gen. Microbiol. 136, 1645–1653.
Dismukes, G.C., Carrieri, D., Bennette, N., Ananyev, G.M., Posewitz, M.C., 2008. Aquatic
phototrophs: efficient alternatives to land-based crops for biofuels. Curr. Opin.
Biotechnol. 19, 235–240.
Durre, P., 2007. Biobutanol: an attractive biofuel. Biotechnol. J. 2, 1525–1534.
Dzelzkalns, V.A., Bogorad, L., 1986. Stable transformation of the cyanobacterium
Synechocystis sp. PCC 6803 induced by UV irradiation. J. Bacteriol. 165,
964–971.
90
K.J. Hellingwerf, M.J. Teixeira de Mattos / Journal of Biotechnology 142 (2009) 87–90
Ezeji, T.C., Qureshi, N., Blaschek, H.P., 2007. Bioproduction of butanol from biomass:
from genes to bioreactors. Curr. Opin. Biotechnol. 18, 220–227.
Fu, P.-C., 2009. Genome-scale modeling of Synechocystis sp. PCC 6803 and prediction
of pathway insertion. J. Chem. Technol. Biotechnol. doi:10.1002/jctb.2065.
Goldemberg, J., 2007. Ethanol for a sustainable energy future. Science 315, 808–810.
Hasty, J., McMillen, D., Collins, J.J., 2002. Engineered gene circuits. Nature 420,
224–230.
Hihara, Y., Kamei, A., Kanehisa, M., Kaplan, A., Ikeuchi, M., 2001. DNA microarray
analysis of cyanobacterial gene expression during acclimation to high light. Plant
Cell 13, 793–806.
Hill, J., Nelson, E., Tilman, D., Polasky, S., Tiffany, D., 2006. Environmental, economic,
and energetic costs and benefits of biodiesel and ethanol biofuels. Proc. Natl.
Acad. Sci. USA 103, 11206–11210.
Janssen, M., Tramper, J., Mur, L.R., Wijffels, R.H., 2003. Enclosed outdoor photobioreactors: light regime, photosynthetic efficiency, scale-up, and future prospects.
Biotechnol. Bioeng. 81, 193–210.
Kruse, O., Rupprecht, J., Bader, K.P., Thomas-Hall, S., Schenk, P.M., Finazzi, G., Hankamer, B., 2005. Improved photobiological H2 production in engineered green
algal cells. J. Biol. Chem. 280, 34170–34177.
Madigan, M.T., Martinko, J.M., Parker, J., 2003. Brock Biology of Microorganisms.
Pearson Education Ltd., London.
Marris, E., 2006. Drink the best and drive the rest. Nature 444, 670–672.
Metzger, P., Largeau, C., 2005. Botryococcus braunii: a rich source for hydrocarbons
and related ether lipids. Appl. Microbiol. Biotechnol. 66, 486–496.
Mussgnug, J.H., Thomas-Hall, S., Rupprecht, J., Foo, A., Klassen, V., McDowall, A.,
Schenk, P.M., Kruse, O., Hankamer, B., 2007. Engineering photosynthetic light
capture: impacts on improved solar energy to biomass conversion. Plant Biotechnol. J. 5, 802–814.
Nobles, J.R., Brown, R.M., in press. Cellulose.
Prince, R.C., Kheshgi, H.S., 2005. The photobiological production of hydrogen: potential efficiency and effectiveness as a renewable fuel. Crit. Rev. Microbiol. 31,
19–31.
Ratledge, C., 2004. Fatty acid biosynthesis in microorganisms being used for Single
Cell Oil production. Biochimie 86, 807–815.
Searchinger, T., Heimlich, R., Houghton, R.A., Dong, F.X., Elobeid, A., Fabiosa, J., Tokgoz,
S., Hayes, D., Yu, T.H., 2008. Use of US croplands for biofuels increases greenhouse
gases through emissions from land-use change. Science 319, 1238–1240.
Shastri, A.A., Morgan, J.A., 2005. Flux balance analysis of photoautotrophic
metabolism. Biotechnol. Prog. 21, 1617–1626.
Shestakov, S.V., Khyen, N.T., 1970. Evidence for genetic transformation in blue-green
alga Anacystis nidulans. Mol. Gen. Genet. 107, 372–375.
Slater, E.C., 1976. Introduction to round-table discussion on bioenergetics of tomorrow. FEBS Lett. 64, 3–5.
Stephanopoulos, G., 2007. Challenges in engineering microbes for biofuels production. Science 315, 801–804.
Sticklen, M.B., 2008. Plant genetic engineering for biofuel production: towards
affordable cellulosic ethanol. Nat. Rev. Genet. 9, 433–443.
Takahama, K., Matsuoka, M., Nagahama, K., Ogawa, T., 2003. Construction and analysis of a recombinant cyanobacterium expressing a chromosomally inserted gene
for an ethylene-forming enzyme at the psbAI locus. J. Biosci. Bioeng. 95, 302–305.
Vandijken, J.P., Weusthuis, R.A., Pronk, J.T., 1993. Kinetics of growth and sugar consumption in yeasts. Antonie Van Leeuwenhoek Int. J. Gen. Mol. Microbiol. 63,
343–352.
Vasudevan, P.T., Briggs, M., 2008. Biodiesel production—current state of the art and
challenges. J. Ind. Microbiol. Biotechnol. 35, 421–430.
Weng, J.K., Li, X., Bonawitz, N.D., Chapple, C., 2008. Emerging strategies of lignin engineering and degradation for cellulosic biofuel production. Curr. Opin. Biotechnol.
19, 166–172.