Steven W. Singer, Harry R. Beller, Swapnil Chhabra, Christopher J. Chang, and Jerry Adler

Abstract We are developing an integrated Microbial-ElectroCatalytic (MEC) system consisting of Ralstonia eutropha as a chemolithoautotrophic host for meta­bolic engineering coupled to a small-molecule electrocatalyst for the production of biofuels from CO2 and H2. R. eutropha is an aerobic bacterium that grows with CO2 as the carbon source and H2 as electron donor while producing copious amounts of poly- hydroxybutyrate. Metabolic flux from existing R. eutropha pathways is being diverted into engineered pathways that produce biofuels. Novel molybdenum electrocatalysts that can convert water to hydrogen in neutral aqueous media will act as chemical mediators to generate H2 from electrodes in the presence of engineered strains of R. eutropha. To increase the local concentration of H2, we are engineering R. eutropha’s outer-membrane proteins to tether the electrocatalysts to the bacterial surface. The integrated MEC system will provide a transformational new source of renewable liquid transportation fuels that extends beyond biomass-derived substrates.

S. W. Singer (H)

Earth Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA e-mail: swsinger@lbl. gov

H. R. Beller

Earth Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720 , USA S. Chhabra

Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA C. J. Chang

Department of Chemistry, University of California-Berkeley, Berkeley, CA 94720, USA J. Adler

Logos Technologies, Arlington, VA 22203, USA

J. W. Lee (ed.), Advanced Biofuels and Bioproducts, DOI 10.1007/978-1-4614-3348-4_40, 1091 © Springer Science+Business Media New York 2013

1 Introduction

Liquid transportation fuels are a critical component of the energy infrastructure of the United States. New sources of liquid fuels are required to replace petroleum — derived fuels because current supplies of petroleum are unstable and CO2 produced by combustion of liquid transportation fuels is a significant contributor to green­house gas emissions. Currently, there is significant interest in transforming lignocel — lulosic biomass into liquid transportation fuels through hydrolysis of the biomass to monomeric sugars and fermentation to fuels, providing a carbon-neutral, renewable source of liquid fuel [20]. However, problems associated with generating engineered crops, efficient use of arable land, development of cost-effective pretreatment pro­cesses, and the cost of deconstructing enzymes still remain largely unsolved [24]. An elegant alternative to the production of cellulosic biofuels would be to transform CO2 directly into liquid fuels, mitigating CO2 emissions and creating a biofuel pro­duction process with the potential to be carbon-neutral [7]. Autotrophic microor­ganisms have evolved multiple pathways to utilize ubiquitous natural reductants to reduce CO2 [23]. Diverting these pathways to produce liquid fuels is an intriguing opportunity to develop fuels to replace petroleum-based fossil fuels. Algal and cyanobacterial species have significant potential for generating biofuels, however these organisms absorb light inefficiently and are expensive to culture at industrial scale [17]. Chemoautotrophic organisms that use inorganic reductants ([S2-, Fe(II), H2]) or electricity have the possibility to overcome these limitations, as they may be able to reduce CO2 more efficiently, are not dependent on available light, and may be adapted readily to industrial conditions [7] . However, these chemoautotrophic organisms, which are often isolated from extreme environments, tend to grow to low cell densities and are very difficult to manipulate genetically.

One class of chemoautotrophic bacteria, “Knallgas” bacteria that grow with H2/ CO2 under aerobic conditions, does not have these limitations. The model species of this class, Ralstonia eutropha, can grow to very high cell densities ( >200 g/L) and has been extensively manipulated genetically [16]. Under nutrient limitation, R. eutropha directs most of the reduced carbon flux generated by the Calvin cycle to synthesis of polyhydroxybutyrate (PHB), a biopolymeric compound stored in granules. Under growth with H2/CO2 , 61 g/L of PHB was formed in 40 h, which represents ~70% of total cell weight [10] . PHB and related polyhydroxyalkonate polymers have been produced at industrial scale and marketed as Biopol™ (Monsanto) and Mircel™ (Metabolix) [16].

The PHB synthesis pathway in R. eutropha involves three genes expressed as an operon (Fig. 1) [15]. The gene products of this operon are PhaA, a b-ketothiolase, PhaB, an acetoacetyl-CoA reductase, and PhaC, the PHB synthase (Fig. 1). Numerous mutants have been generated that are impaired in PHB synthesis and these mutagenesis studies have demonstrated that PHB synthesis can be blocked with minimal effects on cellular function [19, 22]. An R. eutropha strain generated by chemical mutagenesis that is impaired in PHB synthesis has been shown to secrete large amounts of pyruvate into the medium under autotrophic conditions,

PhaA = p-ketothiolase PhaB = NADPH-dependent reductase PhaC = PHB synthase

Fig. 1 Polyhydroxybutyrate (PHB) synthesis pathway in Ralstonia eutropha suggesting that the mutant maintains a similar magnitude of carbon flux in the absence of PHB synthesis [4]. Metabolic engineering strategies have been success­fully employed to increase carbon flux through the PHB pathway, suggesting that R. eutropha will be a suitable host for synthetic biology applications [5, 13].

Current efforts to produce biofuels using synthetic biology have focused on using model organisms (Escherichia coli and Saccharomyces cerevisiae) as hosts for met­abolic engineering [3, 6]. These efforts have concentrated on using biomass-derived carbohydrates as the sources for renewable biofuel generation [12]. These strategies require redirection of central metabolic pathways by introduction of new pathways that redirect metabolic flux to a desired end-product. This approach has been used to produce alcohols, alkenes, and isoprenoids that may be used as liquid fuel substi­tutes for petroleum products [8]. Rewiring the metabolism of these model organ­isms so that they can utilize CO2 as the carbon input for biofuel production would have substantial benefits in broadening the substrate scope for metabolic engineer­ing and reducing CO2 emissions. R. eutropha is an attractive host for biofuel pro­duction from CO2 as it already has the capability for autotrophic growth, is amenable to metabolic engineering, and expresses a metabolic pathway that supports significant carbon flux.

An inexpensive source of H2 will be essential for the effective development of R. eutropha as a biofuel-producing platform. For fuel production to be sensible, the method of H2 generation must utilize methods that do not themselves consume fossil-derived energy, or draw low-carbon energy away from carbon mitigating uses. Known small-molecule metal catalysts generally require organic acids, addi­tives, and/or solvents that are also incompatible for use with living organisms [9]. Molybdenum polypyridyl complexes (MoPy5) have been shown to be excellent catalysts for the electrochemical reduction H+ in neutral water at rates that approach to that of hydrogenase enzymes (Fig. 2) [11]. These electrocatalysts are stable and evolve H2 in seawater and are compatible with microbial growth media. R. eutropha is an ideal microbe to couple with electrocatalysis, as growth with H2 generated in situ by an electrode has already been demonstrated [18]. Electrocatalysis will be coupled in two ways: the MoPy5 catalyst will be tethered to the electrode surface and H2 generated at the surface will be used for chemoautotrophic growth and

biofuel production by engineered strains of R. eutropha. In the second configuration, MoPy5 catalysts will be tethered directly to the surface of engineered R. eutropha strains and the strains will interact directly with the electrode surface. In this configuration, the tethered catalyst will generate H2 at the electrode, which will be used by engineered R. eutropha strains for growth and biofuel production.

The integrated MEC (Microbial-ElectroCatalytic) system, the combination of a novel catalytic system to generate H2 directly from water coupled to a chemolithoau — totroph, R. eutropha, that is metabolically engineered to produce high titers of bio­fuels from H2 and CO2, will be a novel technology that will provide a new source of renewable liquid transportation fuels that extends beyond biomass-derived sub­strates (Fig. 3).

2 Experimental Approach