James B. McKinlay

Prospects for a bio-based succinate industry

Bio-based succinate is receiving increasing attention as a potential intermediary feedstock for replacing a large petrochemical-based bulk chemical market. The prospective economical and environmental benefits of a bio-based succinate industry have motivated research and development of succinate-producing organisms. Bio-based succinate is still faced with the challenge of becoming cost competitive against petrochemical-based alternatives. High succinate concentrations must be produced at high rates, with little or no by-products to most efficiently use substrates and to simplify purification procedures. Herein are described the current prospects for a bio-based succinate industry, with emphasis on specific bacteria that show the greatest promise for industrial succinate production. The succinate-producing characteristics and the metabolic pathway used by each bacterial species are described, and the advantages and disadvantages of each bacterial system are discussed.

Carbon dioxide fixation as a central redox cofactor recycling mechanism in bacteria

The Calvin-Benson-Bassham cycle (Calvin cycle) catalyzes virtually all primary productivity on Earth and is the major sink for atmospheric CO2. A less appreciated function of CO2 fixation is as an electron-accepting process. It is known that anoxygenic phototrophic bacteria require the Calvin cycle to accept electrons when growing with light as their sole energy source and organic substrates as their sole carbon source. However, it was unclear why and to what extent CO2 fixation is required when the organic substrates are more oxidized than biomass. To address these questions we measured metabolic fluxes in the photosynthetic bacterium Rhodopseudomonas palustris grown with 13C-labeled acetate. R. palustris metabolized 22% of acetate provided to CO2 and then fixed 68% of this CO2 into cell material using the Calvin cycle. This Calvin cycle flux enabled R. palustris to reoxidize nearly half of the reduced cofactors generated during conversion of acetate to biomass, revealing that CO2 fixation plays a major role in cofactor recycling. When H2 production via nitrogenase was used as an alternative cofactor recycling mechanism, a similar amount of CO2 was released from acetate, but only 12% of it was reassimilated by the Calvin cycle. These results underscore that N2 fixation and CO2 fixation have electron-accepting roles separate from their better-known roles in ammonia production and biomass generation. Some nonphotosynthetic heterotrophic bacteria have Calvin cycle genes, and their potential to use CO2 fixation to recycle reduced cofactors deserves closer scrutiny.

Insights into Actinobacillus succinogenes Fermentative Metabolism in a Chemically Defined Growth Medium

ABSTRACT Chemically defined media allow for a variety of metabolic studies that are not possible with undefined media. A defined medium, AM3, was created to expand the experimental opportunities for investigating the fermentative metabolism of succinate-producing Actinobacillus succinogenes. AM3 is a phosphate-buffered medium containing vitamins, minerals, NH4Cl as the main nitrogen source, and glutamate, cysteine, and methionine as required amino acids. A. succinogenes growth trends and end product distributions in AM3 and rich medium fermentations were compared. The effects of NaHCO3 concentration in AM3 on end product distribution, growth rate, and metabolic rates were also examined. The A. succinogenes growth rate was 1.3 to 1.4 times higher at an NaHCO3 concentration of 25 mM than at any other NaHCO3 concentration, likely because both energy-producing metabolic branches (i.e., the succinate-producing branch and the formate-, acetate-, and ethanol-producing branch) were functioning at relatively high rates in the presence of 25 mM bicarbonate. To improve the accuracy of the A. succinogenes metabolic map, the reasons for A. succinogenes glutamate auxotrophy were examined by enzyme assays and by testing the ability of glutamate precursors to support growth. Enzyme activities were detected for glutamate synthesis that required glutamine or α-ketoglutarate. The inability to synthesize α-ketoglutarate from glucose indicates that at least two tricarboxylic acid cycle-associated enzyme activities are absent in A. succinogenes.

Photobiological production of hydrogen gas as a biofuel

Solar energy can be converted into chemical energy in the form of hydrogen gas using oxygenic and anoxygenic photosynthetic microbes. Laboratory-scale measurements suggest that photobiological hydrogen production rates could yield more energy than current crop-based biofuel productivities. Major challenges, such as inhibitory amounts of oxygen produced during oxygenic photosynthesis and inhibition of H(2)-producing nitrogenase by ammonia, are being overcome through genetic engineering. Further advances are expected as the metabolic and regulatory aspects behind photobiological hydrogen production are revealed. Genetic engineering, coculturing, and bioreactor designs making use of immobilized cells have the potential to increase conversion efficiencies of light energy to H(2) and to decrease the land area needed for photobiological H(2) production.

A genomic perspective on the potential of Actinobacillus succinogenes for industrial succinate production

BackgroundSuccinate is produced petrochemically from maleic anhydride to satisfy a small specialty chemical market. If succinate could be produced fermentatively at a price competitive with that of maleic anhydride, though, it could replace maleic anhydride as the precursor of many bulk chemicals, transforming a multi-billion dollar petrochemical market into one based on renewable resources. Actinobacillus succinogenes naturally converts sugars and CO2 into high concentrations of succinic acid as part of a mixed-acid fermentation. Efforts are ongoing to maximize carbon flux to succinate to achieve an industrial process.ResultsDescribed here is the 2.3 Mb A. succinogenes genome sequence with emphasis on A. succinogeness potential for genetic engineering, its metabolic attributes and capabilities, and its lack of pathogenicity. The genome sequence contains 1,690 DNA uptake signal sequence repeats and a nearly complete set of natural competence proteins, suggesting that A. succinogenes is capable of natural transformation. A. succinogenes lacks a complete tricarboxylic acid cycle as well as a glyoxylate pathway, and it appears to be able to transport and degrade about twenty different carbohydrates. The genomes of A. succinogenes and its closest known relative, Mannheimia succiniciproducens, were compared for the presence of known Pasteurellaceae virulence factors. Both species appear to lack the virulence traits of toxin production, sialic acid and choline incorporation into lipopolysaccharide, and utilization of hemoglobin and transferrin as iron sources. Perspectives are also given on the conservation of A. succinogenes genomic features in other sequenced Pasteurellaceae.ConclusionsBoth A. succinogenes and M. succiniciproducens genome sequences lack many of the virulence genes used by their pathogenic Pasteurellaceae relatives. The lack of pathogenicity of these two succinogens is an exciting prospect, because comparisons with pathogenic Pasteurellaceae could lead to a better understanding of Pasteurellaceae virulence. The fact that the A. succinogenes genome encodes uptake and degradation pathways for a variety of carbohydrates reflects the variety of carbohydrate substrates available in the rumen, A. succinogeness natural habitat. It also suggests that many different carbon sources can be used as feedstock for succinate production by A. succinogenes.

Extracellular Iron Reduction Is Mediated in Part by Neutral Red and Hydrogenase in Escherichia coli

ABSTRACT Both microbial iron reduction and microbial reduction of anodes in fuel cells can occur by way of soluble electron mediators. To test whether neutral red (NR) mediates iron reduction, as it does anode reduction, by Escherichia coli, ferrous iron levels were monitored in anaerobic cultures grown with amorphous iron oxide. Ferrous iron levels were 19.4 times higher in cultures fermenting pyruvate in the presence of NR than in the absence of NR. NR did not stimulate iron reduction in cultures respiring with nitrate. To explore the mechanism of NR-mediated iron reduction, cell extracts of E. coli were used. Cell extract-NADH-NR mixtures had an enzymatic iron reduction rate almost 15-fold higher than the chemical NR-mediated iron reduction rate observed in controls with no cell extract. Hydrogen was consumed during stationary phase (in which iron reduction was detectable) especially in cultures containing both NR and iron oxide. An E. coli hypE mutant, with no hydrogenase activity, was also impaired in NR-mediated iron reduction activity. NR-mediated iron reduction rates by cell extracts were 1.5 to 2 times higher with hydrogen or formate as the electron source than with NADH. Our findings suggest that hydrogenase donates electrons to NR for extracellular iron reduction. This process appears to be analogous to those of iron reduction by bacteria that use soluble electron mediators (e.g., humic acids and 2,6-anthraquinone disulfonate) and of anode reduction by bacteria using soluble mediators (e.g., NR and thionin) in microbial fuel cells.

Calvin Cycle Flux, Pathway Constraints, and Substrate Oxidation State Together Determine the H2 Biofuel Yield in Photoheterotrophic Bacteria

ABSTRACT Hydrogen gas (H2) is a possible future transportation fuel that can be produced by anoxygenic phototrophic bacteria via nitrogenase. The electrons for H2 are usually derived from organic compounds. Thus, one would expect more H2 to be produced when anoxygenic phototrophs are supplied with increasingly reduced (electron-rich) organic compounds. However, the H2 yield does not always differ according to the substrate oxidation state. To understand other factors that influence the H2 yield, we determined metabolic fluxes in Rhodopseudomonas palustris grown on 13C-labeled fumarate, succinate, acetate, and butyrate (in order from most oxidized to most reduced). The flux maps revealed that the H2 yield was influenced by two main factors in addition to substrate oxidation state. The first factor was the route that a substrate took to biosynthetic precursors. For example, succinate took a different route to acetyl-coenzyme A (CoA) than acetate. As a result, R. palustris generated similar amounts of reducing equivalents and similar amounts of H2 from both succinate and acetate, even though succinate is more oxidized than acetate. The second factor affecting the H2 yield was the amount of Calvin cycle flux competing for electrons. When nitrogenase was active, electrons were diverted away from the Calvin cycle towards H2, but to various extents, depending on the substrate. When Calvin cycle flux was blocked, the H2 yield increased during growth on all substrates. In general, this increase in H2 yield could be predicted from the initial Calvin cycle flux. IMPORTANCE Photoheterotrophic bacteria, like Rhodopseudomonas palustris, obtain energy from light and carbon from organic compounds during anaerobic growth. Cells can naturally produce the biofuel H2 as a way of disposing of excess electrons. Unexpectedly, feeding cells organic compounds with more electrons does not necessarily result in more H2. Despite repeated observations over the last 40 years, the reasons for this discrepancy have remained unclear. In this paper, we identified two metabolic factors that influence the H2 yield, (i) the route taken to make biosynthetic precursors and (ii) the amount of CO2-fixing Calvin cycle flux that competes against H2 production for electrons. We show that the H2 yield can be improved on all substrates by using a strain that is incapable of Calvin cycle flux. We also contributed quantitative knowledge to the long-standing question of why photoheterotrophs must produce H2 or fix CO2 even on relatively oxidized substrates. Photoheterotrophic bacteria, like Rhodopseudomonas palustris, obtain energy from light and carbon from organic compounds during anaerobic growth. Cells can naturally produce the biofuel H2 as a way of disposing of excess electrons. Unexpectedly, feeding cells organic compounds with more electrons does not necessarily result in more H2. Despite repeated observations over the last 40 years, the reasons for this discrepancy have remained unclear. In this paper, we identified two metabolic factors that influence the H2 yield, (i) the route taken to make biosynthetic precursors and (ii) the amount of CO2-fixing Calvin cycle flux that competes against H2 production for electrons. We show that the H2 yield can be improved on all substrates by using a strain that is incapable of Calvin cycle flux. We also contributed quantitative knowledge to the long-standing question of why photoheterotrophs must produce H2 or fix CO2 even on relatively oxidized substrates.

Production of Hydrogen Gas from Light and the Inorganic Electron Donor Thiosulfate by Rhodopseudomonas palustris

ABSTRACT A challenge for photobiological production of hydrogen gas (H2) as a potential biofuel is to find suitable electron-donating feedstocks. Here, we examined the inorganic compound thiosulfate as a possible electron donor for nitrogenase-catalyzed H2 production by the purple nonsulfur phototrophic bacterium (PNSB) Rhodopseudomonas palustris. Thiosulfate is an intermediate of microbial sulfur metabolism in nature and is also generated in industrial processes. We found that R. palustris grew photoautotrophically with thiosulfate and bicarbonate and produced H2 when nitrogen gas was the sole nitrogen source (nitrogen-fixing conditions). In addition, illuminated nongrowing R. palustris cells converted about 80% of available electrons from thiosulfate to H2. H2 production with acetate and succinate as electron donors was less efficient (40 to 60%), partly because nongrowing cells excreted the intermediary metabolite α-ketoglutarate into the culture medium. The fixABCX operon (RPA4602 to RPA4605) encoding a predicted electron-transfer complex is necessary for growth using thiosulfate under nitrogen-fixing conditions and may serve as a point of engineering to control rates of H2 production. The possibility to use thiosulfate expands the range of electron-donating compounds for H2 production by PNSBs beyond biomass-based electron donors.

Non-growing Rhodopseudomonas palustris increases the hydrogen gas yield from acetate by shifting from the glyoxylate shunt to the tricarboxylic acid cycle.

Background: The metabolism of non-growing microbes is poorly understood. Results: In a nitrogen-starved and non-growing photoheterotrophic bacterium, metabolic flow was diverted to mobilize electrons for H2 production. Conclusion: During starvation bacteria decouple their metabolism from biosynthesis. Significance: An understanding of metabolic activities of non-growing cells can be used to engineer improved biocatalysts. When starved for nitrogen, non-growing cells of the photosynthetic bacterium Rhodopseudomonas palustris continue to metabolize acetate and produce H2, an important industrial chemical and potential biofuel. The enzyme nitrogenase catalyzes H2 formation. The highest H2 yields are obtained when cells are deprived of N2 and thus use available electrons to synthesize H2 as the exclusive product of nitrogenase. To understand how R. palustris responds metabolically to increase H2 yields when it is starved for N2, and thus not growing, we tracked changes in biomass composition and global transcript levels. In addition to a 3.5-fold higher H2 yield by non-growing cells we also observed an accumulation of polyhydroxybutyrate to over 30% of the dry cell weight. The transcriptome of R. palustris showed down-regulation of biosynthetic processes and up-regulation of nitrogen scavenging mechanisms in response to N2 starvation but gene expression changes did not point to metabolic activities that could generate the reductant necessary to explain the high H2 yield. We therefore tracked 13C-labeled acetate through central metabolic pathways. We found that non-growing cells shifted their metabolism to use the tricarboxylic acid cycle to metabolize acetate in contrast to growing cells, which used the glyoxylate cycle exclusively. This shift enabled cells to more fully oxidize acetate, providing the necessary reducing power to explain the high H2 yield.

N2 gas is an effective fertilizer for bioethanol production by Zymomonas mobilis

Significance Recently there has been a surge in ethanol biofuel production from cellulosic feedstocks, offering more environmental benefits than conventional ethanol production from food crops. However, cellulosic feedstocks are low in nitrogen, requiring that millions of dollars be spent on nitrogen supplements to grow the ethanol-producing microbes. Zymomonas mobilis is a bacterium that has long been viewed as a potential rival to Baker’s yeast as an ethanol producer. Contrary to published remarks, we discovered that Z. mobilis can use the most abundant gas in the atmosphere, N2, as a nitrogen source and does so without detriment to the high ethanol yield. Using N2-utilizing Z. mobilis could offset much of the monetary and environmental costs of current industrial nitrogen supplements. A nascent cellulosic ethanol industry is struggling to become cost-competitive against corn ethanol and gasoline. Millions of dollars are spent on nitrogen supplements to make up for the low nitrogen content of the cellulosic feedstock. Here we show for the first time to our knowledge that the ethanol-producing bacterium, Zymomonas mobilis, can use N2 gas in lieu of traditional nitrogen supplements. Despite being an electron-intensive process, N2 fixation by Z. mobilis did not divert electrons away from ethanol production, as the ethanol yield was greater than 97% of the theoretical maximum. In a defined medium, Z. mobilis produced ethanol 50% faster per cell and generated half the unwanted biomass when supplied N2 instead of ammonium. In a cellulosic feedstock-derived medium, Z. mobilis achieved a similar cell density and a slightly higher ethanol yield when supplied N2 instead of the industrial nitrogen supplement, corn steep liquor. We estimate that N2-utilizing Z. mobilis could save a cellulosic ethanol production facility more than