Carboxydotrophic growth of <i>Geobacter sulfurreducens</i>

This study shows that Geobacter sulfurreducensgrows on carbon monoxide (CO) as electron donor with fumarateas electron acceptor. Geobacter sulfurreducens wastolerant to high CO levels, with up to 150 kPa in the headspacetested. During growth, hydrogen was detected in very slightamounts (~5 Pa). In assays with cell-free extract of cellsgrown with CO and fumarate, production of hydrogen fromCO was not observed, and hydrogenase activity with benzylviologen as electron acceptor was very low. Taken together,this suggested that CO is not utilized via hydrogen as intermediate.In the presence of CO, reduction of NADP+ wasobserved at a rate comparable to CO oxidation coupled tofumarate reduction in vivo. The G. sulfurreducens genomecontains a single putative carbon monoxide dehydrogenaseencodinggene. The gene is part of a predicted operon alsocomprising a putative Fe–S cluster-binding subunit (CooF)and a FAD–NAD(P) oxidoreductase and is preceded by aputative CO-sensing transcription factor. This cluster may beinvolved in a novel pathway for CO oxidation, but furtherstudies are necessary to ascertain this. Similar gene clustersare present in several other species belonging to theDeltaproteobacteria and Firmicutes, for which CO utilizationis currently not known

Atypical one-carbon metabolism of an acetogenic and hydrogenogenic Moorella thermoacetica strain

A thermophilic spore-forming bacterium (strain AMP) was isolated from a thermophilic methanogenic bioreactor that was fed with cobalt-deprived synthetic medium containing methanol as substrate. 16S rRNA gene analysis revealed that strain AMP was closely related to the acetogenic bacterium Moorella thermoacetica DSM 521T (98.3% sequence similarity). DNA¿DNA hybridization showed 75.2 ± 4.7% similarity to M. thermoacetica DSM 521T, suggesting that strain AMP is a M. thermoacetica strain. Strain AMP has a unique one-carbon metabolism compared to other Moorella species. In media without cobalt growth of strain AMP on methanol was only sustained in coculture with a hydrogen-consuming methanogen, while in media with cobalt it grew acetogenically in the absence of the methanogen. Addition of thiosulfate led to sulfide formation and less acetate formation. Growth of strain AMP with CO resulted in the formation of hydrogen as the main product, while other CO-utilizing Moorella strains produce acetate as product. Formate supported growth only in the presence of thiosulfate or in coculture with the methanogen. Strain AMP did not grow with H2/CO2, unlike M. thermoacetica (DSM 521T). The lack of growth with H2/CO2 likely is due to the absence of cytochrome b in strain AM

Carbon monoxide conversion by thermophilic sulfate-reducing bacteria in pure culture and in co-culture with Carboxydothermus hydrogenoformans

Biological sulfate (SO4) reduction with carbon monoxide (CO) as electron donor was investigated. Four thermophilic SO4-reducing bacteria, Desulfotomaculum thermoacetoxidans (DSM 5813), Thermodesulfovibrio yellowstonii (ATCC 51303), Desulfotomaculum kuznetsovii (DSM 6115; VKM B-1805), and Desulfotomaculum thermobenzoicum subsp. thermosyntrophicum (DSM 14055), were studied in pure culture and in co-culture with the thermophilic carboxydotrophic bacterium Carboxydothermus hydrogenoformans (DSM 6008). D. thermoacetoxidans and T. yellowstonii were extremely sensitive to CO: their growth on pyruvate was completely inhibited at CO concentrations above 2% in the gas phase. D. kuznetsovii and D. thermobenzoicum subsp. thermosyntrophicum were less sensitive to CO. In pure culture, D. kuznetsovii and D. thermobenzoicum subsp. thermosyntrophicum were able to grow on CO as the only electron donor and, in particular in the presence of hydrogen/carbon dioxide, at CO concentrations as high as 50-70%. The latter SO4 reducers coupled CO oxidation to SO4 reduction, but a large part of the CO was converted to acetate. In co-culture with C. hydrogenoformans, D. kuznetsovii and D. thermobenzoicum subsp. thermosyntrophicum could even grow with 100% CO (P CO=120 kPa)

Desulfotomaculum carboxydivorans sp.nov., a novel sulfate-reducing bacterium capable of growth at 100% CO

A moderately thermophilic, anaerobic, chemolithoheterotrophic, sulfate-reducing bacterium, strain CO-1-SRBT, was isolated from sludge from an anaerobic bioreactor treating paper mill wastewater. Cells were Gram-positive, motile, spore-forming rods. The temperature range for growth was 30¿68 °C, with an optimum at 55 °C. The NaCl concentration range for growth was 0¿17 g l¿1; there was no change in growth rate until the NaCl concentration reached 8 g l¿1. The pH range for growth was 6·0¿8·0, with an optimum of 6·8¿7·2. The bacterium could grow with 100 % CO in the gas phase. With sulfate, CO was converted to H2 and CO2 and part of the H2 was used for sulfate reduction; without sulfate, CO was completely converted to H2 and CO2. With sulfate, strain CO-1-SRBT utilized H2/CO2, pyruvate, glucose, fructose, maltose, lactate, serine, alanine, ethanol and glycerol. The strain fermented pyruvate, lactate, glucose and fructose. Yeast extract was necessary for growth. Sulfate, thiosulfate and sulfite were used as electron acceptors, whereas elemental sulfur and nitrate were not. A phylogenetic analysis of 16S rRNA gene sequences placed strain CO-1-SRBT in the genus Desulfotomaculum, closely resembling Desulfotomaculum nigrificans DSM 574T and Desulfotomaculum sp. RHT-3 (99 and 100 % similarity, respectively). However, the latter strains were completely inhibited above 20 and 50 % CO in the gas phase, respectively, and were unable to ferment CO, lactate or glucose in the absence of sulfate. DNA¿DNA hybridization of strain CO-1-SRBT with D. nigrificans and Desulfotomaculum sp. RHT-3 showed 53 and 60 % relatedness, respectively. On the basis of phylogenetic and physiological features, it is suggested that strain CO-1-SRBT represents a novel species within the genus Desulfotomaculum, for which the name Desulfotomaculum carboxydivorans is proposed. This is the first description of a sulfate-reducing micro-organism that is capable of growth under an atmosphere of pure CO with and without sulfate. The type strain is CO-1-SRBT (=DSM 14880T=VKM B-2319T

CO metabolism of carboxydothermus hydrogenoformans and archaeoglobus fulgidus

Microbial CO metabolism was studied in detail with the ultimate aim to assess the feasibility of a biotechnological process that could replace the existing water gas shift technology in the production of a fuel cell grade hydrogen gas from synthesis gas. It is expected that a biotechnological process is less sensitive to impurities present in synthesis gas and can reach lower CO thresholds, and thus might be more cost effective than conventional catalysts. Low CO thresholds are especially required for polymer electrolyte membrane fuel cells. These fuel cells have a broad application potential in a future hydrogen economy. The bulk of hydrogen produced today is derived from natural gas in a steam reforming process that forms CO besides H 2 . CO is removed by its conversion to H 2 in the water gas shift reaction. However, due to thermodynamic limitations of the process operation temperature, the required CO thresholds are not obtained (&lt;10 ppm). CO conversion at more ambient temperatures is in that respect advantageous for biological CO conversion to obtain a fuel cell grade H 2 .It was demonstrated in Chapter 2 that batch cultures of Carboxydothermus hydrogenoformans converted CO to levels below 2 ppm while accumulating H 2 . In these cultures it was necessary to remove CO 2 from the gas phase. Without removal of CO 2 , CO thresholds approached 100 ppm. CO limits that are generally communicated for PEM-FC, indicate 10 ppm as allowable threshold for CO in H 2 gas. The potential of a biotechnological process is thus supported, however, the time needed to reach these thresholds was considerable and needs attention. Biomass activity and gas/liquid mass transfer are possible rate limiting factors in the biological water gas shift reaction. As demonstrated in Chapter 3, higher conversion rates are possible in batch culture than achieved with the cultivation technique used in Chapter 2. In fact, it appears that only in the early stage of batch cultivation the biomass is limiting CO conversion rates. After some time biomass has grown to sufficient density and gas/mass transfer becomes limiting. Even in the most turbulent shaking regime allowed by the incubator, gas/liquid mass transfer remained rate limiting. Success of a tentative biotechnological process therefore likely depends on gas/liquid mass transfer rates of specific reactor types and associated restrictions dictated by process economics.Physiological PerspectiveIn recent years it has become clear that CO is used as a substrate by a diverse group of strict anaerobic micro-organisms. While it previously was believed that CO predominantly inhibited the growth of many anaerobes, albeit at higher partial pressures than aerobes. Currently research has indicated that CO is a versatile substrate which is effectively used in many microbial metabolisms. The advances that were made especially show that many electron acceptors can be reduced by a wide variety of micro organisms with CO as electron donor. The advances could be made due to the fact that CO is generally neglected as a substrate in physiological studies of novel anaerobic isolates. Genome sequencing projects also demonstrate that the enzymes, CO dehydrogenases, involved in the CO metabolism are present in already well known organisms that have never been tested with CO. An example of this is Archaeoglobus fulgidus , which was tested for its ability to grow with CO in Chapter 5. While it was speculated that A. fulgidus could oxidise CO coupled to the reduction of protons to H 2 , it grew acetogenically with CO instead. In fact, A. fulgidus is the first true homo-acetogenic archaeon known. Remarkable of its metabolism was the intermediate accumulation of formate. It was proposed that A. fulgidus forms acetate via the acetyl-CoA pathway. In this pathway, CO 2 is reduced to form the methyl group of acetate. However, formate is not an intermediate in the expected pathway in which CO 2 is reduced to form formyl-methanofuran and subsequently formyl-tertahydromethanopterin. Besides the formation of acetate, A. fulgidus can reduce sulfate to sulfide with CO as electron donor. It distinguishes itself from bacterial sulfate reducers by its tolerance to CO. While most known sulfate-reducing bacteria are inhibited by elevated levels of CO, A. fulgidus was not noticeably inhibited in the presence of 136 kPa CO. Since A. fulgidus is not capable of growth with H 2 and CO 2 or sulfate as electron acceptors, this organism could be employed to selectively oxidise CO in gas mixtures containing H 2 and remove trace amounts of CO to levels below 10 ppm.Selective oxidation of CO is not possible with C. hydrogenoformans . Although C. hydrogenoformans can reduce various electron acceptors with CO, it also does so with H 2 (Chapter 4). C. hydrogenoformans is a true CO specialist. It is able to grow hydrogenogenically with CO, to reduce various other electron acceptors with CO, and to use CO as sole source of energy and carbon. C. hydrogenoformans contains five CO dehydrogenase genes. Besides the identified activities in H 2 formation, in NADPH generation and in autotrophic carbon fixation for three of these CO dehydrogenase, two CO dehydrogenases are still without function (Chapter 5). Conditions that may lead to expression of these CO dehydrogenases are proposed. In nitrate amended cultures a CO dehydrogenase was present, that was not present in cells grown with other substrates. Wu et al. (2005) already indicated that a CO dehydrogenase of C. hydrogenoformans might be involved in oxidative stress response and be expressed under micro aerophilic conditions. Clearly the microbial physiology regarding CO is still incomplete and is more diverse than thought up to now. Further studies are needed in this respect

Archaeoglobus fulgidus couples CO oxidation to sulfate reduction and acetogenesis with transient formate accumulation

The genome sequence of Archaeoglobus fulgidus VC16 encodes three CO dehydrogenase genes. Here we explore the capacity of A.¿fulgidus to use CO as growth substrate. Archaeoglobus fulgidus VC16 was successfully adapted to growth medium that contained sulfate and CO. In the presence of CO and sulfate the culture OD660 increased to 0.41 and sulfide, carbon dioxide, acetate and formate were formed. Accumulation of formate was transient. Similar results, except that no sulfide was formed, were obtained when sulfate was omitted. Hydrogen was never detected. Under the conditions tested, the observed concentrations of acetate (18¿mM) and formate (8.2¿mM) were highest in cultures without sulfate. Proton NMR spectroscopy indicated that CO2, and not CO, is the precursor of formate and the methyl group of acetate. Methylviologen-dependent formate dehydrogenase activity (1.4¿¿mol formate oxidized min¿1¿mg¿1) was detected in cell-free extracts and expected to have a role in formate reuptake. It is speculated that formate formation proceeds through hydrolysis of formyl-methanofuran or formyl-tetrahydromethanopterin. This study demonstrates that A.¿fulgidus can grow chemolithoautotrophically with CO as acetogen, and is not strictly dependent on the presence of sulfate, thiosulfate or other sulfur compounds as electron acceptor

Microbiology of synthesis gas fermentation for biofuel production

A significant portion of biomass sources like straw and wood is poorly degradable and cannot be converted to biofuels by microorganisms. The gasification of this waste material to produce synthesis gas (or syngas) could offer a solution to this problem, as microorganisms that convert CO and H2 (the essential components of syngas) to multicarbon compounds are available. These are predominantly mesophilic microorganisms that produce short-chain fatty acids and alcohols from CO and H2. Additionally, hydrogen can be produced by carboxydotrophic hydrogenogenic bacteria that convert CO and H2O to H2 and CO2. The production of ethanol through syngas fermentation is already available as a commercial process. The use of thermophilic microorganisms for these processes could offer some advantages; however, to date, few thermophiles are known that grow well on syngas and produce organic compounds. The identification of new isolates that would broaden the product range of syngas fermentations is desirable. Metabolic engineering could be employed to broaden the variety of available products, although genetic tools for such engineering are currently unavailable. Nevertheless, syngas fermenting microorganisms possess advantageous characteristics for biofuel production and hold potential for future engineering efforts

Microbial CO conversions with applications in synthesis gas purification and bio-desulfurization.

Recent advances in the field of microbial physiology demonstrate that carbon monoxide is a readily used substrate by a wide variety of anaerobic micro-organisms, and may be employed in novel biotechnological. processes for production of bulk and fine chemicals or in biological treatment of waste streams. Synthesis gas produced from fossil fuels or biomass is rich in hydrogen and carbon monoxide. Conversion of carbon monoxide to hydrogen allows use of synthesis gas in existing hydrogen utilizing processes and is interesting in view of a transition from hydrogen production from fossil fuels to sustainable (CO2-neutral) biomass. The conversion of CO with H2O to CO2 and H-2 is catalyzed by a rapidly increasing group of micro-organisms. Hydrogen is a preferred electron donor in biotechnological desulfurization of wastewaters and flue gases. Additionally, CO is a good alternative electron donor considering the recent isolation of a CO oxidizing, sulfate reducing bacterium. Here we review CO utilization by various anaerobic micro-organisms and their possible role in biotechnological processes, with a focus on hydrogen production and bio-desulfurization

Sulfidogenesis under extremely haloalkaline conditions by Desulfonatronospira thiodismutans gen. nov., sp. nov., and Desulfonatronospira delicata sp. nov. - a novel lineage of Deltaproteobacteria from hypersaline soda lakes

High rates of sulfidogenesis were observed in sediments from hypersaline soda lakes. Anaerobic enrichment cultures at 2 M Na(+) and pH 10 inoculated with sediment samples from these lakes produced sulfide most actively with sulfite and thiosulfate as electron acceptors, and resulted in the isolation of three pure cultures of extremely natronophilic sulfidogenic bacteria. Strain ASO3-1 was isolated using sulfite as a sole substrate, strain AHT 8 with thiosulfate and formate, and strain AHT 6 with thiosulfate and acetate. All strains grew in a mineral soda-based medium by dismutation of either sulfite or thiosulfate, as well as with sulfite, thiosulfate and sulfate as acceptors, and H(2) and simple organic compounds as electron donors. The acetyl-CoA pathway was identified as the pathway for inorganic carbon assimilation by strain ASO3-1. All strains were obligately alkaliphilic, with an optimum at pH 9.5-10, and grew in soda brines containing 1-4 M total Na(+) (optimum at 1.0-2.0 M). The cells accumulated high amounts of the organic osmolyte glycine betaine. They formed a new lineage within the family Desulfohalobiaceae (Deltaproteobacteria), for which the name Desulfonatronospira gen. nov. is proposed. Strains ASO3-1(T) and AHT 8 from Kulunda Steppe formed Desulfonatronospira thiodismutans sp. nov., and strain AHT 6(T) from Wadi al Natrun is suggested as Desulfonatronospira delicata sp. no

Diversity and ecophysiological features of thermophilic carboxydotrophic anaerobes

Both natural and anthropogenic hot environments contain appreciable levels of carbon monoxide (CO). Anaerobic microbial communities play an important role in CO conversion in such environments. CO is involved in a number of redox reactions. It is biotransformed by thermophilic methanogens, acetogens, hydrogenogens, sulfate reducers, and ferric iron reducers. Most thermophilic CO-oxidizing anaerobes have diverse metabolic capacities, but two hydrogenogenic species are obligate carboxydotrophs. Among known thermophilic carboxydotrophic anaerobes, hydrogenogens are most numerous, and based on available data they are most important in CO biotransformation in hot environment