Michael J. McInerney

Physiology, Ecology, Phylogeny, and Genomics of Microorganisms Capable of Syntrophic Metabolism

Syntrophic metabolism is diverse in two respects: phylogenetically with microorganisms capable of syntrophic metabolism found in the Deltaproteobacteria and in the low G+C gram‐positive bacteria, and metabolically given the wide variety of compounds that can be syntrophically metabolized. The latter includes saturated fatty acids, unsaturated fatty acids, alcohols, and hydrocarbons. Besides residing in freshwater and marine anoxic sediments and soils, microbes capable of syntrophic metabolism also have been observed in more extreme habitats, including acidic soils, alkaline soils, thermal springs, and permanently cold soils, demonstrating that syntrophy is a widely distributed metabolic process in nature. Recent ecological and physiological studies show that syntrophy plays a far larger role in carbon cycling than was previously thought. The availability of the first complete genome sequences for four model microorganisms capable of syntrophic metabolism provides the genetic framework to begin dissecting the biochemistry of the marginal energy economies and interspecies interactions that are characteristic of the syntrophic lifestyle.

Anaerobic microbial metabolism can proceed close to thermodynamic limits

Many fermentative bacteria obtain energy for growth by reactions in which the change in free energy (ΔG′) is less than that needed to synthesize ATP. These bacteria couple substrate metabolism directly to ATP synthesis, however, by classical phosphoryl transfer reactions. An explanation for the energy economy of these organisms is that biological systems conserve energy in discrete amounts, with a minimum, biochemically convertible energy value of about -20 kJ mol-1 (refs 1, 2, 3). This concept predicts that anaerobic substrate decay ceases before the minimum free energy value is reached, and several studies support this prediction. Here we show that metabolism by syntrophic associations, in which the degradation of a substrate by one species is thermodynamically possible only through removal of the end product by another species, can occur at values close to thermodynamic equilibrium (ΔG′ ≈ 0 kJ mol-1). The free energy remaining when substrate metabolism halts is not constant; it depends on the terminal electron-accepting reaction and the amount of energy required for substrate activation. Syntrophic associations metabolize near thermodynamic equilibrium, indicating that bacteria operate extremely efficient catabolic systems.

The genome of Syntrophus aciditrophicus: Life at the thermodynamic limit of microbial growth

Biochemically, the syntrophic bacteria constitute the missing link in our understanding of anaerobic flow of carbon in the biosphere. The completed genome sequence of Syntrophus aciditrophicus SB, a model fatty acid- and aromatic acid-degrading syntrophic bacterium, provides a glimpse of the composition and architecture of the electron transfer and energy-transducing systems needed to exist on marginal energy economies of a syntrophic lifestyle. The genome contains 3,179,300 base pairs and 3,169 genes where 1,618 genes were assigned putative functions. Metabolic reconstruction of the gene inventory revealed that most biosynthetic pathways of a typical Gram-negative microbe were present. A distinctive feature of syntrophic metabolism is the need for reverse electron transport; the presence of a unique Rnf-type ion-translocating electron transfer complex, menaquinone, and membrane-bound Fe-S proteins with associated heterodisulfide reductase domains suggests mechanisms to accomplish this task. Previously undescribed approaches to degrade fatty and aromatic acids, including multiple AMP-forming CoA ligases and acyl-CoA synthetases seem to be present as ways to form and dissipate ion gradients by using a sodium-based energy strategy. Thus, S. aciditrophicus, although nutritionally self-sufficient, seems to be a syntrophic specialist with limited fermentative and respiratory metabolism. Genomic analysis confirms the S. aciditrophicus metabolic and regulatory commitment to a nonconventional mode of life compared with our prevailing understanding of microbiology.

Genomic insights into syntrophy: the paradigm for anaerobic metabolic cooperation.

Syntrophy is a tightly coupled mutualistic interaction between hydrogen-/formate-producing and hydrogen-/formate-using microorganisms that occurs throughout the microbial world. Syntrophy is essential for global carbon cycling, waste decomposition, and biofuel production. Reverse electron transfer, e.g., the input of energy to drive critical redox reactions, is a defining feature of syntrophy. Genomic analyses indicate multiple systems for reverse electron transfer, including ion-translocating ferredoxin:NAD(+) oxidoreductase and hydrogenases, two types of electron transfer flavoprotein:quinone oxidoreductases, and other quinone reactive complexes. Confurcating hydrogenases that couple the favorable production of hydrogen from reduced ferredoxin with the unfavorable production of hydrogen from NADH are present in almost all syntrophic metabolizers, implicating their critical role in syntrophy. Transcriptomic analysis shows upregulation of many genes without assigned functions in the syntrophic lifestyle. High-throughput technologies provide insight into the mechanisms used to establish and maintain syntrophic consortia and conserve energy from reactions that operate close to thermodynamic equilibrium.

Syntrophus aciditrophicus sp. nov., a new anaerobic bacterium that degrades fatty acids and benzoate in syntrophic association with hydrogen-using microorganisms.

Abstract Strain SBT is a new, strictly anaerobic, gram-negative, nonmotile, non-sporeforming, rod-shaped bacterium that degrades benzoate and certain fatty acids in syntrophic association with hydrogen/formate-using microorganisms. Strain SBT produced approximately 3 mol of acetate and 0.6 mol of methane per mol of benzoate in coculture with Methanospirillum hungatei strain JF1. Saturated fatty acids, some unsaturated fatty acids, and methyl esters of butyrate and hexanoate also supported growth of strain SBT in coculture with Desulfovibrio strain G11. Strain SBT grew in pure culture with crotonate, producing acetate, butyrate, caproate, and hydrogen. The molar growth yield was 17 ± 1 g cell dry mass per mol of crotonate. Strain SBT did not grow with fumarate, iron(III), polysulfide, or oxyanions of sulfur or nitrogen as electron acceptors with benzoate as the electron donor. The DNA base composition of strain SBT was 43.1 mol% G+C. Analysis of the 16 S rRNA gene sequence placed strain SBT in the δ-subdivision of the Proteobacteria, with sulfate-reducing bacteria. Strain SBT was most closely related to members of the genus Syntrophus. The clear phenotypic and genotypic differences between strain SBT and the two described species in the genus Syntrophus justify the formation of a new species, Syntrophus aciditrophicus.

Properties of the biosurfactant produced byBacillus licheniformis strain JF-2

SummaryThe activity of the biosurfactant produced byBacillus licheniformis strain JF-2 was quantified using a unit defined as the amount of the acid-precipitated biosurfactant that lowered the surface tension by 10 mN/m. One unit was equivalent to 37 μg/ml of the acid-precipitated biosurfactant. Acid precipitation was very effective in the removal of the biosurfactant from the spent medium. Among the solvents tested methanol was the most efficient in extracting the surfactant activity from acid-precipitated material. Thin-layer chromatography of the acid-precipitated biosurfactant revealed four components, two of which contained a lipid moiety and one of which contained an amino group. The methanol-soluble fraction also contained these four components. Studies suggested that all four components were needed for full activity. The lowest interfacial tensions against octane were observed when NaCl concentrations were 50 g/l or greater. Calcium concentrations greater than 25 g/l significantly increased the interfacial tension

In Situ Biosurfactant Production by Bacillus Strains Injected into a Limestone Petroleum Reservoir

ABSTRACT Biosurfactant-mediated oil recovery may be an economic approach for recovery of significant amounts of oil entrapped in reservoirs, but evidence that biosurfactants can be produced in situ at concentrations needed to mobilize oil is lacking. We tested whether two Bacillus strains that produce lipopeptide biosurfactants can metabolize and produce their biosurfactants in an oil reservoir. Five wells that produce from the same Viola limestone formation were used. Two wells received an inoculum (a mixture of Bacillus strain RS-1 and Bacillus subtilis subsp. spizizenii NRRL B-23049) and nutrients (glucose, sodium nitrate, and trace metals), two wells received just nutrients, and one well received only formation water. Results showed in situ metabolism and biosurfactant production. The average concentration of lipopeptide biosurfactant in the produced fluids of the inoculated wells was about 90 mg/liter. This concentration is approximately nine times the minimum concentration required to mobilize entrapped oil from sandstone cores. Carbon dioxide, acetate, lactate, ethanol, and 2,3-butanediol were detected in the produced fluids of the inoculated wells. Only CO2 and ethanol were detected in the produced fluids of the nutrient-only-treated wells. Microbiological and molecular data showed that the microorganisms injected into the formation were retrieved in the produced fluids of the inoculated wells. We provide essential data for modeling microbial oil recovery processes in situ, including growth rates (0.06 ± 0.01 h−1), carbon balances (107% ± 34%), biosurfactant production rates (0.02 ± 0.001 h−1), and biosurfactant yields (0.015 ± 0.001 mol biosurfactant/mol glucose). The data demonstrate the technical feasibility of microbial processes for oil recovery.

Anaerobic benzene biodegradation--a new era.

Benzene is biodegraded in the absence of oxygen under a variety of terminal electron-accepting conditions. However, the mechanism by which anaerobic benzene degradation occurs is unclear. Phenol and benzoate have been consistently detected as intermediates of anaerobic benzene degradation, suggesting that the hydroxylation of benzene to phenol is one of the initial steps in anaerobic benzene degradation. The conversion of phenol to benzoate could then occur by the carboxylation of phenol to form 4-hydroxybenzoate followed by the reductive removal of the hydroxyl group to form benzoate. 13C-Labeling studies suggest that the carboxyl carbon of benzoate is derived from one of the carbons of benzene. Although the fumarate addition reaction is commonly used to activate many hydrocarbons for anaerobic degradation, the large activation energy required to remove hydrogen from the benzene ring argues against such an approach for anaerobic benzene metabolism. The alkylation of benzene to toluene has been detected in several mammalian tissues, and offers an interesting alternate hypothesis for anaerobic benzene degradation in microbial systems. In support of this, anaerobic benzene degradation by Dechloromonas strain RCB, the only known species to degrade benzene in the absence of oxygen, is stimulated by the addition of vitamin B12 and inhibited by the addition of propyl iodide which is consistent with the involvement of a corrinoid enzymatic step. Alkylation of benzene to toluene is also consistent with labeling data that suggests that the carboxyl carbon of benzoate is derived from one of the benzene carbons. However, it is difficult to envision how phenol would be formed if benzene is alkylated to toluene. As such, it is possible that diverse mechanisms for anaerobic benzene degradation may be operative in different anaerobic microorganisms.

Geovibrio ferrireducens, a phylogenetically distinct dissimilatory Fe(III)-reducing bacterium

Abstract A new, phylogenetically distinct, dissimilatory, Fe(III)-reducing bacterium was isolated from surface sediment of a hydrocarbon-contaminated ditch. The isolate, designated strain PAL-1, was an obligately anaerobic, non-fermentative, motile, gram-negative vibrio. PAL-1 grew in a defined medium with acetate as electron donor and ferric pyrophosphate, ferric oxyhydroxide, ferric citrate, Co(III)-EDTA, or elemental sulfur as sole electron acceptor. PAL-1 also used proline, hydrogen, lactate, propionate, succinate, fumarate, pyruvate, or yeast extract as electron donors for Fe(III) reduction. It is the first bacterium known to couple the oxidation of an amino acid to Fe(III) reduction. PAl-1 did not reduce oxygen, Mn(IV), U(VI), Cr(VI), nitrate, sulfate, sulfite, or thiosulfate with acetate as the electron donor. Cell suspensions of PAL-1 exhibited dithionite-reduced minus air-oxidized difference spectra that were characteristic of c-type cytochromes. Analysis of the 16S rRNA gene sequence of PAL-1 showed that the strain is not related to any of the described metal-reducing bacteria in the Proteobacteria and, together with Flexistipes sinusarabici, forms a separate line of descent within the Bacteria. Phenotypically and phylogenetically, strain PAl-1 differs from all other described bacteria, and represents the type strain of a new genus and species, Geovibrioferrireducens.

The genome sequence of Desulfatibacillum alkenivorans AK-01: a blueprint for anaerobic alkane oxidation

Desulfatibacillum alkenivorans AK-01 serves as a model organism for anaerobic alkane biodegradation because of its distinctive biochemistry and metabolic versatility. The D. alkenivorans genome provides a blueprint for understanding the genetic systems involved in alkane metabolism including substrate activation, CoA ligation, carbon-skeleton rearrangement and decarboxylation. Genomic analysis suggested a route to regenerate the fumarate needed for alkane activation via methylmalonyl-CoA and predicted the capability for syntrophic alkane metabolism, which was experimentally verified. Pathways involved in the oxidation of alkanes, alcohols, organic acids and n-saturated fatty acids coupled to sulfate reduction and the ability to grow chemolithoautotrophically were predicted. A complement of genes for motility and oxygen detoxification suggests that D. alkenivorans may be physiologically adapted to a wide range of environmental conditions. The D. alkenivorans genome serves as a platform for further study of anaerobic, hydrocarbon-oxidizing microorganisms and their roles in bioremediation, energy recovery and global carbon cycling.