Biological Soil Crust Bacterial Communities Vary Along Climatic and Shrub Cover Gradients Within a Sagebrush Steppe Ecosystem.

Numerous studies have examined bacterial communities in biological soil crusts (BSCs) associated with warm arid to semiarid ecosystems. Few, however, have examined bacterial communities in BSCs associated with cold steppe ecosystems, which often span a wide range of climate conditions and are sensitive to trends predicted by relevant climate models. Here, we utilized Illumina sequencing to examine BSC bacterial communities with respect to climatic gradients (elevation), land management practices (grazing vs. non-grazing), and shrub/intershrub patches in a cold sagebrush steppe ecosystem in southwestern Idaho, United States. Particular attention was paid to shifts in bacterial community structure and composition. BSC bacterial communities, including keystone N-fixing taxa, shifted dramatically with both elevation and shrub-canopy microclimates within elevational zones. BSC cover and BSC cyanobacteria abundance were much higher at lower elevation (warmer and drier) sites and in intershrub areas. Shrub-understory BSCs were significantly associated with several non-cyanobacteria diazotrophic genera, including Mesorhizobium and Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium. High elevation (wetter and colder) sites had distinct, highly diverse, but low-cover BSC communities that were significantly indicated by non-cyanobacterial diazotrophic taxa including families in the order Rhizobiales and the family Frankiaceae. Abiotic soil characteristics, especially pH and ammonium, varied with both elevation and shrub/intershrub level, and were strongly associated with BSC community composition. Functional inference using the PICRUSt pipeline identified shifts in putative N-fixing taxa with respect to both the elevational gradient and the presence/absence of shrub canopy cover. These results add to current understanding of biocrust microbial ecology in cold steppe, serving as a baseline for future mechanistic research.

Electron Transfer to Nitrogenase in Different Genomic and Metabolic Backgrounds.

Nitrogenase catalyzes the reduction of dinitrogen (N2) using low-potential electrons from ferredoxin (Fd) or flavodoxin (Fld) through an ATP-dependent process. Since its emergence in an anaerobic chemoautotroph, this oxygen (O2)-sensitive enzyme complex has evolved to operate in a variety of genomic and metabolic backgrounds, including those of aerobes, anaerobes, chemotrophs, and phototrophs. However, whether pathways of electron delivery to nitrogenase are influenced by these different metabolic backgrounds is not well understood. Here, we report the distribution of homologs of Fds, Flds, and Fd-/Fld-reducing enzymes in 359 genomes of putative N2 fixers (diazotrophs). Six distinct lineages of nitrogenase were identified, and their distributions largely corresponded to differences in the host cells' ability to integrate O2 or light into energy metabolism. The predicted pathways of electron transfer to nitrogenase in aerobes, facultative anaerobes, and phototrophs varied from those in anaerobes at the levels of Fds/Flds used to reduce nitrogenase, the enzymes that generate reduced Fds/Flds, and the putative substrates of these enzymes. Proteins that putatively reduce Fd with hydrogen or pyruvate were enriched in anaerobes, while those that reduce Fd with NADH/NADPH were enriched in aerobes, facultative anaerobes, and anoxygenic phototrophs. The energy metabolism of aerobic, facultatively anaerobic, and anoxygenic phototrophic diazotrophs often yields reduced NADH/NADPH that is not sufficiently reduced to drive N2 reduction. At least two mechanisms have been acquired by these taxa to overcome this limitation and to generate electrons with potentials capable of reducing Fd. These include the bifurcation of electrons or the coupling of Fd reduction to reverse ion translocation.IMPORTANCE Nitrogen fixation supplies fixed nitrogen to cells from a variety of genomic and metabolic backgrounds, including those of aerobes, facultative anaerobes, chemotrophs, and phototrophs. Here, using informatics approaches applied to genomic data, we show that pathways of electron transfer to nitrogenase in metabolically diverse diazotrophic taxa have diversified primarily in response to host cells' acquired ability to integrate O2 or light into their energy metabolism. The acquisition of two key enzyme complexes enabled aerobic and facultatively anaerobic phototrophic taxa to generate electrons of sufficiently low potential to reduce nitrogenase: the bifurcation of electrons via the Fix complex or the coupling of Fd reduction to reverse ion translocation via the Rhodobacter nitrogen fixation (Rnf) complex.

A pathway for biological methane production using bacterial iron-only nitrogenase.

Methane (CH4) is a potent greenhouse gas that is released from fossil fuels and is also produced by microbial activity, with at least one billion tonnes of CH4 being formed and consumed by microorganisms in a single year 1 . Complex methanogenesis pathways used by archaea are the main route for bioconversion of carbon dioxide (CO2) to CH4 in nature2-4. Here, we report that wild-type iron-iron (Fe-only) nitrogenase from the bacterium Rhodopseudomonas palustris reduces CO2 simultaneously with nitrogen gas (N2) and protons to yield CH4, ammonia (NH3) and hydrogen gas (H2) in a single enzymatic step. The amount of CH4 produced by purified Fe-only nitrogenase was low compared to its other products, but CH4 production by this enzyme in R. palustris was sufficient to support the growth of an obligate CH4-utilizing Methylomonas strain when the two microorganisms were grown in co-culture, with oxygen (O2) added at intervals. Other nitrogen-fixing bacteria that we tested also formed CH4 when expressing Fe-only nitrogenase, suggesting that this is a general property of this enzyme. The genomes of 9% of diverse nitrogen-fixing microorganisms from a range of environments encode Fe-only nitrogenase. Our data suggest that active Fe-only nitrogenase, present in diverse microorganisms, contributes CH4 that could shape microbial community interactions.

Unraveling the interactions of the physiological reductant flavodoxin with the different conformations of the Fe protein in the nitrogenase cycle.

Nitrogenase reduces dinitrogen (N2) to ammonia in biological nitrogen fixation. The nitrogenase Fe protein cycle involves a transient association between the reduced, MgATP-bound Fe protein and the MoFe protein and includes electron transfer, ATP hydrolysis, release of Pi, and dissociation of the oxidized, MgADP-bound Fe protein from the MoFe protein. The cycle is completed by reduction of oxidized Fe protein and nucleotide exchange. Recently, a kinetic study of the nitrogenase Fe protein cycle involving the physiological reductant flavodoxin reported a major revision of the rate-limiting step from MoFe protein and Fe protein dissociation to release of Pi Because the Fe protein cannot interact with flavodoxin and the MoFe protein simultaneously, knowledge of the interactions between flavodoxin and the different nucleotide states of the Fe protein is critically important for understanding the Fe protein cycle. Here we used time-resolved limited proteolysis and chemical cross-linking to examine nucleotide-induced structural changes in the Fe protein and their effects on interactions with flavodoxin. Differences in proteolytic cleavage patterns and chemical cross-linking patterns were consistent with known nucleotide-induced structural differences in the Fe protein and indicated that MgATP-bound Fe protein resembles the structure of the Fe protein in the stabilized nitrogenase complex structures. Docking models and cross-linking patterns between the Fe protein and flavodoxin revealed that the MgADP-bound state of the Fe protein has the most complementary docking interface with flavodoxin compared with the MgATP-bound state. Together, these findings provide new insights into the control mechanisms in protein-protein interactions during the Fe protein cycle.

Defining Electron Bifurcation in the Electron-Transferring Flavoprotein Family.

Electron bifurcation is the coupling of exergonic and endergonic redox reactions to simultaneously generate (or utilize) low- and high-potential electrons. It is the third recognized form of energy conservation in biology and was recently described for select electron-transferring flavoproteins (Etfs). Etfs are flavin-containing heterodimers best known for donating electrons derived from fatty acid and amino acid oxidation to an electron transfer respiratory chain via Etf-quinone oxidoreductase. Canonical examples contain a flavin adenine dinucleotide (FAD) that is involved in electron transfer, as well as a non-redox-active AMP. However, Etfs demonstrated to bifurcate electrons contain a second FAD in place of the AMP. To expand our understanding of the functional variety and metabolic significance of Etfs and to identify amino acid sequence motifs that potentially enable electron bifurcation, we compiled 1,314 Etf protein sequences from genome sequence databases and subjected them to informatic and structural analyses. Etfs were identified in diverse archaea and bacteria, and they clustered into five distinct well-supported groups, based on their amino acid sequences. Gene neighborhood analyses indicated that these Etf group designations largely correspond to putative differences in functionality. Etfs with the demonstrated ability to bifurcate were found to form one group, suggesting that distinct conserved amino acid sequence motifs enable this capability. Indeed, structural modeling and sequence alignments revealed that identifying residues occur in the NADH- and FAD-binding regions of bifurcating Etfs. Collectively, a new classification scheme for Etf proteins that delineates putative bifurcating versus nonbifurcating members is presented and suggests that Etf-mediated bifurcation is associated with surprisingly diverse enzymes.IMPORTANCE Electron bifurcation has recently been recognized as an electron transfer mechanism used by microorganisms to maximize energy conservation. Bifurcating enzymes couple thermodynamically unfavorable reactions with thermodynamically favorable reactions in an overall spontaneous process. Here we show that the electron-transferring flavoprotein (Etf) enzyme family exhibits far greater diversity than previously recognized, and we provide a phylogenetic analysis that clearly delineates bifurcating versus nonbifurcating members of this family. Structural modeling of proteins within these groups reveals key differences between the bifurcating and nonbifurcating Etfs.

The Electron Bifurcating FixABCX Protein Complex from Azotobacter vinelandii: Generation of Low-Potential Reducing Equivalents for Nitrogenase Catalysis.

The biological reduction of dinitrogen (N2) to ammonia (NH3) by nitrogenase is an energetically demanding reaction that requires low-potential electrons and ATP; however, pathways used to deliver the electrons from central metabolism to the reductants of nitrogenase, ferredoxin or flavodoxin, remain unknown for many diazotrophic microbes. The FixABCX protein complex has been proposed to reduce flavodoxin or ferredoxin using NADH as the electron donor in a process known as electron bifurcation. Herein, the FixABCX complex from Azotobacter vinelandii was purified and demonstrated to catalyze an electron bifurcation reaction: oxidation of NADH (Em = -320 mV) coupled to reduction of flavodoxin semiquinone (Em = -460 mV) and reduction of coenzyme Q (Em = 10 mV). Knocking out fix genes rendered Δrnf A. vinelandii cells unable to fix dinitrogen, confirming that the FixABCX system provides another route for delivery of electrons to nitrogenase. Characterization of the purified FixABCX complex revealed the presence of flavin and iron-sulfur cofactors confirmed by native mass spectrometry, electron paramagnetic resonance spectroscopy, and transient absorption spectroscopy. Transient absorption spectroscopy further established the presence of a short-lived flavin semiquinone radical, suggesting that a thermodynamically unstable flavin semiquinone may participate as an intermediate in the transfer of an electron to flavodoxin. A structural model of FixABCX, generated using chemical cross-linking in conjunction with homology modeling, revealed plausible electron transfer pathways to both high- and low-potential acceptors. Overall, this study informs a mechanism for electron bifurcation, offering insight into a unique method for delivery of low-potential electrons required for energy-intensive biochemical conversions.

Oleaginous yeast platform for producing biofuels via co-solvent hydrothermal liquefaction.

BACKGROUND: Oleaginous microorganisms are attractive feedstock for production of liquid biofuels. Direct hydrothermal liquefaction (HTL) is an efficient route that converts whole, wet biomass into an energy-dense liquid fuel precursor, called 'biocrude'. HTL represents a promising alternative to conventional lipid extraction methods as it does not require a dry feedstock or additional steps for lipid extraction. However, high operating pressure in HTL can pose challenges in reactor sizing and overall operating costs. Through the use of co-solvents the HTL operating pressure can be reduced. The present study investigates low-temperature co-solvent HTL of oleaginous yeast, Cryptococcus curvatus, using laboratory batch reactors. RESULTS: In this study, we report the co-solvent HTL of microbial yeast biomass in an isopropanol-water binary system in the presence or absence of Na2CO3 catalyst. This novel approach proved to be effective and resulted in significantly higher yield of biocrude (56.4 ± 0.1 %) than that of HTL performed without a co-solvent (49.1 ± 0.4 %)(p = 0.001). Addition of Na2CO3 as a catalyst marginally improved the biocrude yield. The energy content of the resulting biocrude (~37 MJ kg(-1)) was only slightly lower than that of petroleum crude (42 MJ kg(-1)). The HTL process was successful in removing carboxyl groups from fatty acids and creating their associated straight-chain alkanes (C17-C21). Experimental results were leveraged to inform techno-economic analysis (TEA) of the baseline HTL conversion pathway to evaluate the commercial feasibility of this process. TEA results showed a renewable diesel fuel price of $5.09 per gallon, with the HTL-processing step accounting for approximately 23 % of the total cost for the baseline pathway. CONCLUSIONS: This study shows the feasibility of co-solvent HTL of oleaginous yeast biomass in producing an energy-dense biocrude, and hence provides a platform for adding value to the current dairy industry. Co-solvents can be used to lower the HTL temperature and hence the operating pressure. This process results in a higher biocrude yield at a lower HTL temperature. A conceptual yeast HTL biofuel platform suggests the use of a dairy waste stream for increasing the productivity and sustainability of rural areas while providing a new feedstock (yeast) for generating biofuels.

Techno-economic feasibility and life cycle assessment of dairy effluent to renewable diesel via hydrothermal liquefaction.

The economic feasibility and environmental impact is investigated for the conversion of agricultural waste, delactosed whey permeate, through yeast fermentation to a renewable diesel via hydrothermal liquefaction. Process feasibility was demonstrated at laboratory-scale with data leveraged to validate systems models used to perform industrial-scale economic and environmental impact analyses. Results show a minimum fuel selling price of $4.78 per gallon of renewable diesel, a net energy ratio of 0.81, and greenhouse gas emissions of 30.0g-CO2-eqMJ(-1). High production costs and greenhouse gas emissions can be attributed to operational temperatures and durations of both fermentation and hydrothermal liquefaction. However, high lipid yields of the yeast counter these operational demands, resulting in a favorable net energy ratio. Results are presented on the optimization of the process based on economy of scale and a sensitivity analysis highlights improvements in conversion efficiency, yeast biomass productivity and hydrotreating efficiency can dramatically improve commercial feasibility.

Biogenic mineral production by a novel arsenic-metabolizing thermophilic bacterium from the Alvord Basin, Oregon.

The Alvord Basin in southeast Oregon contains a variety of hydrothermal features which have never been microbiologically characterized. A sampling of Murky Pot (61 degrees C; pH 7.1) led to the isolation of a novel arsenic-metabolizing organism (YeAs) which produces an arsenic sulfide mineral known as beta-realgar, a mineral that has not previously been observed as a product of bacterial arsenic metabolism. YeAs was grown on a freshwater medium and utilized a variety of organic substrates, particularly carbohydrates and organic acids. The temperature range for growth was 37 to 75 degrees C (optimum, 55 degrees C), and the pH range for growth was 6.0 to 8.0 (optimum, pH 7.0 to 7.5). No growth was observed when YeAs was grown under aerobic conditions. The doubling time when the organism was grown with yeast extract and As(V) was 0.71 h. Microscopic examination revealed Gram stain-indeterminate, non-spore-forming, nonmotile, rod-shaped cells, with dimensions ranging from 0.1 to 0.2 microm wide by 3 to 10 microm long. Arsenic sulfide mineralization of cell walls and extracellular arsenic sulfide particulate deposition were observed with electron microscopy and elemental analysis. 16S rRNA gene analysis placed YeAs in the family Clostridiaceae and indicated that the organism is most closely related to the Caloramator and Thermobrachium species. The G+C content was 35%. YeAs showed no detectable respiratory arsenate reductase but did display significant detoxification arsenate reductase activity. The phylogenetic, physiological, and morphological characteristics of YeAs demonstrate that it is an anaerobic, moderately thermophilic, arsenic-reducing bacterium. This organism and its associated metabolism could have major implications in the search for innovative methods for arsenic waste management and in the search for novel biogenic mineral signatures.