Connecting biodiversity and potential functional role in modern euxinic environments by microbial metagenomics

Stratified sulfurous lakes are appropriate environments for studying the links between composition and functionality in microbial communities and are potentially modern analogs of anoxic conditions prevailing in the ancient ocean. We explored these aspects in the Lake Banyoles karstic area (NE Spain) through metagenomics and in silico reconstruction of carbon, nitrogen and sulfur metabolic pathways that were tightly coupled through a few bacterial groups. The potential for nitrogen fixation and denitrification was detected in both autotrophs and heterotrophs, with a major role for nitrogen and carbon fixations in Chlorobiaceae. Campylobacterales accounted for a large percentage of denitrification genes, while Gallionellales were putatively involved in denitrification, iron oxidation and carbon fixation and may have a major role in the biogeochemistry of the iron cycle. Bacteroidales were also abundant and showed potential for dissimilatory nitrate reduction to ammonium. The very low abundance of genes for nitrification, the minor presence of anammox genes, the high potential for nitrogen fixation and mineralization and the potential for chemotrophic CO₂ fixation and CO oxidation all provide potential clues on the anoxic zones functioning. We observed higher gene abundance of ammonia-oxidizing bacteria than ammonia-oxidizing archaea that may have a geochemical and evolutionary link related to the dominance of Fe in these environments. Overall, these results offer a more detailed perspective on the microbial ecology of anoxic environments and may help to develop new geochemical proxies to infer biology and chemistry interactions in ancient ecosystems.     

Unveiling microbial life in the new deep-sea hypersaline Lake Thetis. Part II: a metagenomic study

So far only little is known about the microbial ecology of Mediterranean deep-sea hypersaline anoxic lakes (DHALs). These brine lakes were formed by evaporite dissolution/brine seeps and are important model environments to provide insights into possible metabolisms and distributions of microorganisms on the early Earth. Our study on the Lake Thetis, a new thalassohaline DHAL located South-East of the Medriff Corridor, has revealed microbial communities of contrasting compositions with a high number of novel prokaryotic candidate divisions. The major finding of our present work is co-occurrence of at least three autotrophic carbon dioxide fixation pathways in the brine-seawater interface that are likely fuelled by an active ramified sulphur cycle. Genes for the reductive acetyl-CoA and reductive TCA pathways were also found in the brine suggesting that these pathways are operational even at extremely elevated salinities and that autotrophy is more important in hypersaline environments than previously assumed. Surprisingly, genes coding for RuBisCo were found in the highly reduced brine. Three types of sulphide oxidation pathways were found in the interface. The first involves a multienzyme Sox complex catalysing the complete oxidation of reduced sulphur compounds to sulphate, the second type recruits SQR sulphide:quinone reductase for oxidation of sulphide to elemental sulphur, which, in the presence of sulphide, could further be reduced by polysulphide reductases in the third pathway. The presence of the latter two allows a maximal energy yield from the oxidation of sulphide and at the same time prevents the acidification and the accumulation of S(0) deposits. Amino acid composition analysis of deduced proteins revealed a significant overrepresentation of acidic residues in the brine compared with the interface. This trait is typical for halophilic organisms as an adaptation to the brine's extreme hypersalinity. This work presents the first metagenomic survey of the microbial communities of the recently discovered Lake Thetis whose brine constitutes one of saltiest water bodies ever reported.     

Computation of metabolic fluxes and efficiencies for biological carbon dioxide fixation

With rising energy prices and concern over the environmental impact of fossil fuel consumption, the push to develop biomass derived fuels has increased significantly. Although most global carbon fixation occurs via the Calvin Benson Bassham cycle, there are currently five other known pathways for carbon fixation; the goal of this study was to determine the thermodynamic efficiencies of all six carbon fixation pathways for the production of biomass using flux balance analysis. The three chemotrophic pathways, the reductive acetyl-CoA pathway, the 3-hydroxypropionate/4-hydroxybutyrate cycle and the dicarboxylate/4-hydroxybutyrate cycle, were found to be more efficient than photoautotrophic carbon fixation pathways. However, as hydrogen is not freely available, the energetic cost of hydrogen production from sunlight was calculated and included in the overall energy demand, which results in a 5 fold increase in the energy demand of chemoautotrophic carbon fixation. Therefore, when the cost of hydrogen production is included, photoautotrophic pathways are more efficient. However, the energetic cost for the production of 12 metabolic precursors was found to vary widely across the different carbon fixation pathways; therefore, different pathways may be more efficient at producing products from a single precursor than others. The results of this study have significant impact on the selection or design of autotrophic organisms for biofuel or biochemical production. Overall biomass production from solar energy is most efficient in organisms using the reductive TCA cycle, however, products derived from one metabolic precursor may be more efficiently produced using other carbon fixation pathways.     

Is the distribution of nitrogen‐fixing cyanobacteria in the oceans related to temperature?

Approximately 50% of the global natural fixation of nitrogen occurs in the oceans supporting a considerable part of the new primary production. Virtually all nitrogen fixation in the ocean occurs in the tropics and subtropics where the surface water temperature is 25°C or higher. It is attributed almost exclusively to cyanobacteria. This is remarkable firstly because diazotrophic cyanobacteria are found in other environments irrespective of temperature and secondly because primary production in temperate and cold oceans is generally limited by nitrogen. Cyanobacteria are oxygenic phototrophic organisms that evolved a variety of strategies protecting nitrogenase from oxygen inactivation. Free-living diazotrophic cyanobacteria in the ocean are of the non-heterocystous type, namely the filamentous Trichodesmium and the unicellular groups A–C. I will argue that warm water is a prerequisite for these diazotrophic organisms because of the low-oxygen solubility and high rates of respiration allowing the organism to maintain anoxic conditions in the nitrogen-fixing cell. Heterocystous cyanobacteria are abundant in freshwater and brackish environments in all climatic zones. The heterocyst cell envelope is a tuneable gas diffusion barrier that optimizes the influx of both oxygen and nitrogen, while maintaining anoxic conditions inside the cell. It is not known why heterocystous cyanobacteria are absent from the temperate and cold oceans and seas.     

Carbon isotopic fractionations associated with thermophilic bacteria Thermotoga maritima and Persephonella marina

Stable carbon isotopes can provide insight into carbon cycling pathways in natural environments. We examined carbon isotope fractionations associated with a hyperthermophilic fermentative bacterium, Thermotoga maritima, and a thermophilic chemolithoautotrophic bacterium Persephonella marina. In T. maritima, phospholipid fatty acids (PLFA) are slightly enriched in 13C relative to biomass (epsilon = 0.1-0.8 per thousand). However, PLFA and biomass are depleted in 13C relative to the substrate glucose by approximately 8 per thousand. In P. marina, PLFA are 1.8-14.5 per thousand enriched in 13C relative to biomass, which suggests that the reversed tricarboxylic acid (TCA) cycle or the 3-hydroxypropionate pathway may be used for CO2 fixation. This is supported by small fractionation between biomass and CO2 (epsilon = -3.8 per thousand to -5.0 per thousand), which is similar to fractionations reported for other organisms using similar CO2 fixation pathways. Identification of the exact pathway will require biochemical assay for specific enzymes associated with the reversed TCA cycle or the 3-hydroxypropionate pathway.     

A survey of carbon fixation pathways through a quantitative lens

While the reductive pentose phosphate cycle is responsible for the fixation of most of the carbon in the biosphere, it has several natural substitutes. In fact, due to the characterization of three new carbon fixation pathways in the last decade, the diversity of known metabolic solutions for autotrophic growth has doubled. In this review, the different pathways are analysed and compared according to various criteria, trying to connect each of the different metabolic alternatives to suitable environments or metabolic goals. The different roles of carbon fixation are discussed; in addition to sustaining autotrophic growth it can also be used for energy conservation and as an electron sink for the recycling of reduced electron carriers. Our main focus in this review is on thermodynamic and kinetic aspects, including thermodynamically challenging reactions, the ATP requirement of each pathway, energetic constraints on carbon fixation, and factors that are expected to limit the rate of the pathways. Finally, possible metabolic structures of yet unknown carbon fixation pathways are suggested and discussed.     

可用于二氧化碳捕获过程的微生物碳酸酐酶的挖掘与改造

二氧化碳排放量的急剧上升引起全球温室效应加剧。碳酸酐酶是地球上反应速率最快的几种酶之一,可以大幅提高CO_2捕获和生物矿化的效率,从而降低大气中CO_2的排放量。但捕获过程在高温条件,而CO_2生物矿化形成CaCO_3的过程则需要碱性条件。因此,迫切需要筛选出既嗜热又耐碱的碳酸酐酶以用于CO_2捕获,极端微生物是这类酶的重要来源之一。文中系统、深入地介绍了目前从极端微生物或利用蛋白质工程技术获取嗜热、耐碱的碳酸酐酶的最新研究进展,同时简要介绍了一些新型固定化碳酸酐酶的方法。最后指出当前研究的重点应致力于拓宽寻找碳酸酐酶的范围,改良蛋白质工程改造技术,研发高效廉价、易于放大的固定化方法,为减轻温室效应、延缓全球变暖这一迫切需要解决的问题提供新思路。     

Genomic resolution of a cold subsurface aquifer community provides metabolic insights for novel microbes adapted to high CO2 concentrations

As in many deep underground environments, the microbial communities in subsurface high‐CO₂ ecosystems remain relatively unexplored. Recent investigations based on single‐gene assays revealed a remarkable variety of organisms from little studied phyla in Crystal Geyser (Utah, USA), a site where deeply sourced CO₂‐saturated fluids are erupted at the surface. To provide genomic resolution of the metabolisms of these organisms, we used a novel metagenomic approach to recover 227 high‐quality genomes from 150 microbial species affiliated with 46 different phylum‐level lineages. Bacteria from two novel phylum‐level lineages have the capacity for CO₂ fixation. Analyses of carbon fixation pathways in all studied organisms revealed that the Wood‐Ljungdahl pathway and the Calvin‐Benson‐Bassham Cycle occurred with the highest frequency, whereas the reverse TCA cycle was little used. We infer that this, and selection for form II RuBisCOs, are adaptions to high CO₂‐concentrations. However, many autotrophs can also grow mixotrophically, a strategy that confers metabolic versatility. The assignment of 156 hydrogenases to 90 different organisms suggests that H₂ is an important inter‐species energy currency even under gaseous CO₂‐saturation. Overall, metabolic analyses at the organism level provided insight into the biochemical cycles that support subsurface life under the extreme condition of CO₂ saturation.     

Ancient biochemistries and the evolution of parasites

The characteristic respiratory metabolism of parasites consists of fermentation to carbon-rich, highly reduced volatile fatty acids which are excreted, and electron transport systems emphasising fumarate reductase and b-type cytochromes. The taxonomic groups that contribute major parasites (the heterogeneous protozoa and the helminths) have their evolutionary origins in environments from which oxygen was absent or present in very low concentrations. The Ediacarian period, about 700 million years ago, contains fossils of the appropriate grade of organisation to be contemporaneous with the ancestors of platyhelmiths, nematodes and acanthocephalans. With the oxygen transition, carbon flow in the biosphere resulted in conservative, anoxic environments together with oxygen rich ones. The organisms of the former retained their emphasis on anaerobic energy generation, while cytochrome systems were as much concerned with oxygen detoxification as energy generation. Metabolic pathways in the modern parasitic groups are echoes of such ancient biochemistries.     

Life in hot acid: pathway analyses in extremely thermoacidophilic archaea

The extremely thermoacidophilic archaea are a particularly intriguing group of microorganisms that must simultaneously cope with biologically extreme pHs (< or = 4) and temperatures (Topt > or = 60 degrees C) in their natural environments. Their expanding biotechnological significance relates to their role in biomining of base and precious metals and their unique mechanisms of survival in hot acid, at both the cellular and biomolecular levels. Recent developments, such as advances in understanding of heavy metal tolerance mechanisms, implementation of a genetic system, and discovery of a new carbon fixation pathway, have been facilitated by the availability of genome sequence data and molecular genetic systems. As a result, new insights into the metabolic pathways and physiological features that define extreme thermoacidophily have been obtained, in some cases suggesting prospects for biotechnological opportunities.     

Thermodynamic constraints shape the structure of carbon fixation pathways

Thermodynamics impose a major constraint on the structure of metabolic pathways. Here, we use carbon fixation pathways to demonstrate how thermodynamics shape the structure of pathways and determine the cellular resources they consume. We analyze the energetic profile of prototypical reactions and show that each reaction type displays a characteristic change in Gibbs energy. Specifically, although carbon fixation pathways display a considerable structural variability, they are all energetically constrained by two types of reactions: carboxylation and carboxyl reduction. In fact, all adenosine triphosphate (ATP) molecules consumed by carbon fixation pathways - with a single exception - are used, directly or indirectly, to power one of these unfavorable reactions. When an indirect coupling is employed, the energy released by ATP hydrolysis is used to establish another chemical bond with high energy of hydrolysis, e.g. a thioester. This bond is cleaved by a downstream enzyme to energize an unfavorable reaction. Notably, many pathways exhibit reduced ATP requirement as they couple unfavorable carboxylation or carboxyl reduction reactions to exergonic reactions other than ATP hydrolysis. In the most extreme example, the reductive acetyl coenzyme A (acetyl-CoA) pathway bypasses almost all ATP-consuming reactions. On the other hand, the reductive pentose phosphate pathway appears to be the least ATP-efficient because it is the only carbon fixation pathway that invests ATP in metabolic aims other than carboxylation and carboxyl reduction. Altogether, our analysis indicates that basic thermodynamic considerations accurately predict the resource investment required to support a metabolic pathway and further identifies biochemical mechanisms that can decrease this requirement.     

Evidence for autotrophic CO2 fixation via the reductive tricarboxylic acid cycle by members of the epsilon subdivision of proteobacteria

Based on 16S rRNA gene surveys, bacteria of the epsilon subdivision of proteobacteria have been identified to be important members of microbial communities in a variety of environments, and quite a few have been demonstrated to grow autotrophically. However, no information exists on what pathway of autotrophic carbon fixation these bacteria might use. In this study, Thiomicrospira denitrificans and Candidatus Arcobacter sulfidicus, two chemolithoautotrophic sulfur oxidizers of the epsilon subdivision of proteobacteria, were examined for activities of the key enzymes of the known autotrophic CO(2) fixation pathways. Both organisms contained activities of the key enzymes of the reductive tricarboxylic acid cycle, ATP citrate lyase, 2-oxoglutarate:ferredoxin oxidoreductase, and pyruvate:ferredoxin oxidoreductase. Furthermore, no activities of key enzymes of other CO(2) fixation pathways, such as the Calvin cycle, the reductive acetyl coenzyme A pathway, and the 3-hydroxypropionate cycle, could be detected. In addition to the key enzymes, the activities of the other enzymes involved in the reductive tricarboxylic acid cycle could be measured. Sections of the genes encoding the alpha- and beta-subunits of ATP citrate lyase could be amplified from both organisms. These findings represent the first direct evidence for the operation of the reductive tricarboxylic acid cycle for autotrophic CO(2) fixation in epsilon-proteobacteria. Since epsilon-proteobacteria closely related to these two organisms are important in many habitats, such as hydrothermal vents, oxic-sulfidic interfaces, or oilfields, these results suggest that autotrophic CO(2) fixation via the reductive tricarboxylic acid cycle might be more important than previously considered.     

How do vascular plants perform photosynthesis in extreme environments? An integrative ecophysiological and biochemical story

In this work, we review the physiological and molecular mechanisms that allow vascular plants to perform photosynthesis in extreme environments, such as deserts, polar and alpine ecosystems. Specifically, we discuss the morpho/anatomical, photochemical and metabolic adaptive processes that enable a positive carbon balance in photosynthetic tissues under extreme temperatures and/or severe water‐limiting conditions in C₃ species. Nevertheless, only a few studies have described the in situ functioning of photoprotection in plants from extreme environments, given the intrinsic difficulties of fieldwork in remote places. However, they cover a substantial geographical and functional range, which allowed us to describe some general trends. In general, photoprotection relies on the same mechanisms as those operating in the remaining plant species, ranging from enhanced morphological photoprotection to increased scavenging of oxidative products such as reactive oxygen species. Much less information is available about the main physiological and biochemical drivers of photosynthesis: stomatal conductance (g_s), mesophyll conductance (g_m) and carbon fixation, mostly driven by RuBisCO carboxylation. Extreme environments shape adaptations in structures, such as cell wall and membrane composition, the concentration and activation state of Calvin–Benson cycle enzymes, and RuBisCO evolution, optimizing kinetic traits to ensure functionality. Altogether, these species display a combination of rearrangements, from the whole‐plant level to the molecular scale, to sustain a positive carbon balance in some of the most hostile environments on Earth.     

Protein-SIP enables time-resolved analysis of the carbon flux in a sulfate-reducing,benzene-degrading microbial consortium

Benzene is a major contaminant in various environments, but the mechanisms behind its biodegradation under strictly anoxic conditions are not yet entirely clear. Here we analyzed a benzene-degrading, sulfate-reducing enrichment culture originating from a benzene-contaminated aquifer by a metagenome-based functional metaproteomic approach, using protein-based stable isotope probing (protein-SIP). The time-resolved, quantitative analysis of carbon fluxes within the community supplied with either ¹³C-labeled benzene or ¹³C-labeled carbonate yielded different functional groups of organisms, with their peptides showing specific time dependencies of ¹³C relative isotope abundance indicating different carbon utilization. Through a detailed analysis of the mass spectrometric (MS) data, it was possible to quantify the utilization of the initial carbon source and the metabolic intermediates. The functional groups were affiliated to Clostridiales, Deltaproteobacteria and Bacteroidetes/Chlorobi. The Clostridiales-related organisms were involved in benzene degradation, putatively by fermentation, and additionally used significant amounts of carbonate as a carbon source. The other groups of organisms were found to perform diverse functions, with Deltaproteobacteria degrading fermentation products and Bacteroidetes/Chlorobi being putative scavengers feeding on dead cells. A functional classification of identified proteins supported this allocation and gave further insights into the metabolic pathways and the interactions between the community members. This example shows how protein-SIP can be applied to obtain temporal and phylogenetic information about functional interdependencies within microbial communities.     

Ecosystem-Scale Oxygen Manipulations Alter Terminal Electron Acceptor Pathways in a Eutrophic Reservoir

Lakes and reservoirs globally are experiencing unprecedented changes in land use and climate, depleting dissolved oxygen (DO) in the bottom waters (hypolimnia) of these ecosystems. Because DO is the most energetically favorable terminal electron acceptor (TEA) for organic carbon mineralization, its availability controls the onset of alternate TEA pathways (for example, denitrification, manganese reduction, iron reduction, sulfate reduction, methanogenesis). Low DO concentrations can trigger organic carbon mineralization via alternate TEA pathways in the water column and sediments, which has important implications for greenhouse gas production [carbon dioxide (CO₂) and methane (CH₄)]. In this study, we experimentally injected supersaturated DO into the hypolimnion of a eutrophic reservoir and measured concentrations of TEAs and terminal electron products (TEPs) in the experimental reservoir and an upstream reference reservoir. We calculated the electron equivalents yielded from each TEA pathway and estimated the contributions of each TEA pathway to organic carbon processing in both reservoirs. DO additions to the hypolimnion of the experimental reservoir promoted aerobic respiration, suppressing most alternate TEA pathways and resulting in elevated CO₂ accumulation. In comparison, organic carbon mineralization in the reference reservoir’s anoxic hypolimnion was dominated by alternate TEA pathways, resulting in both CH₄ and CO₂ accumulation. Our ecosystem-scale experiments demonstrate that the alternate TEA pathways that succeed aerobic respiration in lakes and reservoirs can be manipulated at the ecosystem scale. Moreover, changes in the DO dynamics of freshwater lakes and reservoirs may result in concomitant changes in the redox reactions in the water column that control organic carbon mineralization and greenhouse gas accumulation.

     

Dark aerobic sulfide oxidation by anoxygenic phototrophs in anoxic waters

Anoxygenic phototrophic sulfide oxidation by green and purple sulfur bacteria (PSB) plays a key role in sulfide removal from anoxic shallow sediments and stratified waters. Although some PSB can also oxidize sulfide with nitrate and oxygen, little is known about the prevalence of this chemolithotrophic lifestyle in the environment. In this study, we investigated the role of these phototrophs in light-independent sulfide removal in the chemocline of Lake Cadagno. Our temporally resolved, high-resolution chemical profiles indicated that dark sulfide oxidation was coupled to high oxygen consumption rates of ~9 μM O₂·h⁻¹. Single-cell analyses of lake water incubated with ¹³CO₂ in the dark revealed that Chromatium okenii was to a large extent responsible for aerobic sulfide oxidation and it accounted for up to 40% of total dark carbon fixation. The genome of Chr. okenii reconstructed from the Lake Cadagno metagenome confirms its capacity for microaerophilic growth and provides further insights into its metabolic capabilities. Moreover, our genomic and single-cell data indicated that other PSB grow microaerobically in these apparently anoxic waters. Altogether, our observations suggest that aerobic respiration may not only play an underappreciated role in anoxic environments but also that organisms typically considered strict anaerobes may be involved.     

Biological treatment of transformer oil using commercial mixtures of microorganisms

The biodegradation of six PCB congeners (IUPAC nos. 28, 52, 101, 138, 153, and 168) present in transformer oil using different commercial mixtures of microorganisms (Sybron 1000, Biozyn 301, Biozyn 300, DBC 5, ChZR, NS 20-10, NS 20-20, and NS 20) under anoxic, oxic, or anoxic/oxic treatments was investigated at laboratory scale. Overall, PCB congener biodegradation was observed in all treatments in the ranges of 0–99%, 2–97% and 40–94% under anoxic, oxic, or anoxic/oxic treatments, respectively. The highest biodegradation of total PCB congeners occurred using Sybron under oxic and anoxic conditions (76.0% and 91.3% reduction, respectively; initial PCB concentration, 1417.1 mg l^?1). Also, the highest biodegradation extent of the PCB congeners occurred when the commercially available mixture of microorganisms, Sybron, was used (84.7% reduction) under combined anoxic/oxic conditions. Demonstration that biodegradation of most of the individual PCB congeners was achieved by the commercial mixtures of microorganisms in this study suggests that PCB-impacted environments can sustain populations of these PCB-metabolizing organisms. This is particularly relevant for the development of biostimulation or bioaugmentation strategies for the bioremediation of PCB-contaminated wastes.     

Stable carbon isotopic fractionations associated with inorganic carbon fixation by anaerobic ammonium-oxidizing bacteria

Isotopic analyses of Candidatus "Brocadia anammoxidans," a chemolithoautotrophic bacterium that anaerobically oxidizes ammonium (anammox), show that it strongly fractionates against (13)C; i.e., lipids are depleted by up to 47 per thousand versus CO(2). Similar results were obtained for the anammox bacterium Candidatus "Scalindua sorokinii," which thrives in the anoxic water column of the Black Sea, suggesting that different anammox bacteria use identical carbon fixation pathways, which may be either the Calvin cycle or the acetyl coenzyme A pathway.     

The first metazoa living in permanently anoxic conditions

Background  

Several unicellular organisms (prokaryotes and protozoa) can live under permanently anoxic conditions. Although a few metazoans can survive temporarily in the absence of oxygen, it is believed that multi-cellular organisms cannot spend their entire life cycle without free oxygen. Deep seas include some of the most extreme ecosystems on Earth, such as the deep hypersaline anoxic basins of the Mediterranean Sea. These are permanently anoxic systems inhabited by a huge and partly unexplored microbial biodiversity.     

代谢工程改造异养微生物固定CO2研究进展

环境保护和能源供应是人类关心的两大问题。能源消耗释放出的温室气体对环境造成了严重影响。利用CO_2固定途径可将CO_2转化成燃料或化学品。天然固碳生物通常存在生长缓慢、固碳效率低等问题。通过在模式微生物中增强或重构CO_2固定途径,实现CO_2的再循环,可提高燃料或化学品的产量,减少温室气体排放。文中详细介绍了通过代谢工程手段改造CO_2固定途径改善化学品生产以及糖合成,阐述了相关代谢途径及其中的关键酶在CO_2固定中的作用,介绍了电生化合成系统的应用,显示出CO_2固定的巨大潜力,并展望了未来CO_2固定的研究方向。