Environmental Breviatea harbour mutualistic Arcobacter epibionts.

Breviatea form a lineage of free living, unicellular protists, distantly related to animals and fungi. This lineage emerged almost one billion years ago, when the oceanic oxygen content was low, and extant Breviatea have evolved or retained an anaerobic lifestyle. Here we report the cultivation of Lenisia limosa, gen. et sp. nov., a newly discovered breviate colonized by relatives of animal-associated Arcobacter. Physiological experiments show that the association of L. limosa with Arcobacter is driven by the transfer of hydrogen and is mutualistic, providing benefits to both partners. With whole-genome sequencing and differential proteomics, we show that an experimentally observed fitness gain of L. limosa could be explained by the activity of a so far unknown type of NAD(P)H-accepting hydrogenase, which is expressed in the presence, but not in the absence, of Arcobacter. Differential proteomics further reveal that the presence of Lenisia stimulates expression of known 'virulence' factors by Arcobacter. These proteins typically enable colonization of animal cells during infection, but may in the present case act for mutual benefit. Finally, re-investigation of two currently available transcriptomic data sets of other Breviatea reveals the presence and activity of related hydrogen-consuming Arcobacter, indicating that mutualistic interaction between these two groups of microbes might be pervasive. Our results support the notion that molecular mechanisms involved in virulence can also support mutualism, as shown here for Arcobacter and Breviatea.

Metaproteomics method to determine carbon sources and assimilation pathways of species in microbial communities.

Measurements of stable carbon isotope ratios (δ13C) are widely used in biology to address questions regarding food sources and metabolic pathways used by organisms. The analysis of these so-called stable isotope fingerprints (SIFs) for microbes involved in biogeochemical cycling and microbiota of plants and animals has led to major discoveries in environmental microbiology. Currently, obtaining SIFs for microbial communities is challenging as the available methods either only provide low taxonomic resolution, such as the use of lipid biomarkers, or are limited in throughput, such as nanoscale secondary ion MS imaging of single cells. Here we present "direct protein-SIF" and the Calis-p software package (https://sourceforge.net/projects/calis-p/), which enable high-throughput measurements of accurate δ13C values for individual species within a microbial community. We benchmark the method using 20 pure culture microorganisms and show that the method reproducibly provides SIF values consistent with gold-standard bulk measurements performed with an isotope ratio mass spectrometer. Using mock community samples, we demonstrate that SIF values can also be obtained for individual species within a microbial community. Finally, a case study of an obligate bacteria-animal symbiosis shows that direct protein-SIF confirms previous physiological hypotheses and can provide unexpected insights into the symbionts' metabolism. This confirms the usefulness of this approach to accurately determine δ13C values for different species in microbial community samples.

Methane oxidation and methylotroph population dynamics in groundwater mesocosms.

Extraction of natural gas from unconventional hydrocarbon reservoirs by hydraulic fracturing raises concerns about methane migration into groundwater. Microbial methane oxidation can be a significant methane sink. Here, we inoculated replicated, sand-packed, continuous mesocosms with groundwater from a field methane release experiment. The mesocosms experienced thirty-five weeks of dynamic methane, oxygen and nitrate concentrations. We determined concentrations and stable isotope signatures of methane, carbon dioxide and nitrate and monitored microbial community composition of suspended and attached biomass. Methane oxidation was strictly dependent on oxygen availability and led to enrichment of 13 C in residual methane. Nitrate did not enhance methane oxidation under oxygen limitation. Methylotrophs persisted for weeks in the absence of methane, making them a powerful marker for active as well as past methane leaks. Thirty-nine distinct populations of methylotrophic bacteria were observed. Methylotrophs mainly occurred attached to sediment particles. Abundances of methanotrophs and other methylotrophs were roughly similar across all samples, pointing at transfer of metabolites from the former to the latter. Two populations of Gracilibacteria (Candidate Phyla Radiation) displayed successive blooms, potentially triggered by a period of methane famine. This study will guide interpretation of future field studies and provides increased understanding of methylotroph ecophysiology.

Critical Assessment of Metagenome Interpretation-a benchmark of metagenomics software.

Methods for assembly, taxonomic profiling and binning are key to interpreting metagenome data, but a lack of consensus about benchmarking complicates performance assessment. The Critical Assessment of Metagenome Interpretation (CAMI) challenge has engaged the global developer community to benchmark their programs on highly complex and realistic data sets, generated from ∼700 newly sequenced microorganisms and ∼600 novel viruses and plasmids and representing common experimental setups. Assembly and genome binning programs performed well for species represented by individual genomes but were substantially affected by the presence of related strains. Taxonomic profiling and binning programs were proficient at high taxonomic ranks, with a notable performance decrease below family level. Parameter settings markedly affected performance, underscoring their importance for program reproducibility. The CAMI results highlight current challenges but also provide a roadmap for software selection to answer specific research questions.

Direct capture and conversion of CO2 from air by growing a cyanobacterial consortium at pH up to 11.2.

Bioenergy with carbon capture and storage (BECCS) is recognized as a potential negative emission technology, needed to keep global warming within safe limits. With current technologies, large-scale implementation of BECCS would compromise food production. Bioenergy derived from phototrophic microorganisms, with direct capture of CO2 from air, could overcome this challenge and become a sustainable way to realize BECCS. Here we present an alkaline capture and conversion system that combines high atmospheric CO2 transfer rates with high and robust phototrophic biomass productivity (15.2 ± 1.0 g/m 2 /d). The system is based on a cyanobacterial consortium, that grows at high alkalinity (0.5 mol/L) and a pH swing between 10.4 and 11.2 during growth and harvest cycles.

The rates and players of denitrification, dissimilatory nitrate reduction to ammonia (DNRA) and anaerobic ammonia oxidation (anammox) in mangrove soils.

Mangroves are ecosystems located in the transition zone between land and sea, characterized by periodic flooding that confer to its unique characteristics. Little is known about the transformation of nutrients that occur during the organic matter degradation in this system. In this study, we monitor the nitrogen transformations in soils from three mangroves with distinct levels of contamination using labeled 15NO3-. We also screened the mangroves metagenomes for the presence of genes that encode enzymes involved in denitrification (nirS, nirK, nosZ, norB and narG), anaerobic oxidation of ammonia (anammox) (hh, hao and hzo) and dissimilatory nitrate reduction to ammonium (DNRA) (nrfA). The transformations of 15NO3- indicated the balance of denitrification over anammox and DNRA in all three mangroves, with lower rates of processes in the mangrove affected by oil contamination. The metagenomic analysis detected 56 sequences related to denitrification, 19 with anammox and 6 with DNRA. Genes related with denitrification were phylogenetically distributed among several groups of bacteria (mainly Gammaproteobacteria). Anammox and DNRA related sequences were affiliated with Planctomycetes and Gammaproteobacteria, respectively. Thus, metagenomic and functional approaches supported the description of denitrification, anammox and DNRA rates in mangrove soils, and identified the major bacterial groups involved in these processes.

Metabolic specialization of denitrifiers in permeable sediments controls N2 O emissions.

Coastal oceans receive large amounts of anthropogenic fixed nitrogen (N), most of which is denitrified in the sediment before reaching the open ocean. Sandy sediments, which are common in coastal regions, seem to play an important role in catalysing this N-loss. Permeable sediments are characterized by advective porewater transport, which supplies high fluxes of organic matter into the sediment, but also leads to fluctuations in oxygen and nitrate concentrations. Little is known about how the denitrifying communities in these sediments are adapted to such fluctuations. Our combined results indicate that denitrification in eutrophied sandy sediments from the world's largest tidal flat system, the Wadden Sea, is carried out by different groups of microorganisms. This segregation leads to the formation of N2 O which is advectively transported to the overlying waters and thereby emitted to the atmosphere. At the same time, the production of N2 O within the sediment supports a subset of Flavobacteriia which appear to be specialized on N2 O reduction. If the mechanisms shown here are active in other coastal zones, then denitrification in eutrophied sandy sediments may substantially contribute to current marine N2 O emissions.

Transient exposure to oxygen or nitrate reveals ecophysiology of fermentative and sulfate-reducing benthic microbial populations.

For the anaerobic remineralization of organic matter in marine sediments, sulfate reduction coupled to fermentation plays a key role. Here, we enriched sulfate-reducing/fermentative communities from intertidal sediments under defined conditions in continuous culture. We transiently exposed the cultures to oxygen or nitrate twice daily and investigated the community response. Chemical measurements, provisional genomes and transcriptomic profiles revealed trophic networks of microbial populations. Sulfate reducers coexisted with facultative nitrate reducers or aerobes enabling the community to adjust to nitrate or oxygen pulses. Exposure to oxygen and nitrate impacted the community structure, but did not suppress fermentation or sulfate reduction as community functions, highlighting their stability under dynamic conditions. The most abundant sulfate reducer in all cultures, related to Desulfotignum balticum, appeared to have coupled both acetate- and hydrogen oxidation to sulfate reduction. We describe a novel representative of the widespread uncultured candidate phylum Fermentibacteria (formerly candidate division Hyd24-12). For this strictly anaerobic, obligate fermentative bacterium, we propose the name 'U Sabulitectum silens' and identify it as a partner of sulfate reducers in marine sediments. Overall, we provide insights into the function of fermentative, as well as sulfate-reducing microbial communities and their adaptation to a dynamic environment.

Molecular mechanism of anaerobic ammonium oxidation.

Two distinct microbial processes, denitrification and anaerobic ammonium oxidation (anammox), are responsible for the release of fixed nitrogen as dinitrogen gas (N(2)) to the atmosphere. Denitrification has been studied for over 100 years and its intermediates and enzymes are well known. Even though anammox is a key biogeochemical process of equal importance, its molecular mechanism is unknown, but it was proposed to proceed through hydrazine (N(2)H(4)). Here we show that N(2)H(4) is produced from the anammox substrates ammonium and nitrite and that nitric oxide (NO) is the direct precursor of N(2)H(4). We resolved the genes and proteins central to anammox metabolism and purified the key enzymes that catalyse N(2)H(4) synthesis and its oxidation to N(2). These results present a new biochemical reaction forging an N-N bond and fill a lacuna in our understanding of the biochemical synthesis of the N(2) in the atmosphere. Furthermore, they reinforce the role of nitric oxide in the evolution of the nitrogen cycle.

Nitrite-driven anaerobic methane oxidation by oxygenic bacteria.

Only three biological pathways are known to produce oxygen: photosynthesis, chlorate respiration and the detoxification of reactive oxygen species. Here we present evidence for a fourth pathway, possibly of considerable geochemical and evolutionary importance. The pathway was discovered after metagenomic sequencing of an enrichment culture that couples anaerobic oxidation of methane with the reduction of nitrite to dinitrogen. The complete genome of the dominant bacterium, named 'Candidatus Methylomirabilis oxyfera', was assembled. This apparently anaerobic, denitrifying bacterium encoded, transcribed and expressed the well-established aerobic pathway for methane oxidation, whereas it lacked known genes for dinitrogen production. Subsequent isotopic labelling indicated that 'M. oxyfera' bypassed the denitrification intermediate nitrous oxide by the conversion of two nitric oxide molecules to dinitrogen and oxygen, which was used to oxidize methane. These results extend our understanding of hydrocarbon degradation under anoxic conditions and explain the biochemical mechanism of a poorly understood freshwater methane sink. Because nitrogen oxides were already present on early Earth, our finding opens up the possibility that oxygen was available to microbial metabolism before the evolution of oxygenic photosynthesis.

Use of highly alkaline conditions to improve cost-effectiveness of algal biotechnology.

Phototrophic microorganisms have been proposed as an alternative to capture carbon dioxide (CO2) and to produce biofuels and other valuable products. Low CO2 absorption rates, low volumetric productivities, and inefficient downstream processing, however, currently make algal biotechnology highly energy intensive, expensive, and not economically competitive to produce biofuels. This mini-review summarizes advances made regarding the cultivation of phototrophic microorganisms at highly alkaline conditions, as well as other innovations oriented toward reducing the energy input into the cultivation and processing stages. An evaluation, in terms of energy requirements and energy return on energy invested, is performed for an integrated high-pH, high-alkalinity growth process that uses biofilms. Performance in terms of productivity and expected energy return on energy invested is presented for this process and is compared to previously reported life cycle assessments (LCAs) for systems at near-neutral pH. The cultivation of alkaliphilic phototrophic microorganisms in biofilms is shown to have a significant potential to reduce both energy requirements and capital costs.

Metagenome from a Spirulina digesting biogas reactor: analysis via binning of contigs and classification of short reads.

BACKGROUND: Anaerobic digestion is a biological process in which a consortium of microorganisms transforms a complex substrate into methane and carbon dioxide. A good understanding of the interactions between the populations that form this consortium can contribute to a successful anaerobic digestion of the substrate. In this study we combine the analysis of the biogas production in a laboratory anaerobic digester fed with the microalgae Spirulina, a protein rich substrate, with the analysis of the metagenome of the consortium responsible for digestion, obtained by high-throughput DNA sequencing. The obtained metagenome was also compared with a metagenome from a full scale biogas plant fed with cellulose rich material. RESULTS: The optimal organic loading rate for the anaerobic digestion of Spirulina was determined to be 4.0 g Spirulina L(-1) day(-1) with a specific biogas production of 350 mL biogas g Spirulina (-1) with a methane content of 68 %. Firmicutes dominated the microbial consortium at 38 % abundance followed by Bacteroidetes, Chloroflexi and Thermotogae. Euryarchaeota represented 3.5 % of the total abundance. The most abundant organism (14.9 %) was related to Tissierella, a bacterium known to use proteinaceous substrates for growth. Methanomicrobiales and Methanosarcinales dominated the archaeal community. Compared to the full scale cellulose-fed digesters, Pfam domains related to protein degradation were more frequently detected and Pfam domains related to cellulose degradation were less frequent in our sample. CONCLUSIONS: The results presented in this study suggest that Spirulina is a suitable substrate for the production of biogas. The proteinaceous substrate appeared to have a selective impact on the bacterial community that performed anaerobic digestion. A direct influence of the substrate on the selection of specific methanogenic populations was not observed.

Denitrification and aerobic respiration, hybrid electron transport chains and co-evolution.

This paper explores the bioenergetics and potential co-evolution of denitrification and aerobic respiration. The advantages and disadvantages of combining these two pathways in a single, hybrid respiratory chain are discussed and the experimental evidence for the co-respiration of nitrate and oxygen is critically reviewed. A scenario for the co-evolution of the two pathways is presented. This article is part of a Special Issue entitled: The evolutionary aspects of bioenergetic systems.

Rapid succession of uncultured marine bacterial and archaeal populations in a denitrifying continuous culture.

Marine denitrification constitutes an important part of the global nitrogen cycle and the diversity, abundance and process rates of denitrifying microorganisms have been the focus of many studies. Still, there is little insight in the ecophysiology of marine denitrifying communities. In this study, a heterotrophic denitrifying community from sediments of a marine intertidal flat active in nitrogen cycling was selected in a chemostat and monitored over a period of 50 days. The chemostat enabled the maintenance of constant and well-defined experimental conditions over the time-course of the experiment. Analysis of the microbial community composition by automated ribosomal intergenic spacer analysis (ARISA), Illumina sequencing and catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) revealed strong dynamics in community composition over time, while overall denitrification by the enrichment culture was stable. Members of the genera Arcobacter, Pseudomonas, Pseudovibrio, Rhodobacterales and of the phylum Bacteroidetes were identified as the dominant denitrifiers. Among the fermenting organisms co-enriched with the denitrifiers was a novel archaeon affiliated with the recently proposed DPANN-superphylum. The pan-genome of populations affiliated to Pseudovibrio encoded a NirK as well as a NirS nitrite reductase, indicating the rare co-occurrence of both evolutionary unrelated nitrite reductases within coexisting subpopulations.

Phylogenetics and environmental distribution of nitric oxide-forming nitrite reductases reveal their distinct functional and ecological roles.

The two evolutionarily unrelated nitric oxide-producing nitrite reductases, NirK and NirS, are best known for their redundant role in denitrification. They are also often found in organisms that do not perform denitrification. To assess the functional roles of the two enzymes and to address the sequence and structural variation within each, we reconstructed robust phylogenies of both proteins with sequences recovered from 6973 isolate and metagenome-assembled genomes and identified 32 well-supported clades of structurally distinct protein lineages. We then inferred the potential niche of each clade by considering other functional genes of the organisms carrying them as well as the relative abundances of each nir gene in 4082 environmental metagenomes across diverse aquatic, terrestrial, host-associated, and engineered biomes. We demonstrate that Nir phylogenies recapitulate ecology distinctly from the corresponding organismal phylogeny. While some clades of the nitrite reductase were equally prevalent across biomes, others had more restricted ranges. Nitrifiers make up a sizeable proportion of the nitrite-reducing community, especially for NirK in marine waters and dry soils. Furthermore, the two reductases showed distinct associations with genes involved in oxidizing and reducing other compounds, indicating that the NirS and NirK activities may be linked to different elemental cycles. Accordingly, the relative abundance and diversity of NirS versus NirK vary between biomes. Our results show the divergent ecological roles NirK and NirS-encoding organisms may play in the environment and provide a phylogenetic framework to distinguish the traits associated with organisms encoding the different lineages of nitrite reductases.

High diversity, abundance, and expression of hydrogenases in groundwater.

Hydrogen may be the most important electron donor available in the subsurface. Here we analyse the diversity, abundance and expression of hydrogenases in 5 proteomes, 25 metagenomes, and 265 amplicon datasets of groundwaters with diverse geochemistry. A total of 1545 new [NiFe]-hydrogenase gene sequences were recovered, which considerably increased the number of sequences (1999) in a widely used database. [NiFe]-hydrogenases were highly abundant, as abundant as the DNA-directed RNA polymerase. The abundance of hydrogenase genes increased with depth from 0 to 129 m. Hydrogenases were present in 481 out of 1245 metagenome-assembled genomes. The relative abundance of microbes with hydrogenases accounted for ~50% of the entire community. Hydrogenases were actively expressed, making up as much as 5.9% of methanogen proteomes. Most of the newly discovered diversity of hydrogenases was in "Group 3b", which has been associated with sulfur metabolism. "Group 3d", facilitating the interconversion of electrons between hydrogen and NAD, was the most abundant and mainly observed in methanotrophs and chemoautotrophs. "Group 3a", associated with methanogenesis, was the most abundant in proteomes. Two newly discovered groups of [NiFe]-hydrogenases, observed in Methanobacteriaceae and Anaerolineaceae, further expanded diversity. Our results highlight the vast diversity, abundance and expression of hydrogenases in groundwaters, suggesting a high potential for hydrogen oxidation in subsurface habitats.

Viral diversity and dynamics and CRISPR-Cas-mediated immunity in a robust alkaliphilic cyanobacterial consortium.

IMPORTANCE: Biotechnology applications utilizing the function of microbial communities have become increasingly important solutions as we strive for sustainable applications. Although viral infections are known to have a significant impact on microbial turnover and nutrient cycling, viral dynamics have remained largely overlooked in these engineered communities. Predatory perturbations to the functional stability of these microbial biotechnology applications must be investigated in order to design more robust applications. In this study, we closely examine virus-microbe dynamics in a model microbial community used in a biotechnology application. Our findings suggest that viral dynamics change significantly with environmental conditions and that microbial immunity may play an important role in maintaining functional stability. We present this study as a comprehensive template for other researchers interested in exploring predatory dynamics in engineered microbial communities.

Autofermentation of alkaline cyanobacterial biomass to enable biorefinery approach.

BACKGROUND: Carbon capture using alkaliphilic cyanobacteria can be an energy-efficient and environmentally friendly process for producing bioenergy and bioproducts. The inefficiency of current harvesting and downstream processes, however, hinders large-scale feasibility. The high alkalinity of the biomass also introduces extra challenges, such as potential corrosion, inhibitory effects, or contamination of the final products. Thus, it is critical to identify low cost and energy-efficient downstream processes. RESULTS: Autofermentation was investigated as an energy-efficient and low-cost biomass pre-treatment method to reduce pH to levels suitable for downstream processes, enabling the conversion of cyanobacterial biomass into hydrogen and organic acids using cyanobacteria's own fermentative pathways. Temperature, initial biomass concentration, and oxygen presence were found to affect yield and distribution of organic acids. Autofermentation of alkaline cyanobacterial biomass was found to be a viable approach to produce hydrogen and organic acids simultaneously, while enabling the successful conversion of biomass to biogas. Between 5.8 and 60% of the initial carbon was converted into organic acids, 8.7-25% was obtained as soluble protein, and 16-72% stayed in the biomass. Interestingly, we found that extensive dewatering is not needed to effectively process the alkaline cyanobacterial biomass. Using natural settling as the only harvesting and dewatering method resulted in a slurry with relatively low biomass concentration. Nevertheless, autofermentation of this slurry led to the maximum total organic acid yield (60% C mol/C mol biomass) and hydrogen yield (326.1 µmol/g AFDM). CONCLUSION: Autofermentation is a simple, but highly effective pretreatment that can play a significant role within a cyanobacterial-based biorefinery platform by enabling the conversion of alkaline cyanobacterial biomass into organic acids, hydrogen, and methane via anaerobic digestion without the addition of energy or chemicals.

Frequency of change determines effectiveness of microbial response strategies.

Nature challenges microbes with change at different frequencies and demands an effective response for survival. Here, we used controlled laboratory experiments to investigate the effectiveness of different response strategies, such as post-translational modification, transcriptional regulation, and specialized versus adaptable metabolisms. For this, we inoculated replicated chemostats with an enrichment culture obtained from sulfidic stream microbiomes 16 weeks prior. The chemostats were submitted to alternatingly oxic and anoxic conditions at three frequencies, with periods of 1, 4 and 16 days. The microbial response was recorded with 16S rRNA gene amplicon sequencing, shotgun metagenomics, transcriptomics and proteomics. Metagenomics resolved provisional genomes of all abundant bacterial populations, mainly affiliated with Proteobacteria and Bacteroidetes. Almost all these populations maintained a steady growth rate under both redox conditions at all three frequencies of change. Our results supported three conclusions: (1) Oscillating oxic/anoxic conditions selected for generalistic species, rather than species specializing in only a single condition. (2) A high frequency of change selected for strong codon usage bias. (3) Alignment of transcriptomes and proteomes required multiple generations and was dependent on a low frequency of change.

Ultra-sensitive isotope probing to quantify activity and substrate assimilation in microbiomes.

BACKGROUND: Stable isotope probing (SIP) approaches are a critical tool in microbiome research to determine associations between species and substrates, as well as the activity of species. The application of these approaches ranges from studying microbial communities important for global biogeochemical cycling to host-microbiota interactions in the intestinal tract. Current SIP approaches, such as DNA-SIP or nanoSIMS allow to analyze incorporation of stable isotopes with high coverage of taxa in a community and at the single cell level, respectively, however they are limited in terms of sensitivity, resolution or throughput. RESULTS: Here, we present an ultra-sensitive, high-throughput protein-based stable isotope probing approach (Protein-SIP), which cuts cost for labeled substrates by 50-99% as compared to other SIP and Protein-SIP approaches and thus enables isotope labeling experiments on much larger scales and with higher replication. The approach allows for the determination of isotope incorporation into microbiome members with species level resolution using standard metaproteomics liquid chromatography-tandem mass spectrometry (LC-MS/MS) measurements. At the core of the approach are new algorithms to analyze the data, which have been implemented in an open-source software ( https://sourceforge.net/projects/calis-p/ ). We demonstrate sensitivity, precision and accuracy using bacterial cultures and mock communities with different labeling schemes. Furthermore, we benchmark our approach against two existing Protein-SIP approaches and show that in the low labeling range used our approach is the most sensitive and accurate. Finally, we measure translational activity using 18O heavy water labeling in a 63-species community derived from human fecal samples grown on media simulating two different diets. Activity could be quantified on average for 27 species per sample, with 9 species showing significantly higher activity on a high protein diet, as compared to a high fiber diet. Surprisingly, among the species with increased activity on high protein were several Bacteroides species known as fiber consumers. Apparently, protein supply is a critical consideration when assessing growth of intestinal microbes on fiber, including fiber-based prebiotics. CONCLUSIONS: We demonstrate that our Protein-SIP approach allows for the ultra-sensitive (0.01 to 10% label) detection of stable isotopes of elements found in proteins, using standard metaproteomics data.