Long-Term Enrichment on Cellulose or Xylan Causes Functional and Taxonomic Convergence of Microbial Communities from Anaerobic Digesters

Cellulose and xylan are two major components of lignocellulosic biomass, which represents a potentially important energy source, as it is abundant and can be converted to methane by microbial action. However, it is recalcitrant to hydrolysis, and the establishment of a complete anaerobic digestion system requires a specific repertoire of microbial functions. In this study, we maintained 2-year enrichment cultures of anaerobic digestion sludge amended with cellulose or xylan to investigate whether a cellulose- or xylan-digesting microbial system could be assembled from sludge previously used to treat neither of them. While efficient methane-producing communities developed under mesophilic (35°C) incubation, they did not under thermophilic (55°C) conditions. Illumina amplicon sequencing results of the archaeal and bacterial 16S rRNA genes revealed that the mature cultures were much lower in richness than the inocula and were dominated by single archaeal (genus Methanobacterium) and bacterial (order Clostridiales) groups, although at finer taxonomic levels the bacteria were differentiated by substrates. Methanogenesis was primarily via the hydrogenotrophic pathway under all conditions, although the identity and growth requirements of syntrophic acetate-oxidizing bacteria were unclear. Incubation conditions (substrate and temperature) had a much greater effect than inoculum source in shaping the mature microbial community, although analysis based on unweighted UniFrac distance found that the inoculum still determined the pool from which microbes could be enriched. Overall, this study confirmed that anaerobic digestion sludge treating nonlignocellulosic material is a potential source of microbial cellulose- and xylan-digesting functions given appropriate enrichment conditions.     

Effect of warming and drought on grassland microbial communities

The soil microbiome is responsible for mediating key ecological processes; however, little is known about its sensitivity to climate change. Observed increases in global temperatures and alteration to rainfall patterns, due to anthropogenic release of greenhouse gases, will likely have a strong influence on soil microbial communities and ultimately the ecosystem services they provide. Therefore, it is vital to understand how soil microbial communities will respond to future climate change scenarios. To this end, we surveyed the abundance, diversity and structure of microbial communities over a 2-year period from a long-term in situ warming experiment that experienced a moderate natural drought. We found the warming treatment and soil water budgets strongly influence bacterial population size and diversity. In normal precipitation years, the warming treatment significantly increased microbial population size 40–150% but decreased diversity and significantly changed the composition of the community when compared with the unwarmed controls. However during drought conditions, the warming treatment significantly reduced soil moisture thereby creating unfavorable growth conditions that led to a 50–80% reduction in the microbial population size when compared with the control. Warmed plots also saw an increase in species richness, diversity and evenness; however, community composition was unaffected suggesting that few phylotypes may be active under these stressful conditions. Our results indicate that under warmed conditions, ecosystem water budget regulates the abundance and diversity of microbial populations and that rainfall timing is critical at the onset of drought for sustaining microbial populations.     

Heterotrophic Archaea Contribute to Carbon Cycling in Low-pH,Suboxic Biofilm Communities

Archaea are widely distributed and yet are most often not the most abundant members of microbial communities. Here, we document a transition from Bacteria- to Archaea-dominated communities in microbial biofilms sampled from the Richmond Mine acid mine drainage (AMD) system (∼pH 1.0, ∼38°C) and in laboratory-cultivated biofilms. This transition occurs when chemoautotrophic microbial communities that develop at the air-solution interface sink to the sediment-solution interface and degrade under microaerobic and anaerobic conditions. The archaea identified in these sunken biofilms are from the class Thermoplasmata, and in some cases, the highly divergent ARMAN nanoarchaeal lineage. In several of the sunken biofilms, nanoarchaea comprise 10 to 25% of the community, based on fluorescent in situ hybridization and metagenomic analyses. Comparative community proteomic analyses show a persistence of bacterial proteins in sunken biofilms, but there is clear evidence for amino acid modifications due to acid hydrolysis. Given the low representation of bacterial cells in sunken biofilms based on microscopy, we infer that hydrolysis reflects proteins derived from lysed cells. For archaea, we detected ∼2,400 distinct proteins, including a subset involved in proteolysis and peptide uptake. Laboratory cultivation experiments using complex carbon substrates demonstrated anaerobic enrichment of Ferroplasma and Aplasma coupled to the reduction of ferric iron. These findings indicate dominance of acidophilic archaea in degrading biofilms and suggest that they play roles in anaerobic nutrient cycling at low pH.     

Metabolically active microbial communities in marine sediment under high-CO2 and low-pH extremes

Sediment-hosting hydrothermal systems in the Okinawa Trough maintain a large amount of liquid, supercritical and hydrate phases of CO₂ in the seabed. The emission of CO₂ may critically impact the geochemical, geophysical and ecological characteristics of the deep-sea sedimentary environment. So far it remains unclear whether microbial communities that have been detected in such high-CO₂ and low-pH habitats are metabolically active, and if so, what the biogeochemical and ecological consequences for the environment are. In this study, RNA-based molecular approaches and radioactive tracer-based respiration rate assays were combined to study the density, diversity and metabolic activity of microbial communities in CO₂-seep sediment at the Yonaguni Knoll IV hydrothermal field of the southern Okinawa Trough. In general, the number of microbes decreased sharply with increasing sediment depth and CO₂ concentration. Phylogenetic analyses of community structure using reverse-transcribed 16S ribosomal RNA showed that the active microbial community became less diverse with increasing sediment depth and CO₂ concentration, indicating that microbial activity and community structure are sensitive to CO₂ venting. Analyses of RNA-based pyrosequences and catalyzed reporter deposition-fluorescence in situ hybridization data revealed that members of the SEEP-SRB2 group within the Deltaproteobacteria and anaerobic methanotrophic archaea (ANME-2a and -2c) were confined to the top seafloor, and active archaea were not detected in deeper sediments (13–30 cm in depth) characterized by high CO₂. Measurement of the potential sulfate reduction rate at pH conditions of 3–9 with and without methane in the headspace indicated that acidophilic sulfate reduction possibly occurs in the presence of methane, even at very low pH of 3. These results suggest that some members of the anaerobic methanotrophs and sulfate reducers can adapt to the CO₂-seep sedimentary environment; however, CO₂ and pH in the deep-sea sediment were found to severely impact the activity and structure of the microbial community.     

Metabolic activity of subterranean microbial communities in deep granitic groundwater supplemented with methane and H2

It was previously concluded that opposing gradients of sulphate and methane, observations of 16S ribosomal DNA sequences displaying great similarity to those of anaerobic methane-oxidizing Archaea and a peak in sulphide concentration in groundwater from a depth of 250–350 m in Olkiluoto, Finland, indicated proper conditions for methane oxidation with sulphate. In the present research, pressure-resistant, gas-tight circulating systems were constructed to enable the investigation of attached and unattached anaerobic microbial populations from a depth of 327 m in Olkiluoto under in situ pressure (2.4 MPa), diversity, dissolved gas and chemistry conditions. Three parallel flow cell cabinets were configured to allow observation of the influence on microbial metabolic activity of 11 mℳ methane, 11 mℳ methane plus 10 mℳ H₂ or 2.1 mℳ O₂ plus 7.9 mℳ N₂ (that is, air). The concentrations of these gases and of organic acids and carbon, sulphur chemistry, pH and E_h, ATP, numbers of cultivable micro-organisms, and total numbers of cells and bacteriophages were subsequently recorded under batch conditions for 105 days. The system containing H₂ and methane displayed microbial reduction of 0.7 mℳ sulphate to sulphide, whereas the system containing only methane resulted in 0.2 mℳ reduced sulphate. The system containing added air became inhibited and displayed no signs of microbial activity. Added H₂ and methane induced increasing numbers of lysogenic bacteriophages per cell. It appears likely that a microbial anaerobic methane-oxidizing process coupled to acetate formation and sulphate reduction may be ongoing in aquifers at a depth of 250–350 m in Olkiluoto.     

Two-way microscale interactions between immigrant bacteria and plant leaf microbiota as revealed by live imaging

The phyllosphere – the aerial parts of plants – is an important microbial habitat that is home to diverse microbial communities. The spatial organization of bacterial cells on leaf surfaces is non-random, and correlates with leaf microscopic features. Yet, the role of microscale interactions between bacterial cells therein is not well understood. Here, we ask how interactions between immigrant bacteria and resident microbiota affect the spatial organization of the combined community. By means of live imaging in a simplified in vitro system, we studied the spatial organization, at the micrometer scale, of the biocontrol agent Pseudomonas fluorescens A506 and the plant pathogen P. syringae B728a when introduced to pear and bean leaf microbiota (the corresponding native plants of these strains). We found significant co-localization of immigrant and resident microbial cells at distances of a few micrometers, for both strains. Interestingly, this co-localization was in part due to preferential attachment of microbiota cells near newly formed P. fluorescens aggregates. Our results indicate that two-way immigrant bacteria – resident microbiota interactions affect the microscale spatial organization of leaf microbiota, and possibly that of other surface-related microbial communities.Subject terms: Microbial ecology, Microbial ecology     

Plasma membrane protrusions mediate host cell–cell fusion induced by Burkholderia thailandensis

Cell–cell fusion is important for biological processes including fertilization, development, immunity, and microbial pathogenesis. Bacteria in the pseudomallei group of the Burkholderia species, including B. thailandensis, spread between host cells by inducing cell–cell fusion. Previous work showed that B. thailandensis-induced cell–cell fusion requires intracellular bacterial motility and a bacterial protein secretion apparatus called the type VI secretion system-5 (T6SS-5), including the T6SS-5 protein VgrG5. However, the cellular-level mechanism of and T6SS-5 proteins important for bacteria-induced cell–cell fusion remained incompletely described. Using live-cell imaging, we found bacteria used actin-based motility to push on the host cell plasma membrane to form plasma membrane protrusions that extended into neighboring cells. Then, membrane fusion occurred within membrane protrusions either proximal to the bacterium at the tip or elsewhere within protrusions. Expression of VgrG5 by bacteria within membrane protrusions was required to promote cell–cell fusion. Furthermore, a second predicted T6SS-5 protein, TagD5, was also required for cell–cell fusion. In the absence of VgrG5 or TagD5, bacteria in plasma membrane protrusions were engulfed into neighboring cells. Our results suggest that the T6SS-5 effectors VgrG5 and TagD5 are secreted within membrane protrusions and act locally to promote membrane fusion.     

Host Gut Motility Promotes Competitive Exclusion within a Model Intestinal Microbiota

The gut microbiota is a complex consortium of microorganisms with the ability to influence important aspects of host health and development. Harnessing this “microbial organ” for biomedical applications requires clarifying the degree to which host and bacterial factors act alone or in combination to govern the stability of specific lineages. To address this issue, we combined bacteriological manipulation and light sheet fluorescence microscopy to monitor the dynamics of a defined two-species microbiota within a vertebrate gut. We observed that the interplay between each population and the gut environment produces distinct spatiotemporal patterns. As a consequence, one species dominates while the other experiences sudden drops in abundance that are well fit by a stochastic mathematical model. Modeling revealed that direct bacterial competition could only partially explain the observed phenomena, suggesting that a host factor is also important in shaping the community. We hypothesized the host determinant to be gut motility, and tested this mechanism by measuring colonization in hosts with enteric nervous system dysfunction due to a mutation in the ret locus, which in humans is associated with the intestinal motility disorder known as Hirschsprung disease. In mutant hosts we found reduced gut motility and, confirming our hypothesis, robust coexistence of both bacterial species. This study provides evidence that host-mediated spatial structuring and stochastic perturbation of communities can drive bacterial population dynamics within the gut, and it reveals a new facet of the intestinal host–microbe interface by demonstrating the capacity of the enteric nervous system to influence the microbiota. Ultimately, these findings suggest that therapeutic strategies targeting the intestinal ecosystem should consider the dynamic physical nature of the gut environment.     

Phylogenetic Analysis of an Anaerobic, Trichlorobenzene-Transforming Microbial Consortium

A culture-independent phylogenetic survey for an anaerobic trichlorobenzene-transforming microbial community was carried out. Small-subunit rRNA genes were PCR amplified from community DNA by using primers specific for Bacteria or Euryarchaeota and were subsequently cloned. Application of a new hybridization-based screening approach revealed 51 bacterial clone families, one of which was closely related to dechlorinating Dehalobacter species. Several clone sequences clustered to rDNA sequences obtained from a molecular study of an anaerobic aquifer contaminated with hydrocarbons and chlorinated solvents (Dojka et al., Appl. Env. Microbiol. 64:3869–3877, 1998).     

A crucial role for profilin-actin in the intracellular motility of Listeria monocytogenes

We have examined the effect of covalently crosslinked profilin–actin (PxA), which closely matches the biochemical properties of ordinary profilin–actin and interferes with actin polymerization in vitro and in vivo, on Listeria monocytogenes motility. PxA caused a marked reduction in bacterial motility, which was accompanied by the detachment of bacterial tails. The effect of PxA was dependent on its binding to proline-rich sequences, as shown by the inability of P_H133SxA, which cannot interact with such sequences, to impair Listeria motility. PxA did not alter the motility of a Listeria mutant that is unable to recruit Ena (Enabled)/VASP (vasodilator-stimulated phosphoprotein) proteins and profilin to its surface. Finally, PxA did not block the initiation of actin-tail formation, indicating that profilin–actin is only required for the elongation of actin filaments at the bacterial surface. Our findings provide further evidence that profilin–actin is important for actin-based processes, and show that it has a key function in Listeria motility.     

Microbial community dynamics and effect of environmental microbial reservoirs on red-backed salamanders (Plethodon cinereus)

Beneficial cutaneous bacteria on amphibians can protect against the lethal disease chytridiomycosis, which has devastated many amphibian species and is caused by the fungus Batrachochytrium dendrobatidis. We describe the diversity of bacteria on red-backed salamanders (Plethodon cinereus) in the wild and the stability of these communities through time in captivity using culture-independent Illumina 16S rRNA gene sequencing. After field sampling, salamanders were housed with soil from the field or sterile media. The captive conditions led to different trajectories of bacterial communities. Eight OTUs present on >90% of salamanders in the field, through time, and in both treatments were defined as the core community, suggesting that some bacteria are closely associated with the host and are independent of an environmental reservoir. One of these taxa, a Pseudomonas sp., was previously cultured from amphibians and found to be antifungal. As all host-associated bacteria were found in the soil reservoir, environmental microbes strongly influence host–microbial diversity and likely regulate the core community. Using PICRUSt, an exploratory bioinformatics tool to predict gene functions, we found that core skin bacteria provided similar gene functions to the entire community. We suggest that future experiments focus on testing whether core bacteria on salamander skin contribute to the observed resistance to chytridiomycosis in this species even under hygenic captive conditions. For disease-susceptible hosts, providing an environmental reservoir with defensive bacteria in captive-rearing programs may improve outcomes by increasing bacterial diversity on threatened amphibians or increasing the likelihood that defensive bacteria are available for colonization.     

Design and Application of a Biosensor for Monitoring Toxicity of Compounds to Eukaryotes

Here we describe an alternative approach to currently used cytotoxicity analyses through applying eukaryotic microbial biosensors. The yeast Saccharomyces cerevisiae was genetically modified to express firefly luciferase, generating a bioluminescent yeast strain. The presence of any toxic chemical that interfered with the cells' metabolism resulted in a quantitative decrease in bioluminescence. In this study, it was demonstrated that the luminescent yeast strain senses chemicals known to be toxic to eukaryotes in samples assessed as nontoxic by prokaryotic biosensors. As the cell wall and adaptive mechanisms of S. cerevisiae cells enhance stability and protect from extremes of pH, solvent exposure, and osmotic shock, these inherent properties were exploited to generate a biosensor that should detect a wide range of both organic and inorganic toxins under extreme conditions.     

Characterization of wheat straw-degrading anaerobic alkali-tolerant mixed cultures from soda lake sediments by molecular and cultivation techniques

Alkaline pretreatment has the potential to enhance the anaerobic digestion of lignocellulosic biomass to biogas. However, the elevated pH of the substrate may require alkalitolerant microbial communities for an effective digestion. Three mixed anaerobic lignocellulolytic cultures were enriched from sediments from two soda lakes with wheat straw as substrate under alkaline (pH 9) mesophilic (37°C) and thermophilic (55°C) conditions. The gas production of the three cultures ceased after 4 to 5 weeks, and the produced gas was composed of carbon dioxide and methane. The main liquid intermediates were acetate and propionate. The physiological behavior of the cultures was stable even after several transfers. The enrichment process was also followed by molecular fingerprinting (terminal restriction fragment length polymorphism) of the bacterial 16S rRNA gene and of the mcrA/mrtA functional gene for methanogens. The main shift in the microbial community composition occurred between the sediment samples and the first enrichment, whereas the structure was stable in the following transfers. The bacterial communities mainly consisted of Sphingobacteriales, Clostridiales and Spirochaeta, but differed at genus level. Methanothermobacter and Methanosarcina genera and the order Methanomicrobiales were predominant methanogenes in the obtained cultures. Additionally, single cellulolytic microorganisms were isolated from enrichment cultures and identified as members of the alkaliphilic or alkalitolerant genera. The results show that anaerobic alkaline habitats harbor diverse microbial communities, which can degrade lignocellulose effectively and are therefore a potential resource for improving anaerobic digestion.     

Cryptic speciation of a pelagic Roseobacter population varying at a few thousand nucleotide sites

A drop of seawater contains numerous microspatial niches at the scale relevant to microbial activities. Examples are abiotic niches such as detrital particles that show different sizes and organic contents, and biotic niches resulting from bacteria–phage and bacteria–phytoplankton interactions. A common practice to investigate the impact of microenvironments on bacterial evolution is to separate the microenvironments physically and compare the bacterial inhabitants from each. It remains poorly understood, however, which microenvironment primarily drives bacterioplankton evolution in the pelagic ocean. By applying a dilution cultivation approach to an undisturbed coastal water sample, we isolate a bacterial population affiliated with the globally dominant Roseobacter group. Although varying at just a few thousand nucleotide sites across the whole genomes, members of this clonal population are diverging into two genetically separated subspecies. Genes underlying speciation are not unique to subspecies but instead clustered at the shared regions that represent ~6% of the genomic DNA. They are primarily involved in vitamin synthesis, motility, oxidative defense, carbohydrate, and amino acid utilization, consistent with the known strategies that roseobacters take to interact with phytoplankton and particles. Physiological assays corroborate that one subspecies outcompetes the other in these traits. Our results indicate that the microenvironments in the pelagic ocean represented by phytoplankton and organic particles are likely important niches that drive the cryptic speciation of the Roseobacter population, though microhabitats contributed by other less abundant pelagic hosts cannot be ruled out.Subject terms: Microbial ecology, Population genetics     

Distinct microbial communities associated with buried soils in the Siberian tundra

Cryoturbation, the burial of topsoil material into deeper soil horizons by repeated freeze–thaw events, is an important storage mechanism for soil organic matter (SOM) in permafrost-affected soils. Besides abiotic conditions, microbial community structure and the accessibility of SOM to the decomposer community are hypothesized to control SOM decomposition and thus have a crucial role in SOM accumulation in buried soils. We surveyed the microbial community structure in cryoturbated soils from nine soil profiles in the northeastern Siberian tundra using high-throughput sequencing and quantification of bacterial, archaeal and fungal marker genes. We found that bacterial abundances in buried topsoils were as high as in unburied topsoils. In contrast, fungal abundances decreased with depth and were significantly lower in buried than in unburied topsoils resulting in remarkably low fungal to bacterial ratios in buried topsoils. Fungal community profiling revealed an associated decrease in presumably ectomycorrhizal (ECM) fungi. The abiotic conditions (low to subzero temperatures, anoxia) and the reduced abundance of fungi likely provide a niche for bacterial, facultative anaerobic decomposers of SOM such as members of the Actinobacteria, which were found in significantly higher relative abundances in buried than in unburied topsoils. Our study expands the knowledge on the microbial community structure in soils of Northern latitude permafrost regions, and attributes the delayed decomposition of SOM in buried soils to specific microbial taxa, and particularly to a decrease in abundance and activity of ECM fungi, and to the extent to which bacterial decomposers are able to act as their functional substitutes.     

BACTERIAL CELL METABOLISM UNDER ANAEROBIC CONDITIONS

Escherichia coli has been cultivated in a peptone water medium saturated continuously with nitrogen by use of a gas train so as to produce anaerobic conditions. Under these circumstances growth was greatly inhibited. Cultures which originally contained 11 million bacteria per cc. showed on the average only 32 million after 5 hours (as compared with 655 million in similar cultures saturated with air). The metabolic activity of the cells in such a culture was greatly reduced by the anaerobic conditions. It actually fell off from 42 mg. x 10^–11 per cell per hour during the 1st hour to 27 mg. during the 2nd hour and rose only to a maximum of 68 during the 3rd hour. Similar cultures saturated with air showed a rise from 37 mg. x 10^–11 during the 1st hour to 123 during the 2nd hour. The addition of glucose to the medium, under aerobic conditions, has been shown in previous studies to cause only a slight increase in bacterial numbers (861 instead of 655 million after the 5th hour). In the cultures aerated with nitrogen, the addition of glucose has no effect during the first hours. There is again a long lag period and a reduced metabolic rate. After the 2nd hour, however, a wholly different phenomenon manifests itself. The bacterial population increases more rapidly than in the anaerobic peptone medium (reaching a maximum of 142 million after 5 hours). This growth is accompanied by an enormous increase in the rate of CO₂ yield, which reaches 211 mg. x 10^–11 per cell per hour during the 4th hour (nearly double the maximum values recorded under aerobic conditions). The same phenomenon is, of course, illustrated by the enormous yield of CO₂ produced by the action of fermenting organisms in carbohydrate media recorded by Anderson (1924) and other students of the obligate anaerobes. We have here, however, a somewhat striking illustration of the distinct type of metabolic activity manifested by a facultative organism under anaerobic conditions in the presence of sugar measured on a cell-per-hour basis. This is a quantitative illustration of the "life without air" described by Pasteur.     

Gut microbiome composition and function in experimental colitis during active disease and treatment-induced remission

Dysregulated immune responses to gut microbes are central to inflammatory bowel disease (IBD), and gut microbial activity can fuel chronic inflammation. Examining how IBD-directed therapies influence gut microbiomes may identify microbial community features integral to mitigating disease and maintaining health. However, IBD patients often receive multiple treatments during disease flares, confounding such analyses. Preclinical models of IBD with well-defined disease courses and opportunities for controlled treatment exposures provide a valuable solution. Here, we surveyed the gut microbiome of the T-bet^−/− Rag2^−/− mouse model of colitis during active disease and treatment-induced remission. Microbial features modified among these conditions included altered potential for carbohydrate and energy metabolism and bacterial pathogenesis, specifically cell motility and signal transduction pathways. We also observed an increased capacity for xenobiotics metabolism, including benzoate degradation, a pathway linking host adrenergic stress with enhanced bacterial virulence, and found decreased levels of fecal dopamine in active colitis. When transferred to gnotobiotic mice, gut microbiomes from mice with active disease versus treatment-induced remission elicited varying degrees of colitis. Thus, our study provides insight into specific microbial clades and pathways associated with health, active disease and treatment interventions in a mouse model of colitis.     

Functionally redundant but dissimilar microbial communities within biogas reactors treating maize silage in co-fermentation with sugar beet silage

Numerous observations indicate a high flexibility of microbial communities in different biogas reactors during anaerobic digestion. Here, we describe the functional redundancy and structural changes of involved microbial communities in four lab-scale continuously stirred tank reactors (CSTRs, 39°C, 12 L volume) supplied with different mixtures of maize silage (MS) and sugar beet silage (SBS) over 80 days. Continuously stirred tank reactors were fed with mixtures of MS and SBS in volatile solid ratios of 1:0 (Continuous Fermenter (CF) 1), 6:1 (CF2), 3:1 (CF3), 1:3 (CF4) with equal organic loading rates (OLR 1.25 kgVS m⁻³ d⁻¹) and showed similar biogas production rates in all reactors. The compositions of bacterial and archaeal communities were analysed by 454 amplicon sequencing approach based on 16S rRNA genes. Both bacterial and archaeal communities shifted with increasing amounts of SBS. Especially pronounced were changes in the archaeal composition towards Methanosarcina with increasing proportion of SBS, while Methanosaeta declined simultaneously. Compositional shifts within the microbial communities did not influence the respective biogas production rates indicating that these communities adapted to environmental conditions induced by different feedstock mixtures. The diverse microbial communities optimized their metabolism in a way that ensured efficient biogas production.