Stephanie A. Eichorst

Advancements in the application of NanoSIMS and Raman microspectroscopy to investigate the activity of microbial cells in soils.

The combined approach of incubating environmental samples with stable isotope-labeled substrates followed by single-cell analyses through high-resolution secondary ion mass spectrometry (NanoSIMS) or Raman microspectroscopy provides insights into the in situ function of microorganisms. This approach has found limited application in soils presumably due to the dispersal of microbial cells in a large background of particles. We developed a pipeline for the efficient preparation of cell extracts from soils for subsequent single-cell methods by combining cell detachment with separation of cells and soil particles followed by cell concentration. The procedure was evaluated by examining its influence on cell recoveries and microbial community composition across two soils. This approach generated a cell fraction with considerably reduced soil particle load and of sufficient small size to allow single-cell analysis by NanoSIMS, as shown when detecting active N2-fixing and cellulose-responsive microorganisms via 15N2 and 13C-UL-cellulose incubations, respectively. The same procedure was also applicable for Raman microspectroscopic analyses of soil microorganisms, assessed via microcosm incubations with a 13C-labeled carbon source and deuterium oxide (D2O, a general activity marker). The described sample preparation procedure enables single-cell analysis of soil microorganisms using NanoSIMS and Raman microspectroscopy, but should also facilitate single-cell sorting and sequencing.

Community dynamics of cellulose‐adapted thermophilic bacterial consortia

Enzymatic hydrolysis of cellulose is a key process in the global carbon cycle and the industrial conversion of biomass to biofuels. In natural environments, cellulose hydrolysis is predominately performed by microbial communities. However, detailed understanding of bacterial cellulose hydrolysis is primarily confined to a few model isolates. Developing models for cellulose hydrolysis by mixed microbial consortia will complement these isolate studies and may reveal new mechanisms for cellulose deconstruction. Microbial communities were adapted to microcrystalline cellulose under aerobic, thermophilic conditions using green waste compost as the inoculum to study cellulose hydrolysis in a microbial consortium. This adaptation selected for three dominant taxa--the Firmicutes, Bacteroidetes and Thermus. A high-resolution profile of community development during the enrichment demonstrated a community transition from Firmicutes to a novel Bacteroidetes population that clusters in the Chitinophagaceae family. A representative strain of this population, strain NYFB, was successfully isolated, and sequencing of a nearly full-length 16S rRNA gene demonstrated that it was only 86% identical compared with other validated strains in the phylum Bacteroidetes. Strain NYFB grew well on soluble polysaccharide substrates, but grew poorly on insoluble polysaccharide substrates. Similar communities were observed in companion thermophilic enrichments on insoluble wheat arabinoxylan, a hemicellulosic substrate, suggesting a common model for deconstruction of plant polysaccharides. Combining observations of community dynamics and the physiology of strain NYFB, a cooperative successional model for polysaccharide hydrolysis by the Firmicutes and Bacteroidetes in the thermophilic cellulolytic consortia is proposed.

Substrate-Specific Development of Thermophilic Bacterial Consortia by Using Chemically Pretreated Switchgrass

ABSTRACT Microbial communities that deconstruct plant biomass have broad relevance in biofuel production and global carbon cycling. Biomass pretreatments reduce plant biomass recalcitrance for increased efficiency of enzymatic hydrolysis. We exploited these chemical pretreatments to study how thermophilic bacterial consortia adapt to deconstruct switchgrass (SG) biomass of various compositions. Microbial communities were adapted to untreated, ammonium fiber expansion (AFEX)-pretreated, and ionic-liquid (IL)-pretreated SG under aerobic, thermophilic conditions using green waste compost as the inoculum to study biomass deconstruction by microbial consortia. After microbial cultivation, gravimetric analysis of the residual biomass demonstrated that both AFEX and IL pretreatment enhanced the deconstruction of the SG biomass approximately 2-fold. Two-dimensional nuclear magnetic resonance (2D-NMR) experiments and acetyl bromide-reactive-lignin analysis indicated that polysaccharide hydrolysis was the dominant process occurring during microbial biomass deconstruction, and lignin remaining in the residual biomass was largely unmodified. Small-subunit (SSU) rRNA gene amplicon libraries revealed that although the dominant taxa across these chemical pretreatments were consistently represented by members of the Firmicutes, the Bacteroidetes, and Deinococcus-Thermus, the abundance of selected operational taxonomic units (OTUs) varied, suggesting adaptations to the different substrates. Combining the observations of differences in the community structure and the chemical and physical structure of the biomass, we hypothesize specific roles for individual community members in biomass deconstruction.

Refining the phylum Chlorobi by resolving the phylogeny and metabolic potential of the representative of a deeply branching, uncultivated lineage

Recent studies have expanded the phylum Chlorobi, demonstrating that the green sulfur bacteria (GSB), the original cultured representatives of the phylum, are a part of a broader lineage whose members have more diverse metabolic capabilities that overlap with members of the phylum Bacteroidetes. The 16S rRNA gene of an uncultivated clone, OPB56, distantly related to the phyla Chlorobi and Bacteroidetes, was recovered from Obsidian Pool in Yellowstone National Park; however, the detailed phylogeny and function of OPB56 and related clones have remained unknown. Culturing of thermophilic bacterial consortia from compost by adaptation to grow on ionic-liquid pretreated switchgrass provided a consortium in which one of the most abundant members, NICIL-2, clustered with OPB56-related clones. Phylogenetic analysis using the full-length 16S rRNA gene from NICIL-2 demonstrated that it was part of a monophyletic clade, referred to as OPB56, distinct from the Bacteroidetes and Chlorobi. A near complete draft genome (>95% complete) was recovered from metagenomic data from the culture adapted to grow on ionic-liquid pretreated switchgrass using an automated binning algorithm, and this genome was used for marker gene-based phylogenetic analysis and metabolic reconstruction. Six additional genomes related to NICIL-2 were reconstructed from metagenomic data sets obtained from thermal springs at Yellowstone National Park and Nevada Great Boiling Spring. In contrast to the 16S rRNA gene phylogenetic analysis, protein phylogenetic analysis was most consistent with the clustering of the Chlorobea, Ignavibacteria and OPB56 into a single phylum level clade. Metabolic reconstruction of NICIL-2 demonstrated a close linkage with the class Ignavibacteria and the family Rhodothermaceae, a deeply branching Bacteroidetes lineage. The combined phylogenetic and functional analysis of the NICIL-2 genome has refined the membership in the phylum Chlorobi and emphasized the close evolutionary and metabolic relationship between the phyla Chlorobi and the Bacteroidetes.

Substrate perturbation alters the glycoside hydrolase activities and community composition of switchgrass‐adapted bacterial consortia

Bacteria modulate glycoside hydrolase expression in response to the changes in the composition of lignocellulosic biomass. The response of switchgrass‐adapted thermophilic bacterial consortia to perturbation with a variety of biomass substrates was characterized to determine if bacterial consortia also responded to changes in biomass composition. Incubation of the switchgrass‐adapted consortia with these alternative substrates produced shifts in glycoside hydrolase activities and bacterial community composition. Substantially increased endoglucanase activity was observed upon incubation with microcrystalline cellulose and trifluororacetic acid‐pretreated switchgrass. In contrast, culturing the microbial consortia with ionic liquid‐pretreated switchgrass increased xylanase activity dramatically. Microbial community analyses of these cultures indicated that the increased endoglucanase activity correlated with an increase in bacteria related to Rhodothermus marinus. Inclusion of simple organic substrates in the culture medium abrogated glycoside hydrolase activity and enriched for bacteria related to Thermus thermophilus. These results demonstrate that the composition of biomass substrates influences the glycoside hydrolase activities and community composition of biomass‐deconstructing bacterial consortia. Biotechnol. Bioeng. 2012; 109:1140–1145.

Peatland Acidobacteria with a dissimilatory sulfur metabolism

Sulfur-cycling microorganisms impact organic matter decomposition in wetlands and consequently greenhouse gas emissions from these globally relevant environments. However, their identities and physiological properties are largely unknown. By applying a functional metagenomics approach to an acidic peatland, we recovered draft genomes of seven novel Acidobacteria species with the potential for dissimilatory sulfite (dsrAB, dsrC, dsrD, dsrN, dsrT, dsrMKJOP) or sulfate respiration (sat, aprBA, qmoABC plus dsr genes). Surprisingly, the genomes also encoded DsrL, which so far was only found in sulfur-oxidizing microorganisms. Metatranscriptome analysis demonstrated expression of acidobacterial sulfur-metabolism genes in native peat soil and their upregulation in diverse anoxic microcosms. This indicated an active sulfate respiration pathway, which, however, might also operate in reverse for dissimilatory sulfur oxidation or disproportionation as proposed for the sulfur-oxidizing Desulfurivibrio alkaliphilus. Acidobacteria that only harbored genes for sulfite reduction additionally encoded enzymes that liberate sulfite from organosulfonates, which suggested organic sulfur compounds as complementary energy sources. Further metabolic potentials included polysaccharide hydrolysis and sugar utilization, aerobic respiration, several fermentative capabilities, and hydrogen oxidation. Our findings extend both, the known physiological and genetic properties of Acidobacteria and the known taxonomic diversity of microorganisms with a DsrAB-based sulfur metabolism, and highlight new fundamental niches for facultative anaerobic Acidobacteria in wetlands based on exploitation of inorganic and organic sulfur molecules for energy conservation.

Application of stable-isotope labelling techniques for the detection of active diazotrophs

Summary Investigating active participants in the fixation of dinitrogen gas is vital as N is often a limiting factor for primary production. Biological nitrogen fixation is performed by a diverse guild of bacteria and archaea (diazotrophs), which can be free‐living or symbionts. Free‐living diazotrophs are widely distributed in the environment, yet our knowledge about their identity and ecophysiology is still limited. A major challenge in investigating this guild is inferring activity from genetic data as this process is highly regulated. To address this challenge, we evaluated and improved several 15N‐based methods for detecting N2 fixation activity (with a focus on soil samples) and studying active diazotrophs. We compared the acetylene reduction assay and the 15N2 tracer method and demonstrated that the latter is more sensitive in samples with low activity. Additionally, tracing 15N into microbial RNA provides much higher sensitivity compared to bulk soil analysis. Active soil diazotrophs were identified with a 15N‐RNA‐SIP approach optimized for environmental samples and benchmarked to 15N‐DNA‐SIP. Lastly, we investigated the feasibility of using SIP‐Raman microspectroscopy for detecting 15N‐labelled cells. Taken together, these tools allow identifying and investigating active free‐living diazotrophs in a highly sensitive manner in diverse environments, from bulk to the single‐cell level.

Genomic Analysis of Xylose Metabolism in Members of the Deinoccocus-Thermus Phylum from Thermophilic Biomass-Deconstructing Bacterial Consortia

Members of the phylum Deinoccocus-Thermus are adapted to grow under extremes of temperature and radiation. Some of these members have broad applications in biotechnology. However, the specific role of members of Deinoccocus-Thermus in plant biomass deconstruction remains largely unknown. Adaptations of thermophilic communities to grow on plant biomass substrates as the sole carbon source have consistently produced consortia with abundant populations affiliated with the Deinoccocus-Thermus. One of these populations was closely related to cultured isolates of Thermus thermophilus, while the second population, termed NIC-1, was distantly related to Truepera radiovictrix. NIC-1 was abundant in adapted cultures grown on xylan-rich substrates, while the T. thermophilus was virtually absent. To begin to understand the origin of this selection, genomic comparisons of xylan and xylose metabolism were undertaken between NIC-1, recovered from the metagenome obtained from an ammonia fiber expansion (AFEX)-pretreated switchgrass-adapted consortium and a T. thermophilus isolate from a related high temperature switchgrass adaptation. While both genomes indicated relatively limited capabilities to hydrolyze xylan, the NIC-1 genome had a putative operon for xylose utilization, while xylose metabolism genes were absent from the T. thermophilus genome. Comparison of multiple T. thermophilus genomes indicated that the genes for xylose metabolism were present on a plasmid in only one strain. Inspection of metagenomic dataset for adapted communities that contain T. thermophilus indicated that the plasmid is present in the T. thermophilus populations but may be lost upon isolation.

A bacterial pioneer produces cellulase complexes that persist through community succession

Cultivation of microbial consortia provides low-complexity communities that can serve as tractable models to understand community dynamics. Time-resolved metagenomics demonstrated that an aerobic cellulolytic consortium cultivated from compost exhibited community dynamics consistent with the definition of an endogenous heterotrophic succession. The genome of the proposed pioneer population, ‘Candidatus Reconcilibacillus cellulovorans’, possessed a gene cluster containing multidomain glycoside hydrolases (GHs). Purification of the soluble cellulase activity from a 300litre cultivation of this consortium revealed that ~70% of the activity arose from the ‘Ca. Reconcilibacillus cellulovorans’ multidomain GHs assembled into cellulase complexes through glycosylation. These remarkably stable complexes have supramolecular structures for enzymatic cellulose hydrolysis that are distinct from cellulosomes. The persistence of these complexes during cultivation indicates that they may be active through multiple cultivations of this consortium and act as public goods that sustain the community. The provision of extracellular GHs as public goods may influence microbial community dynamics in native biomass-deconstructing communities relevant to agriculture, human health and biotechnology.Cultivation of a cellulolytic consortium reveals successional community dynamics and the presence of multidomain glycoside hydrolases assembled into stable complexes distinct from cellulosomes, which are produced by a potential pioneer population.

Genomic insights into the Acidobacteria reveal strategies for their success in terrestrial environments

Summary Members of the phylum Acidobacteria are abundant and ubiquitous across soils. We performed a large‐scale comparative genome analysis spanning subdivisions 1, 3, 4, 6, 8 and 23 (n = 24) with the goal to identify features to help explain their prevalence in soils and understand their ecophysiology. Our analysis revealed that bacteriophage integration events along with transposable and mobile elements influenced the structure and plasticity of these genomes. Low‐ and high‐affinity respiratory oxygen reductases were detected in multiple genomes, suggesting the capacity for growing across different oxygen gradients. Among many genomes, the capacity to use a diverse collection of carbohydrates, as well as inorganic and organic nitrogen sources (such as via extracellular peptidases), was detected – both advantageous traits in environments with fluctuating nutrient environments. We also identified multiple soil acidobacteria with the potential to scavenge atmospheric concentrations of H2, now encompassing mesophilic soil strains within the subdivision 1 and 3, in addition to a previously identified thermophilic strain in subdivision 4. This large‐scale acidobacteria genome analysis reveal traits that provide genomic, physiological and metabolic versatility, presumably allowing flexibility and versatility in the challenging and fluctuating soil environment.