Dynamic changes in anaerobic digester metabolic pathways and microbial populations during acclimatisation to increasing ammonium concentrations.

Transitions in microbial community structure in response to increasing ammonia concentrations were determined by monitoring mesophilic anaerobic digesters seeded with a predominantly acetoclastic methanogenic community from a sewage sludge digester. Ammonia concentration was raised by switching the feed to source segregated domestic food waste and applying two organic loading rates (OLR) and hydraulic retention times (HRT) in paired digesters. One of each pair was dosed with trace elements (TE) known to be essential to the transition, with the other unsupplemented digester acting as a control. Samples taken during the trial were used to determine the metabolic pathway to methanogenesis using 14C labelled acetate. Partitioning of 14C between the product gases was interpreted via an equation to indicate the proportion produced by acetoclastic and hydrogenotrophic routes. Archaeal and selected bacterial groups were identified by 16S rRNA sequencing, to determine relative abundance and diversity. Acclimatisation for digesters with TE was relatively smooth, but OLR and HRT influenced both metabolic route and community structure. The 14C ratio could be used quantitatively and, when interpreted alongside archaeal community structure, showed that at longer HRT and lower loading Methanobacteriaceae were dominant and hydrogenotrophic activity accounted for 77% of methane production. At the higher OLR and shorter HRT, Methanosarcinaceae were dominant with the 14C ratio indicating simultaneous production of methane by acetoclastic and hydrogenotrophic pathways: the first reported observation of this in digestion under mesophilic conditions. Digesters without TE supplementation showed similar initial changes but, as expected failed to complete the transition to stable operation.

A multi-omics approach to lignocellulolytic enzyme discovery reveals a new ligninase activity from Parascedosporium putredinis NO1.

Lignocellulose, the structural component of plant cells, is a major agricultural byproduct and the most abundant terrestrial source of biopolymers on Earth. The complex and insoluble nature of lignocellulose limits its conversion into value-added commodities, and currently, efficient transformation requires expensive pretreatments and high loadings of enzymes. Here, we report on a fungus from the Parascedosporium genus, isolated from a wheat-straw composting community, that secretes a large and diverse array of carbohydrate-active enzymes (CAZymes) when grown on lignocellulosic substrates. We describe an oxidase activity that cleaves the major ß-ether units in lignin, thereby releasing the flavonoid tricin from monocot lignin and enhancing the digestion of lignocellulose by polysaccharidase mixtures. We show that the enzyme, which holds potential for the biorefining industry, is widely distributed among lignocellulose-degrading fungi from the Sordariomycetes phylum.

Mechanistic strategies of microbial communities regulating lignocellulose deconstruction in a UK salt marsh.

BACKGROUND: Salt marshes are major natural repositories of sequestered organic carbon with high burial rates of organic matter, produced by highly productive native flora. Accumulated carbon predominantly exists as lignocellulose which is metabolised by communities of functionally diverse microbes. However, the organisms that orchestrate this process and the enzymatic mechanisms employed that regulate the accumulation, composition and permanence of this carbon stock are not yet known. We applied meta-exo-proteome proteomics and 16S rRNA gene profiling to study lignocellulose decomposition in situ within the surface level sediments of a natural established UK salt marsh. RESULTS: Our studies revealed a community dominated by Gammaproteobacteria, Bacteroidetes and Deltaproteobacteria that drive lignocellulose degradation in the salt marsh. We identify 42 families of lignocellulolytic bacteria of which the most active secretors of carbohydrate-active enzymes were observed to be Prolixibacteracea, Flavobacteriaceae, Cellvibrionaceae, Saccharospirillaceae, Alteromonadaceae, Vibrionaceae and Cytophagaceae. These families secreted lignocellulose-active glycoside hydrolase (GH) family enzymes GH3, GH5, GH6, GH9, GH10, GH11, GH13 and GH43 that were associated with degrading Spartina biomass. While fungi were present, we did not detect a lignocellulolytic contribution from fungi which are major contributors to terrestrial lignocellulose deconstruction. Oxidative enzymes such as laccases, peroxidases and lytic polysaccharide monooxygenases that are important for lignocellulose degradation in the terrestrial environment were present but not abundant, while a notable abundance of putative esterases (such as carbohydrate esterase family 1) associated with decoupling lignin from polysaccharides in lignocellulose was observed. CONCLUSIONS: Here, we identify a diverse cohort of previously undefined bacteria that drive lignocellulose degradation in the surface sediments of the salt marsh environment and describe the enzymatic mechanisms they employ to facilitate this process. Our results increase the understanding of the microbial and molecular mechanisms that underpin carbon sequestration from lignocellulose within salt marsh surface sediments in situ and provide insights into the potential enzymatic mechanisms regulating the enrichment of polyphenolics in salt marsh sediments. Video Abstract.

A Rapid, Sensitive, Low-Cost Assay for Detecting Hydrogenotrophic Methanogens in Anaerobic Digesters Using Loop-Mediated Isothermal Amplification.

Understanding how the presence, absence, and abundance of different microbial genera supply specific metabolic functions for anaerobic digestion (AD) and how these impact on gas production is critical for a long-term understanding and optimization of the AD process. The strictly anaerobic methanogenic archaea are essential for methane production within AD microbial communities. Methanogens are a phylogenetically diverse group that can be classified into three metabolically distinct lineages based on the substrates they use to produce methane. While process optimization based on physicochemical parameters is well established in AD, measurements that could allow manipulation of the underlying microbial community are seldom used as they tend to be non-specific, expensive, or time-consuming, or a combination of all three. Loop-mediated isothermal amplification (LAMP) assays combine a simple, rapid, low-cost detection technique with high sensitivity and specificity. Here, we describe the optimization of LAMP assays for the detection of four different genera of hydrogenotrophic methanogens: Methanoculleus, Methanothermobacter, Methanococcus, and Methanobrevibacter spp. By targeting archaeal elongation factor 2 (aEF2), these LAMP assays provide a rapid, low-cost, presence/absence indication of hydrogenotrophic methanogens that could be used as a real-time measure of process conditions. The assays were shown to be sensitive to 1 pg of DNA from most tested methanogen species, providing a route to a quantitative measure through simple serial dilution of samples. The LAMP assays described here offer a simple, fast, and affordable method for the specific detection of four different genera of hydrogenotrophic methanogens. Our results indicate that this approach could be developed into a quantitative measure that could provide rapid, low-cost insight into the functioning and optimization of AD and related systems.

ß-Glucan is a major growth substrate for human gut bacteria related to Coprococcus eutactus.

A clone encoding carboxymethyl cellulase activity was isolated during functional screening of a human gut metagenomic library using Lactococcus lactis MG1363 as heterologous host. The insert carried a glycoside hydrolase family 9 (GH9) catalytic domain with sequence similarity to a gene from Coprococcus eutactus ART55/1. Genome surveys indicated a limited distribution of GH9 domains among dominant human colonic anaerobes. Genomes of C. eutactus-related strains harboured two GH9-encoding and four GH5-encoding genes, but the strains did not appear to degrade cellulose. Instead, they grew well on ß-glucans and one of the strains also grew on galactomannan, galactan, glucomannan and starch. Coprococcus comes and Coprococcus catus strains did not harbour GH9 genes and were not able to grow on ß-glucans. Gene expression and proteomic analysis of C. eutactus ART55/1 grown on cellobiose, ß-glucan and lichenan revealed similar changes in expression in comparison to glucose. On ß-glucan and lichenan only, one of the four GH5 genes was strongly upregulated. Growth on glucomannan led to a transcriptional response of many genes, in particular a strong upregulation of glycoside hydrolases involved in mannan degradation. Thus, ß-glucans are a major growth substrate for species related to C. eutactus, with glucomannan and galactans alternative substrates for some strains.

Mining the biomass deconstructing capabilities of rice yellow stem borer symbionts.

BACKGROUND: Efficient deconstruction of lignocellulosic biomass into simple sugars in an economically viable manner is a prerequisite for its global acceptance as a feedstock in bioethanol production. This is achieved in nature by suites of enzymes with the capability of efficiently depolymerizing all the components of lignocellulose. Here, we provide detailed insight into the repertoire of enzymes produced by microorganisms enriched from the gut of the crop pathogen rice yellow stem borer (Scirpophaga incertulas). RESULTS: A microbial community was enriched from the gut of the rice yellow stem borer for enhanced rice straw degradation by sub-culturing every 10 days, for 1 year, in minimal medium with rice straw as the main carbon source. The enriched culture demonstrated high cellulolytic and xylanolytic activity in the culture supernatant. Metatranscriptomic and metaexoproteomic analysis revealed a large array of enzymes potentially involved in rice straw deconstruction. The consortium was found to encode genes ascribed to all five classes of carbohydrate-active enzymes (GHs, GTs, CEs, PLs, and AAs), including carbohydrate-binding modules (CBMs), categorized in the carbohydrate-active enzymes (CAZy) database. The GHs were the most abundant class of CAZymes. Predicted enzymes from these CAZy classes have the potential to digest each cell-wall components of rice straw, i.e., cellulose, hemicellulose, pectin, callose, and lignin. Several identified CAZy proteins appeared novel, having an unknown or hypothetical catalytic counterpart with a known class of CBM. To validate the findings, one of the identified enzymes that belong to the GH10 family was functionally characterized. The enzyme expressed in E. coli efficiently hydrolyzed beechwood xylan, and pretreated and untreated rice straw. CONCLUSIONS: This is the first report describing the enrichment of lignocellulose degrading bacteria from the gut of the rice yellow stem borer to deconstruct rice straw, identifying a plethora of enzymes secreted by the microbial community when growing on rice straw as a carbon source. These enzymes could be important candidates for biorefineries to overcome the current bottlenecks in biomass processing.

Defining functional diversity for lignocellulose degradation in a microbial community using multi-omics studies.

BACKGROUND: Lignocellulose is one of the most abundant forms of fixed carbon in the biosphere. Current industrial approaches to the degradation of lignocellulose employ enzyme mixtures, usually from a single fungal species, which are only effective in hydrolyzing polysaccharides following biomass pre-treatments. While the enzymatic mechanisms of lignocellulose degradation have been characterized in detail in individual microbial species, the microbial communities that efficiently breakdown plant materials in nature are species rich and secrete a myriad of enzymes to perform "community-level" metabolism of lignocellulose. Single-species approaches are, therefore, likely to miss important aspects of lignocellulose degradation that will be central to optimizing commercial processes. RESULTS: Here, we investigated the microbial degradation of wheat straw in liquid cultures that had been inoculated with wheat straw compost. Samples taken at selected time points were subjected to multi-omics analysis with the aim of identifying new microbial mechanisms for lignocellulose degradation that could be applied in industrial pre-treatment of feedstocks. Phylogenetic composition of the community, based on sequenced bacterial and eukaryotic ribosomal genes, showed a gradual decrease in complexity and diversity over time due to microbial enrichment. Taxonomic affiliation of bacterial species showed dominance of Bacteroidetes and Proteobacteria and high relative abundance of genera Asticcacaulis, Leadbetterella and Truepera. The eukaryotic members of the community were enriched in peritrich ciliates from genus Telotrochidium that thrived in the liquid cultures compared to fungal species that were present in low abundance. A targeted metasecretome approach combined with metatranscriptomics analysis, identified 1127 proteins and showed the presence of numerous carbohydrate-active enzymes extracted from the biomass-bound fractions and from the culture supernatant. This revealed a wide array of hydrolytic cellulases, hemicellulases and carbohydrate-binding modules involved in lignocellulose degradation. The expression of these activities correlated to the changes in the biomass composition observed by FTIR and ssNMR measurements. CONCLUSIONS: A combination of mass spectrometry-based proteomics coupled with metatranscriptomics has enabled the identification of a large number of lignocellulose degrading enzymes that can now be further explored for the development of improved enzyme cocktails for the treatment of plant-based feedstocks. In addition to the expected carbohydrate-active enzymes, our studies reveal a large number of unknown proteins, some of which may play a crucial role in community-based lignocellulose degradation.

A practical introduction to microbial molecular ecology through the use of isolation chips.

In the context of antimicrobial resistance as one of the most serious issues faced globally by health providers, we explored a practical introduction to molecular microbial ecology. We designed field work and practical experiments for third year members of a 4 year undergraduate Masters Program, in which the students employed traditional and novel isolation techniques to identify antimicrobial activities from soil dwelling microorganisms. Students gained experience in isolating DNA from complex microbial communities, amplifying 16S rRNA genes and applied richness/diversity indices as well as principal coordinate analyses to the interpretation of the data they obtained from high throughput sequencing. Our results confirmed that isolation chips facilitate the growth of a greater diversity and different species subset from the complex soil microorganism community than traditional plate spreading techniques. However, rarefaction of 16S rRNA amplicon sequencing data showed that the majority of observed species in soil remain unculturable by current methods. Based on the written reports produced by the students carrying out the work, we concluded that the described protocols are robust and informative, that these activities provide a good practical introduction to the theories and practice of molecular ecology and can be easily deployed to groups of six or more students in a cost-effective manner.

Targeted metatranscriptomics of compost-derived consortia reveals a GH11 exerting an unusual exo-1,4-ß-xylanase activity.

BACKGROUND: Using globally abundant crop residues as a carbon source for energy generation and renewable chemicals production stand out as a promising solution to reduce current dependency on fossil fuels. In nature, such as in compost habitats, microbial communities efficiently degrade the available plant biomass using a diverse set of synergistic enzymes. However, deconstruction of lignocellulose remains a challenge for industry due to recalcitrant nature of the substrate and the inefficiency of the enzyme systems available, making the economic production of lignocellulosic biofuels difficult. Metatranscriptomic studies of microbial communities can unveil the metabolic functions employed by lignocellulolytic consortia and identify novel biocatalysts that could improve industrial lignocellulose conversion. RESULTS: In this study, a microbial community from compost was grown in minimal medium with sugarcane bagasse sugarcane bagasse as the sole carbon source. Solid-state nuclear magnetic resonance was used to monitor lignocellulose degradation; analysis of metatranscriptomic data led to the selection and functional characterization of several target genes, revealing the first glycoside hydrolase from Carbohydrate Active Enzyme family 11 with exo-1,4-ß-xylanase activity. The xylanase crystal structure was resolved at 1.76 Å revealing the structural basis of exo-xylanase activity. Supplementation of a commercial cellulolytic enzyme cocktail with the xylanase showed improvement in Avicel hydrolysis in the presence of inhibitory xylooligomers. CONCLUSIONS: This study demonstrated that composting microbiomes continue to be an excellent source of biotechnologically important enzymes by unveiling the diversity of enzymes involved in in situ lignocellulose degradation.

Revealing the insoluble metasecretome of lignocellulose-degrading microbial communities.

Microbial communities metabolize plant biomass using secreted enzymes; however, identifying extracellular proteins tightly bound to insoluble lignocellulose in these microbiomes presents a challenge, as the rigorous extraction required to elute these proteins also lyses the microbes associated with the plant biomass releasing intracellular proteins that contaminate the metasecretome. Here we describe a technique for targeting the extracellular proteome, which was used to compare the metasecretome and meta-surface-proteome of two lignocellulose-degrading communities grown on wheat straw and rice straw. A combination of mass spectrometry-based proteomics coupled with metatranscriptomics enabled the identification of a unique secretome pool from these lignocellulose-degrading communities. This method enabled us to efficiently discriminate the extracellular proteins from the intracellular proteins by improving detection of actively secreted and transmembrane proteins. In addition to the expected carbohydrate active enzymes, our new method reveals a large number of unknown proteins, supporting the notion that there are major gaps in our understanding of how microbial communities degrade lignocellulosic substrates.

Defining the functional traits that drive bacterial decomposer community productivity.

Microbial communities are essential to a wide range of ecologically and industrially important processes. To control or predict how these communities function, we require a better understanding of the factors which influence microbial community productivity. Here, we combine functional resource use assays with a biodiversity-ecosystem functioning (BEF) experiment to determine whether the functional traits of constituent species can be used to predict community productivity. We quantified the abilities of 12 bacterial species to metabolise components of lignocellulose and then assembled these species into communities of varying diversity and composition to measure their productivity growing on lignocellulose, a complex natural substrate. A positive relationship between diversity and community productivity was caused by a selection effect whereby more diverse communities were more likely to contain two species that significantly improved community productivity. Analysis of functional traits revealed that the observed selection effect was primarily driven by the abilities of these species to degrade ß-glucan. Our results indicate that by identifying the key functional traits underlying microbial community productivity we could improve industrial bioprocessing of complex natural substrates.

Nutrient availability shapes the microbial community structure in sugarcane bagasse compost-derived consortia.

Microbial communities (MCs) create complex metabolic networks in natural habitats and respond to environmental changes by shifts in the community structure. Although members of MCs are often not amenable for cultivation in pure culture, it is possible to obtain a greater diversity of species in the laboratory setting when microorganisms are grown as mixed cultures. In order to mimic the environmental conditions, an appropriate growth medium must be applied. Here, we examined the hypothesis that a greater diversity of microorganisms can be sustained under nutrient-limited conditions. Using a 16 S rRNA amplicon metagenomic approach, we explored the structure of a compost-derived MC. During a five-week time course the MC grown in minimal medium with sugarcane bagasse (SCB) as a sole carbon source showed greater diversity and enrichment in lignocellulose-degrading microorganisms. In contrast, a MC grown in nutrient rich medium with addition of SCB had a lower microbial diversity and limited number of lignocellulolytic species. Our approach provides evidence that factors such as nutrient availability has a significant selective pressure on the biodiversity of microorganisms in MCs. Consequently, nutrient-limited medium may displace bacterial generalist species, leading to an enriched source for mining novel enzymes for biotechnology applications.