Non-destructive quantification of anaerobic gut fungi and methanogens in co-culture reveals increased fungal growth rate and changes in metabolic flux relative to mono-culture

Background Quantification of individual species in microbial co-cultures and consortia is critical to understanding and designing communities with prescribed functions. However, it is difficult to physically separate species or measure species-specific attributes in most multi-species systems. Anaerobic gut fungi (AGF) (Neocallimastigomycetes) are native to the rumen of large herbivores, where they exist as minority members among a wealth of prokaryotes. AGF have significant biotechnological potential owing to their diverse repertoire of potent lignocellulose-degrading carbohydrate-active enzymes (CAZymes), which indirectly bolsters activity of other rumen microbes through metabolic exchange. While decades of literature suggest that polysaccharide degradation and AGF growth are accelerated in co-culture with prokaryotes, particularly methanogens, methods have not been available to measure concentrations of individual species in co-culture. New methods to disentangle the contributions of AGF and rumen prokaryotes are sorely needed to calculate AGF growth rates and metabolic fluxes to prove this hypothesis and understand its causality for predictable co-culture design. Results We present a simple, microplate-based method to measure AGF and methanogen concentrations in co-culture based on fluorescence and absorbance spectroscopies. Using samples of < 2% of the co-culture volume, we demonstrate significant increases in AGF growth rate and xylan and glucose degradation rates in co-culture with methanogens relative to mono-culture. Further, we calculate significant differences in AGF metabolic fluxes in co-culture relative to mono-culture, namely increased flux through the energy-generating hydrogenosome organelle. While calculated fluxes highlight uncertainties in AGF primary metabolism that preclude definitive explanations for this shift, our method will enable steady-state fluxomic experiments to probe AGF metabolism in greater detail. Conclusions The method we present to measure AGF and methanogen concentrations enables direct growth measurements and calculation of metabolic fluxes in co-culture. These metrics are critical to develop a quantitative understanding of interwoven rumen metabolism, as well as the impact of co-culture on polysaccharide degradation and metabolite production. The framework presented here can inspire new methods to probe systems beyond AGF and methanogens. Simple modifications to the method will likely extend its utility to co-cultures with more than two organisms or those grown on solid substrates to facilitate the design and deployment of microbial communities for bioproduction and beyond.

Small RNAs go global in human gut Bacteroides

The last two decades have seen numerous studies connecting physiological behaviors in Bacteroides —including polysaccharide degradation and capsule production—with elements of global regulation, but a complete model is still elusive. A new study by Adams et al. in this issue of the Journal of Bacteriology reveals another layer of regulation by describing a novel family of RNA-binding proteins (Rbps) in Bacteroides thetaiotaomicron that modify expression of genes involved in carbohydrate utilization and capsule expression, among others.

Structural changes of cellulosic polysaccharides in sesame hull during roasting at various temperatures

This article reports a study of the degradation of roasted sesame hulls cellulosic polysaccharides contribution to the Maillard and caramelization reaction. In the present study, cellulosic polysaccharides were extracted from sesame hulls before and after roasting at various temperatures (160, 180, 200, and 220 °C). The structural vari-ations of the cellulosic polysaccharides were elucidated by using the techniques: scanning electron microscope (SEM), high-performance anion-exchange chromatography, Fourier transform (FT-IR) spectrometer, carbon-13 nuclear magnetic resonance (CP/MAS 13C-NMR), and thermal gravimetric analysis. The pyrolysisgas chromatography-mass spectrometry (Py-GC/MS) characterized and verified the chemical composition obtained from the polysaccharide degradation during roasting. The sugar analysis results showed that galacturonic acid, xylose, and rhamnose were more easily degraded than arabinose, galactose, glucose, and mannose. The morphology of the cellulosic polysaccharides shows irregular dispersed globular fragments after roasting by SEM observation. FT-IR and CP/MAS 13C-NMR spectra indicated the crystalline structure and linkages of the cellulose did not break down in comparison to amorphous cellulose that partly degraded. Abundant acetic acid and 3-furaldehyde were among the polysaccharide degradation products identified by Py-GC/MS. These chemical compounds were likely the significant contributors to caramelization and the Maillard reaction in sesame seed roasting.

Hydrogen Peroxide Effects on Natural-Sourced Polysacchrides: Free Radical Formation/Production, Degradation Process, and Reaction Mechanism—A Critical Synopsis

Numerous reactive oxygen species (ROS) entities exist, and hydrogen peroxide (H2O2) is very key among them as it is well known to possess a stable but poor reactivity capable of generating free radicals. Considered among reactive atoms, molecules, and compounds with electron-rich sites, free radicals emerging from metabolic reactions during cellular respirations can induce oxidative stress and cause cellular structure damage, resulting in diverse life-threatening diseases when produced in excess. Therefore, an antioxidant is needed to curb the overproduction of free radicals especially in biological systems (in vivo and in vitro). Despite the inherent properties limiting its bioactivities, polysaccharides from natural sources increasingly gain research attention given their position as a functional ingredient. Improving the functionality and bioactivity of polysaccharides have been established through degradation of their molecular integrity. In this critical synopsis; we articulate the effects of H2O2 on the degradation of polysaccharides from natural sources. Specifically, the synopsis focused on free radical formation/production, polysaccharide degradation processes with H2O2, the effects of polysaccharide degradation on the structural characteristics; physicochemical properties; and bioactivities; in addition to the antioxidant capability. The degradation mechanisms involving polysaccharide’s antioxidative property; with some examples and their respective sources are briefly summarised.

Bacteroidetes contribute to the carbon and nutrient cycling of deep sea through breaking down diverse glycans

Bacteroidetes are thought to be specialized for the degradation of algae-derived ocean polysaccharides and are a major contributor to the marine carbon and nutrient cycling. Here, we first show Bacteroidetes are the second most abundant phylum bacteria in deep-sea cold seep and possess more genes associated with polysaccharides degradation than other bacteria through metagenomics methods. We further isolate a novel Bacteroidetes species, Maribellus comscasis WC007T, which can efficiently degrade numerous different polysaccharides including: cellulose, pectin, fucoidan, mannan, xylan and starch. These results are verified by transcriptomic analyses and growth assays. Notably, we find cellulose promotes abundant bacterial growth, and using transcriptomics and metabolomics we finally report on the underlying mechanisms of cellulose degradation and utilization, as well as potential contributions to the carbon cycling. Overall, our results suggest Bacteroidetes play key roles in the deep-sea carbon and nutrient cycling, likely due to their high abundance and prominent polysaccharide degradation capabilities.One Sentence SummaryBacteroidetes contribute to ocean carbon and nutrient cycle.

Mercury methylation trait dispersed across diverse anaerobic microbial guilds in a eutrophic sulfate-enriched lake

Mercury (Hg) methylation is a microbially mediated process that converts inorganic Hg into the bioaccumulative neurotoxin methylmercury (MeHg). Exploring the diversity and metabolic potential of the dominant Hg-methylating microorganisms can provide insights into how biogeochemical cycles and water quality parameters underlie MeHg production. However, our understanding of the ecophysiology of methylators in natural ecosystems is still limited. Here, we used shotgun metagenomics paired with biogeochemical data to identify likely hotspots for MeHg production in a lake with elevated sulfate levels and characterize the microbial methylators and the flanking microbial community. Identified putative methylators were dominated by hgcA sequences divergent from those in canonical, experimentally confirmed methylators. Using genome-resolved metagenomics, these sequences were identified within genomes associated with Bacteroidetes and the recently described phylum Kiritimatiellaeota. Over half of the hgcA abundance comes from genomes corresponding to obligately fermentative organisms, many of which have a large number of glucoside hydrolases for polysaccharide degradation. Sulfate-reducing genomes encoding hgcA were also identified, but only accounted for 22% of the abundance of hgcA+ genomes. This work highlights the diverse dispersal of the methylation trait across the microbial anoxic food web.