Long-term litter decomposition controlled by manganese redox cycling.

Litter decomposition is a keystone ecosystem process impacting nutrient cycling and productivity, soil properties, and the terrestrial carbon (C) balance, but the factors regulating decomposition rate are still poorly understood. Traditional models assume that the rate is controlled by litter quality, relying on parameters such as lignin content as predictors. However, a strong correlation has been observed between the manganese (Mn) content of litter and decomposition rates across a variety of forest ecosystems. Here, we show that long-term litter decomposition in forest ecosystems is tightly coupled to Mn redox cycling. Over 7 years of litter decomposition, microbial transformation of litter was paralleled by variations in Mn oxidation state and concentration. A detailed chemical imaging analysis of the litter revealed that fungi recruit and redistribute unreactive Mn(2+) provided by fresh plant litter to produce oxidative Mn(3+) species at sites of active decay, with Mn eventually accumulating as insoluble Mn(3+/4+) oxides. Formation of reactive Mn(3+) species coincided with the generation of aromatic oxidation products, providing direct proof of the previously posited role of Mn(3+)-based oxidizers in the breakdown of litter. Our results suggest that the litter-decomposing machinery at our coniferous forest site depends on the ability of plants and microbes to supply, accumulate, and regenerate short-lived Mn(3+) species in the litter layer. This observation indicates that biogeochemical constraints on bioavailability, mobility, and reactivity of Mn in the plant-soil system may have a profound impact on litter decomposition rates.

Correlated SEM, FIB-SEM, TEM, and NanoSIMS imaging of microbes from the hindgut of a lower termite: methods for in situ functional and ecological studies of uncultivable microbes.

The hindguts of lower termites harbor highly diverse, endemic communities of symbiotic protists, bacteria, and archaea essential to the termite's ability to digest wood. Despite over a century of experimental studies, ecological roles of many of these microbes are unknown, partly because almost none can be cultivated. Many of the protists associate with bacterial symbionts, but hypotheses for their respective roles in nutrient exchange are based on genomes of only two such bacteria. To show how the ecological roles of protists and nutrient transfer with symbiotic bacteria can be elucidated by direct imaging, we combined stable isotope labeling (13C-cellulose) of live termites with analysis of fixed hindgut microbes using correlated scanning electron microscopy, focused ion beam-scanning electron microscopy (FIB-SEM), transmission electron microscopy, and high resolution imaging mass spectrometry (NanoSIMS). We developed methods to prepare whole labeled cells on solid substrates, whole labeled cells milled with a FIB-SEM instrument to reveal cell interiors, and ultramicrotome sections of labeled cells for NanoSIMS imaging of 13C enrichment in protists and associated bacteria. Our results show these methods have the potential to provide direct evidence for nutrient flow and suggest the oxymonad protist Oxymonas dimorpha phagocytoses and enzymatically degrades ingested wood fragments, and may transfer carbon derived from this to its surface bacterial symbionts.

Gut anatomical properties and microbial functional assembly promote lignocellulose deconstruction and colony subsistence of a wood-feeding beetle.

Beneficial microbial associations enhance the fitness of most living organisms, and wood-feeding insects offer some of the most striking examples of this. Odontotaenius disjunctus is a wood-feeding beetle that possesses a digestive tract with four main compartments, each of which contains well-differentiated microbial populations, suggesting that anatomical properties and separation of these compartments may enhance energy extraction from woody biomass. Here, using integrated chemical analyses, we demonstrate that lignocellulose deconstruction and fermentation occur sequentially across compartments, and that selection for microbial groups and their metabolic pathways is facilitated by gut anatomical features. Metaproteogenomics showed that higher oxygen concentration in the midgut drives lignocellulose depolymerization, while a thicker gut wall in the anterior hindgut reduces oxygen diffusion and favours hydrogen accumulation, facilitating fermentation, homoacetogenesis and nitrogen fixation. We demonstrate that depolymerization continues in the posterior hindgut, and that the beetle excretes an energy- and nutrient-rich product on which its offspring subsist and develop. Our results show that the establishment of beneficial microbial partners within a host requires both the acquisition of the microorganisms and the formation of specific habitats within the host to promote key microbial metabolic functions. Together, gut anatomical properties and microbial functional assembly enable lignocellulose deconstruction and colony subsistence on an extremely nutrient-poor diet.

Light Regimes Shape Utilization of Extracellular Organic C and N in a Cyanobacterial Biofilm.

UNLABELLED: Although it is becoming clear that many microbial primary producers can also play a role as organic consumers, we know very little about the metabolic regulation of photoautotroph organic matter consumption. Cyanobacteria in phototrophic biofilms can reuse extracellular organic carbon, but the metabolic drivers of extracellular processes are surprisingly complex. We investigated the metabolic foundations of organic matter reuse by comparing exoproteome composition and incorporation of (13)C-labeled and (15)N-labeled cyanobacterial extracellular organic matter (EOM) in a unicyanobacterial biofilm incubated using different light regimes. In the light and the dark, cyanobacterial direct organic C assimilation accounted for 32% and 43%, respectively, of all organic C assimilation in the community. Under photosynthesis conditions, we measured increased excretion of extracellular polymeric substances (EPS) and proteins involved in micronutrient transport, suggesting that requirements for micronutrients may drive EOM assimilation during daylight hours. This interpretation was supported by photosynthesis inhibition experiments, in which cyanobacteria incorporated N-rich EOM-derived material. In contrast, under dark, C-starved conditions, cyanobacteria incorporated C-rich EOM-derived organic matter, decreased excretion of EPS, and showed an increased abundance of degradative exoproteins, demonstrating the use of the extracellular domain for C storage. Sequence-structure modeling of one of these exoproteins predicted a specific hydrolytic activity that was subsequently detected, confirming increased EOM degradation in the dark. Associated heterotrophic bacteria increased in abundance and upregulated transport proteins under dark relative to light conditions. Taken together, our results indicate that biofilm cyanobacteria are successful competitors for organic C and N and that cyanobacterial nutrient and energy requirements control the use of EOM. IMPORTANCE: Cyanobacteria are globally distributed primary producers, and the fate of their fixed C influences microbial biogeochemical cycling. This fate is complicated by cyanobacterial degradation and assimilation of organic matter, but because cyanobacteria are assumed to be poor competitors for organic matter consumption, regulation of this process is not well tested. In mats and biofilms, this is especially relevant because cyanobacteria produce an extensive organic extracellular matrix, providing the community with a rich source of nutrients. Light is a well-known regulator of cyanobacterial metabolism, so we characterized the effects of light availability on the incorporation of organic matter. Using stable isotope tracing at the single-cell level, we quantified photoautotroph assimilation under different metabolic conditions and integrated the results with proteomics to elucidate metabolic status. We found that cyanobacteria effectively compete for organic matter in the light and the dark and that nutrient requirements and community interactions contribute to cycling of extracellular organic matter.

Compartmentalized microbial composition, oxygen gradients and nitrogen fixation in the gut of Odontotaenius disjunctus.

Coarse woody debris is an important biomass pool in forest ecosystems that numerous groups of insects have evolved to take advantage of. These insects are ecologically important and represent useful natural analogs for biomass to biofuel conversion. Using a range of molecular approaches combined with microelectrode measurements of oxygen, we have characterized the gut microbiome and physiology of Odontotaenius disjunctus, a wood-feeding beetle native to the eastern United States. We hypothesized that morphological and physiological differences among gut regions would correspond to distinct microbial populations and activities. In fact, significantly different communities were found in the foregut (FG), midgut (MG)/posterior hindgut (PHG) and anterior hindgut (AHG), with Actinobacteria and Rhizobiales being more abundant toward the FG and PHG. Conversely, fermentative bacteria such as Bacteroidetes and Clostridia were more abundant in the AHG, and also the sole region where methanogenic Archaea were detected. Although each gut region possessed an anaerobic core, micron-scale profiling identified radial gradients in oxygen concentration in all regions. Nitrogen fixation was confirmed by (15)N2 incorporation, and nitrogenase gene (nifH) expression was greatest in the AHG. Phylogenetic analysis of nifH identified the most abundant transcript as related to Ni-Fe nitrogenase of a Bacteroidetes species, Paludibacter propionicigenes. Overall, we demonstrate not only a compartmentalized microbiome in this beetle digestive tract but also sharp oxygen gradients that may permit aerobic and anaerobic metabolism to occur within the same regions in close proximity. We provide evidence for the microbial fixation of N2 that is important for this beetle to subsist on woody biomass.