Genes and genome-resolved metagenomics reveal the microbial functional make up of Amazon peatlands under geochemical gradients.

The Pastaza-Marañón Foreland Basin (PMFB) holds the most extensive tropical peatland area in South America. PMFB peatlands store ~7.07 Gt of organic carbon interacting with multiple microbial heterotrophic, methanogenic, and other aerobic/anaerobic respirations. Little is understood about the contribution of distinct microbial community members inhabiting tropical peatlands. Here, we studied the metagenomes of three geochemically distinct peatlands spanning minerotrophic, mixed, and ombrotrophic conditions. Using gene- and genome-centric approaches, we evaluate the functional potential of the underlying microbial communities. Abundance analyses show significant differences in C, N, P, and S acquisition genes. Furthermore, community interactions mediated by toxin-antitoxin and CRISPR-Cas systems were enriched in oligotrophic soils, suggesting that non-metabolic interactions may exert additional controls in low-nutrient environments. Additionally, we reconstructed 519 metagenome-assembled genomes spanning 28 phyla. Our analyses detail key differences across the geochemical gradient in the predicted microbial populations involved in degradation of organic matter, and the cycling of N and S. Notably, we observed differences in the nitric oxide (NO) reduction strategies between sites with high and low N2 O fluxes and found phyla putatively capable of both NO and sulfate reduction. Our findings detail how gene abundances and microbial populations are influenced by geochemical differences in tropical peatlands.

Methanogenesis in biogas reactors under inhibitory ammonia concentration requires community-wide tolerance.

Ammonia (NH3) inhibition represents a major limitation to methane production during anaerobic digestion of organic material in biogas reactors. This process relies on co-operative metabolic interactions between diverse taxa at the community-scale. Despite this, most investigations have focused singularly on how methanogenic Archaea respond to NH3 stress. With a high-NH3 pre-adapted and un-adapted community, this study investigated responses to NH3 inhibition both at the community-scale and down to individual taxa. The pre-adapted community performed methanogenesis under inhibitory NH3 concentrations better than the un-adapted. While many functionally important phyla were shared between the two communities, only taxa from the pre-adapted community were robust to NH3. Functionally important phyla were mostly comprised of sensitive taxa (≥ 50%), yet all groups, including methanogens, also possessed tolerant individuals (10-50%) suggesting that potential mechanisms for tolerance are non-specific and widespread. Hidden Markov Model-based phylogenetic analysis of methanogens confirmed that NH3 tolerance was not restricted to specific taxonomic groups, even at the genus level. By reconstructing covarying growth patterns via network analyses, methanogenesis by the pre-adapted community was best explained by continued metabolic interactions (edges) between tolerant methanogens and other tolerant taxa (nodes). However, under non-inhibitory conditions, sensitive taxa re-emerged to dominate the pre-adapted community, suggesting that mechanisms of NH3 tolerance can be disadvantageous to fitness without selection pressure. This study demonstrates that methanogenesis under NH3 inhibition depends on broad-scale tolerance throughout the prokaryotic community. Mechanisms for tolerance seem widespread and non-specific, which has practical significance for the development of robust methanogenic biogas communities. KEY POINTS: • Ammonia pre-adaptation allows for better methanogenesis under inhibitory conditions. • All functionally important prokaryote phyla have some ammonia tolerant individuals. • Methanogenesis was likely dependent on interactions between tolerant individuals.

Delineating the Drivers and Functionality of Methanogenic Niches within an Arid Landfill.

Microbial communities mediate the transformation of organic matter within landfills into methane (CH4). Yet their ecological role in CH4 production is rarely evaluated. To characterize the microbiome associated with this biotransformation, the overall community and methanogenic Archaea were surveyed in an arid landfill using leachate collected from distinctly aged landfill cells (i.e., younger, intermediate, and older). We hypothesized that distinct methanogenic niches exist within an arid landfill, driven by geochemical gradients that developed under extended and age-dependent waste biodegradation stages. Using 16S rRNA and mcrA gene amplicon sequencing, we identified putative methanogenic niches as follows. The order Methanomicrobiales was the most abundant order in leachate from younger cells, where leachate temperature and propionate concentrations were measured at 41.8°C ± 1.7°C and 57.1 ± 10.7 mg L-1. In intermediate-aged cells, the family Methanocellaceae was identified as a putative specialist family under intermediate-temperature and -total dissolved solid (TDS) conditions, wherein samples had a higher alpha diversity index and near CH4 concentrations. In older-aged cells, accumulating metals and TDS supported Methanocorpusculaceae, "Candidatus Bathyarchaeota," and "Candidatus Verstraetearchaeota" operational taxonomic units (OTUs). Consistent with the mcrA data, we assayed methanogenic activity across the age gradient through stable isotopic measurements of δ13C of CH4 and δ13C of CO2. The majority (80%) of the samples' carbon fractionation was consistent with hydrogenotrophic methanogenesis. Together, we report age-dependent geochemical gradients detected through leachate in an arid landfill seemingly influencing CH4 production, niche partitioning, and methanogenic activity. IMPORTANCE Microbiome analysis is becoming common in select municipal and service ecosystems, including wastewater treatment and anaerobic digestion, but its potential as a microbial-status-informative tool to promote or mitigate CH4 production has not yet been evaluated in landfills. Methanogenesis mediated by Archaea is highly active in solid-waste microbiomes but is commonly neglected in studies employing next-generation sequencing techniques. Identifying methanogenic niches within a landfill offers detail into operations that positively or negatively impact the commercial production of methane known as biomethanation. We provide evidence that the geochemistry of leachate and its microbiome can be a variable accounting for ecosystem-level (coarse) variation of CH4 production, where we demonstrate through independent assessments of leachate and gas collection that the functional variability of an arid landfill is linked to the composition of methanogenic Archaea.

Nitrogen-rich microbial products provide new organo-mineral associations for the stabilization of soil organic matter.

Understanding the cycling of C and N in soils is important for maintaining soil fertility while also decreasing greenhouse gas emissions, but much remains unknown about how organic matter (OM) is stabilized in soils. We used nano-scale secondary ion mass spectrometry (NanoSIMS) to investigate the changes in C and N in a Vertisol and an Alfisol incubated for 365 days with 13 C and 15 N pulse labeled lucerne (Medicago sativa L.) to discriminate new inputs of OM from the existing soil OM. We found that almost all OM within the free stable microaggregates of the soil was associated with mineral particles, emphasizing the importance of organo-mineral interactions for the stabilization of C. Of particular importance, it was also found that 15 N-rich microbial products originating from decomposition often sorbed directly to mineral surfaces not previously associated with OM. Thus, we have shown that N-rich microbial products preferentially attach to distinct areas of mineral surfaces compared to C-dominated moieties, demonstrating the ability of soils to store additional OM in newly formed organo-mineral associations on previously OM-free mineral surfaces. Furthermore, differences in 15 N enrichment were observed between the Vertisol and Alfisol presumably due to differences in mineralogy (smectite-dominated compared to kaolinite-dominated), demonstrating the importance of mineralogy in regulating the sorption of microbial products. Overall, our findings have important implications for the fundamental understanding of OM cycling in soils, including the immobilization and storage of N-rich compounds derived from microbial decomposition and subsequent N mineralization to sustain plant growth.

Global changes in soil stocks of carbon, nitrogen, phosphorus, and sulphur as influenced by long-term agricultural production.

Quantifying changes in stocks of C, N, P, and S in agricultural soils is important not only for managing these soils sustainably as required to feed a growing human population, but for C and N, they are also important for understanding fluxes of greenhouse gases from the soil environment. In a global meta-analysis, 102 studies were examined to investigate changes in soil stocks of organic C, total N, total P, and total S associated with long-term land-use changes. Conversion of native vegetation to cropping resulted in substantial losses of C (-1.6 kg m-2 , -43%), N (-0.15 kg m-2 , -42%), P (-0.029 kg m-2 , -27%), and S (-0.015 kg m-2 , -33%). The subsequent conversion of conventional cropping systems to no-till, organic agriculture, or organic amendment systems subsequently increased stocks, but the magnitude of this increase (average of +0.47 kg m-2 for C and +0.051 kg m-2 for N) was small relative to the initial decrease. We also examined the conversion of native vegetation to pasture, with changes in C (-11%), N (+4.1%), and P (+25%) generally being modest relative to changes caused by conversion to cropping. The C:N ratio remained relatively constant irrespective of changes in land use, whilst in contrast, the C:S ratio decreased by 21% in soils converted to cropping - this suggesting that biochemical mineralization is of importance for S. The data presented here will assist in the assessment of different agricultural production systems on soil stocks of C, N, P, and S - this information assisting not only in quantifying the effects of existing agricultural production on these stocks, but also allowing for informed decision-making regarding the potential effects of future land-use changes.

A metagenomic alpha-diversity index for microbial functional biodiversity.

Alpha-diversity indices are an essential tool for describing and comparing biodiversity. Microbial ecologists apply indices originally intended for, or adopted by, macroecology to address questions relating to taxonomy (conserved marker) and function (metagenome-based data). In this Perspective piece, I begin by discussing the nature and mathematical quirks important for interpreting routinely employed alpha-diversity indices. Secondly, I propose a metagenomic alpha-diversity index (MD) that measures the (dis)similarity of protein-encoding genes within a community. MD has defined limits, whereby a community comprised mostly of similar, poorly diverse protein-encoding genes pulls the index to the lower limit, while a community rich in divergent homologs and unique genes drives it toward the upper limit. With data acquired from an in silico and three in situ metagenome studies, I derive MD and typical alpha-diversity indices applied to taxonomic (ribosomal rRNA) and functional (all protein-encoding) genes, and discuss their relationships with each other. Not all alpha-diversity indices detect biological trends, and taxonomic does not necessarily follow functional biodiversity. Throughout, I explain that protein Richness and MD provide complementary and easily interpreted information, while probability-based indices do not. Finally, considerations regarding the unique nature of microbial metagenomic data and its relevance for describing functional biodiversity are discussed.

Broiler farming practices using new or re-used bedding, inclusive of free-range, have no impact on Campylobacter levels, species diversity, Campylobacter community profiles and Campylobacter bacteriophages.

A multi-stage option to address food-safety can be produced by a clearer understanding of Campylobacter's persistence through the broiler production chain, its environmental niche and its interaction with bacteriophages. This study addressed Campylobacter levels, species, genotype, bacteriophage composition/ levels in caeca, litter, soil and carcasses across commercial broiler farming practices to inform on-farm management, including interventions. Broilers were sequentially collected as per company slaughter schedules over two-years from 17 farms, which represented four commercially adopted farming practices, prior to the final bird removal (days 39-53). The practices were conventional full clean-out, conventional litter re-use, free-range-full cleanout and free-range-litter re-use. Caeca, litter and soil collected on-farm, and representative carcases collected at the processing plant, were tested for Campylobacter levels, species dominance and Campylobacter bacteriophages. General community profiling via denaturing gradient gel electrophoresis of the flaA gene was used to establish the population relationships between various farming practices on representative Campylobacter isolates. The farming practice choices did not influence the high caeca Campylobacter levels (log 7.5 to log 8.5 CFU/g), the carcass levels (log 2.5 to log 3.2 CFU/carcass), the C. jejuni/C. coli dominance and the on-farm bacteriophage presence/levels. A principal coordinate analysis of the flaA distribution for farm and litter practices showed strong separation but no obvious farming practice related grouping of Campylobacter. Bacteriophages originated from select farms, were not practice-dependent, and were detected in the environment (litter) only if present in the birds (caeca). This multifaceted study showed no influence of farming practices on on-farm Campylobacter dynamics. The significance of this study means that a unified on-farm risk-management could be adopted irrespective of commercial practice choices to collectively address caeca Campylobacter levels, as well as the potential to include Campylobacter bacteriophage biocontrol. The impact of this study means that there are no constraints in re-using bedding or adopting free-range farming, thus contributing to environmentally sustainable (re-use) and emerging (free-range) broiler farming choices.

One-step PCR amplicon sequencing libraries perform better than two-step when assessing soil microbial diversity and community profiles.

Despite adoption of high-throughput sequencing of PCR-amplified microbial taxonomic markers for ecological analyses, distinct approaches for preparing amplicon libraries exist. One approach utilises long fusion primers and a single PCR (one-step) while another utilises shorter primers in a first reaction, before transferring diluted amplicons to a second reaction for barcode index incorporation (two-step). We investigated whether transferring diluted amplicons risked creating artificially simplified, poorly diverse communities. In soils from three sites with paired cropland and forest, one-step yielded higher alpha-diversity indices, including detection of two-four times more unique taxa. Modelling expected taxa per sequence observation predicted that one-step reaches full coverage by 104 sequences per sample while two-step needs 105-109. Comparisons of rank abundance demonstrated that two-step covered only 38%-69% of distributions. Beta-diversity showed better separation of communities in response to land use change under one-step, although both approaches showed a significant effect. Driving differences was underestimation of relatively minor taxa with the two-step procedure. These taxa were low in abundance, yet play important roles in carbon cycling, secondary metabolite production, anaerobic metabolism, and bacterial predation. We conclude that one-step amplicon libraries are advisable for studies focussed on diversity or relatively minor yet functionally important taxa.

Reconciling concepts of black queen and tragedy of the commons in simulated bulk soil and rhizosphere prokaryote communities.

The Black Queen hypothesis describes the evolutionary strategy to lose costly functions in favour of improving growth efficiency. This results in mutants (cheaters) becoming obligately dependent upon a provider (black queen) to produce a necessary resource. Previous analyses demonstrate black queens and cheaters reach a state of equilibrium in pair-wise systems. However, in complex communities, accumulation of cheaters likely poses a serious burden on shared resources. This should result in a Tragedy of the Commons (ToC), whereby over-utilisation of public resources risks making them growth-limiting. With a collection of differential equations, microbial communities composed of twenty prokaryote 'species' either from rhizosphere, characterised by abundant carbon and energy sources, or bulk soil, with limited carbon and energy supply, were simulated. Functional trait groups differed based on combinations of cellulase and amino acid production, growth and resource uptake. Randomly generated communities were thus composed of species that acted as cellulolytic prototrophic black queens, groups that were either cellulolytic or prototrophic, or non-cellulolytic auxotrophic cheaters. Groups could evolve to lose functions over time. Biomass production and biodiversity were tracked in 8,000 Monte Carlo simulations over 500 generations. Bulk soil favoured oligotrophic co-operative communities where biodiversity was positively associated with growth. Rhizosphere favoured copiotrophic cheaters. The most successful functional group across both environments was neither black queens nor cheaters, but those that balanced providing an essential growth-limiting function at a relatively low maintenance cost. Accumulation of loss of function mutants in bulk soil risked resulting in loss of cumulative growth by ToC, while cumulative growth increased in the rhizosphere. In the bulk soil, oligotrophic adaptations assisted species in avoiding extinction. This demonstrated that loss of function by mutation is a successful evolutionary strategy in host-associated and/or resource-rich environments, but poses a risk to communities that must co-operate with each other for mutual co-existence. It was concluded that microbial communities must follow different evolutionary and community assembly strategies in bulk soil versus rhizosphere, with bulk soil communities more dependent on traits that promote co-operative interactions between microbial species.

Water Vapor Adsorption Provides Daily, Sustainable Water to Soils of the Hyperarid Atacama Desert.

Water is necessary for all life on Earth. Water is so critical that organisms have developed strategies to survive in hyperarid environments. These regions with extremely low water availability are also unique analogs in which to study the physico-chemical conditions of extraterrestrial environments such as Mars. We have identified a daily, sustainable cycle of water vapor adsorption (WVA) and desorption that measurably affects soil water content (SWC) in the hyperarid region of the Atacama Desert in southern Perú. We pair field-based soil temperature and relative humidity soil profiles with laboratory simulations to provide evidence for a daily WVA cycle. Using our WVA model, we estimate that one adsorptive period-one night-increases SWC by 0.2-0.3 mg/g of soil (∼30 µm rainfall). We can plausibly rule out other water inputs during our field campaign that could account for this water input, and we provide evidence that this WVA cycle is driven by solar heating and maintained by atmospheric water vapor. The WVA may also serve to retain water from infrequent rain events in these soils. If the water provided by WVA in these soils is bioavailable, it could have significant implications for the microorganisms that are endemic to hyperarid environments.

Cropping systems impact changes in soil fungal, but not prokaryote, alpha-diversity and community composition stability over a growing season in a long-term field trial.

Crop harvest followed by a fallow period can act as a disturbance on soil microbial communities. Cropping systems intended to improve alpha-diversity of communities may also confer increased compositional stability during succeeding growing seasons. Over a single growing season in a long-term (18 year) agricultural field experiment incorporating conventional (CON), conservation (CA), organic (ORG) and integrated (INT) cropping systems, temporal changes in prokaryote, fungal and arbuscular mycorrhizal fungi (AMF) communities were investigated overwinter, during crop growth and at harvest. While certain prokaryote phyla were influenced by cropping system (e.g. Acidobacteria), the community as a whole was primarily driven by temporal changes over the growing season as distinct overwinter and crop-associated communities, with the same trend observed regardless of cropping system. Species-rich prokaryote communities were most stable over the growing season. Cropping system exerted a greater effect on fungal communities, with alpha-diversity highest and temporal changes most stable under CA. CON was particularly detrimental for alpha-diversity in AMF communities, with AMF alpha-diversity and stability improved under all other cropping systems. Practices that promoted alpha-diversity tended to also increase the similarity and temporal stability of soil fungal (and AMF) communities during a growing season, while prokaryote communities were largely insensitive to management.

Functional trait relationships demonstrate life strategies in terrestrial prokaryotes.

Functional, physiological traits are the underlying drivers of niche differentiation. A common framework related to niches occupied by terrestrial prokaryotes is based on copiotrophy or oligotrophy, where resource investment is primarily in either rapid growth or stress tolerance, respectively. A quantitative trait-based approach sought relationships between taxa, traits and niche in terrestrial prokaryotes. With 175 taxa from 11 Phyla and 35 Families (n = 5 per Family), traits were considered as discrete counts of shared genome-encoded proteins. Trait composition strongly supported non-random functional distributions as preferential clustering of related taxa via unweighted pair-group method with arithmetic mean. Trait similarity between taxa increased as taxonomic rank decreased. A suite of Random Forest models identified traits significantly enriched or depleted in taxonomic groups. These traits conveyed functions related to rapid growth, nutrient acquisition and stress tolerance consistent with their presence in copiotroph-oligotroph niches. Hierarchical clustering of traits identified a clade of competitive, copiotrophic Families resilient to oxidative stress versus glycosyltransferase-enriched oligotrophic Families resistant to antimicrobials and environmental stress. However, the formation of five clades suggested a more nuanced view to describe niche differentiation in terrestrial systems is necessary. We suggest considering traits involved in both resource investment and acquisition when predicting niche.

Microbial Communities and Interactions of Nitrogen Oxides With Methanogenesis in Diverse Peatlands of the Amazon Basin.

Tropical peatlands are hotspots of methane (CH4) production but present high variation and emission uncertainties in the Amazon region. This is because the controlling factors of methane production in tropical peats are not yet well documented. Although inhibitory effects of nitrogen oxides (NO x ) on methanogenic activity are known from pure culture studies, the role of NO x in the methane cycling of peatlands remains unexplored. Here, we investigated the CH4 content, soil geochemistry and microbial communities along 1-m-soil profiles and assessed the effects of soil NO x and nitrous oxide (N2O) on methanogenic abundance and activity in three peatlands of the Pastaza-Marañón foreland basin. The peatlands were distinct in pH, DOC, nitrate pore water concentrations, C/N ratios of shallow soils, redox potential, and 13C enrichment in dissolved inorganic carbon and CH4 pools, which are primarily contingent on H2-dependent methanogenesis. Molecular 16S rRNA and mcrA gene data revealed diverse and novel methanogens varying across sites. Importantly, we also observed a strong stratification in relative abundances of microbial groups involved in NO x cycling, along with a concordant stratification of methanogens. The higher relative abundance of ammonia-oxidizing archaea (Thaumarchaeota) in acidic oligotrophic peat than ammonia-oxidizing bacteria (Nitrospira) is noteworthy as putative sources of NO x . Experiments testing the interaction of NO x species and methanogenesis found that the latter showed differential sensitivity to nitrite (up to 85% reduction) and N2O (complete inhibition), which would act as an unaccounted CH4 control in these ecosystems. Overall, we present evidence of diverse peatlands likely differently affected by inhibitory effects of nitrogen species on methanogens as another contributor to variable CH4 fluxes.

Agricultural practices drive biological loads, seasonal patterns and potential pathogens in the aerobiome of a mixed-land-use dryland.

Air carries a diverse load of particulate microscopic biological matter in suspension, either aerosolized or aggregated with dust particles, the aerobiome, which is dispersed by winds from sources to sinks. The aerobiome is known to contain microbes, including pathogens, as well as debris or small-sized propagules from plants and animals, but its variability and composition has not been studied comprehensibly. To gain a dynamic insight into the aerobiome existing over a mixed-use dryland setting, we conducted a biologically comprehensive, year-long survey of its composition and dynamics for particles less than 10 µm in diameter based on quantitative analyses of DNA content coupled to genomic sequencing. Airborne biological loads were more dependent on seasonal events than on meteorological conditions and only weakly correlated with dust loads. Core aerobiome species could be understood as a mixture of high elevation (e.g. Microbacteriaceae, Micrococcaceae, Deinococci), and local plant and soil sources (e.g. Sphingomonas, Streptomyces, Acinetobacter). Despite the mixed used of the land surrounding the sampling site, taxa that contributed to high load events were largely traceable to proximal agricultural practices like cotton and livestock farming. This included not only the predominance of specific crop plant signals over those of native vegetation, but also that of their pathogens (bacterial, viral and eukaryotic). Faecal bacterial loads were also seasonally important, possibly sourced in intensive animal husbandry or manure fertilization activity, and this microbial load was enriched in tetracycline resistance genes. The presence of the native opportunistic pathogen, Coccidioides spp., by contrast, was detected only with highly sensitive techniques, and only rarely. We conclude that agricultural activity exerts a much stronger influence that the native vegetation as a mass loss factor to the land system and as an input to dryland aerobiomes, including in the dispersal of plant, animal and human pathogens and their genetic resistance characteristics.

Methanogens and Methanotrophs Show Nutrient-Dependent Community Assemblage Patterns Across Tropical Peatlands of the Pastaza-Marañón Basin, Peruvian Amazonia.

Tropical peatlands are globally important carbon reservoirs that play a crucial role in fluxes of atmospheric greenhouse gases. Amazon peatlands are expected to be large source of atmospheric methane (CH4) emissions, however little is understood about the rates of CH4 flux or the microorganisms that mediate it in these environments. Here we studied a mineral nutrient gradient across peatlands in the Pastaza-Marañón Basin, the largest tropical peatland in South America, to describe CH4 fluxes and environmental factors that regulate species assemblages of methanogenic and methanotrophic microorganisms. Peatlands were grouped as minerotrophic, mixed and ombrotrophic categories by their general water source leading to different mineral nutrient content (rich, mixed and poor) quantified by trace elements abundance. Microbial communities clustered dependent on nutrient content (ANOSIM p < 0.001). Higher CH4 flux was associated with minerotrophic communities compared to the other categories. The most dominant methanogens and methanotrophs were represented by Methanobacteriaceae, and Methylocystaceae, respectively. Weighted network analysis demonstrated tight clustering of most methanogen families with minerotrophic-associated microbial families. Populations of Methylocystaceae were present across all peatlands. Null model testing for species assemblage patterns and species rank distributions confirmed non-random aggregations of Methylococcacae methanotroph and methanogen families (p < 0.05). We conclude that in studied amazon peatlands increasing mineral nutrient content provides favorable habitats for Methanobacteriaceae, while Methylocystaceae populations seem to broadly distribute independent of nutrient content.