Metagenomic-based discovery and comparison of the lignin degrading potential of microbiomes in aquatic and terrestrial ecosystems via the LCdb database.

Lignin, as an abundant organic carbon, plays a vital role in the global carbon cycle. However, our understanding of the global lignin-degrading microbiome remains elusive. The greatest barrier has been absence of a comprehensive and accurate functional gene database. Here, we first developed a curated functional gene database (LCdb) for metagenomic profiling of lignin degrading microbial consortia. Via the LCdb, we draw a clear picture describing the global biogeography of communities with lignin-degrading potential. They exhibit clear niche differentiation at the levels of taxonomy and functional traits. The terrestrial microbiomes showed the highest diversity, yet the lowest correlations. In particular, there were few correlations between genes involved in aerobic and anaerobic degradation pathways, showing a clear functional redundancy property. In contrast, enhanced correlations, especially closer inter-connections between anaerobic and aerobic groups, were observed in aquatic consortia in response to the lower diversity. Specifically, dypB and dypA, are widespread on Earth, indicating their essential roles in lignin depolymerization. Estuarine and marine consortia featured the laccase and mnsod genes, respectively. Notably, the roles of archaea in lignin degradation were revealed in marine ecosystems. Environmental factors strongly influenced functional traits, but weakly shaped taxonomic groups. Null mode analysis further verified that composition of functional traits was deterministic, while taxonomic composition was highly stochastic, demonstrating that the environment selects functional genes rather than taxonomic groups. Our study not only develops a useful tool to study lignin degrading microbial communities via metagenome sequencing but also advances our understanding of ecological traits of these global microbiomes.

The mechanism of low blue light-induced leaf senescence mediated by GmCRY1s in soybean.

Leaf senescence is a crucial trait that has a significant impact on crop quality and yield. Previous studies have demonstrated that light is a key factor in modulating the senescence process. However, the precise mechanism by which plants sense light and control senescence remains largely unknown, particularly in crop species. In this study, we reveal that the reduction in blue light under shading conditions can efficiently induce leaf senescence in soybean. The blue light receptors GmCRY1s rather than GmCRY2s, primarily regulate leaf senescence in response to blue light signals. Our results show that GmCRY1s interact with DELLA proteins under light-activated conditions, stabilizing them and consequently suppressing the transcription of GmWRKY100 to delay senescence. Conversely, LBL reduces the interaction between GmCRY1s and the DELLA proteins, leading to their degradation and premature senescence of leaves. Our findings suggest a GmCRY1s-GmDELLAs-GmWRKY100 regulatory cascade that is involved in mediating LBL-induced leaf senescence in soybean, providing insight into the mechanism of how light signals regulate leaf senescence. Additionally, we generate GmWRKY100 knockout soybeans that show delayed leaf senescence and improved yield under natural field conditions, indicating potential applications in enhancing soybean production by manipulating the leaf senescence trait.

Discordant patterns between nitrogen-cycling functional traits and taxa in distant coastal sediments reveal important community assembly mechanisms.

A central question in microbial ecology is how immense microbes are assembled in changing natural environments while executing critical ecosystem functions. Over the past decade, effort has been made to unravel the contribution of stochasticity and determinism to the compositional of microbial communities. However, most studies focus on microbial taxa, ignoring the importance of functional traits. By employing shotgun metagenomic sequencing and state-of-the-art bioinformatics approaches, this study comprehensively investigated the microbially mediated nitrogen (N) cycling processes in two geographically distant coastal locations. Both shotgun and 16S rRNA gene amplicon sequencing demonstrated significantly differed taxonomic compositions between the two sites. The relative abundance of major microbial phyla, such as Pseudomonadota, Thaumarchaeota, and Bacteroidota, significantly differed. In contrast, high homogeneity was observed for N-cycling functional traits. Statistical analyses suggested that N-cycling taxonomic groups were more related to geographic distance, whereas microbial functional traits were more influenced by environmental factors. Multiple community assembly models demonstrated that determinism strongly governed the microbial N-cycling functional traits, whereas their carrying taxonomic groups were highly stochastic. Such discordant patterns between N-cycling functional traits and taxa demonstrated an important mechanism in microbial ecology in which essential ecosystem functions are stably maintained despite geographic distance and stochastic community assembly.

Unraveling the roles of coastal bacterial consortia in degradation of various lignocellulosic substrates.

Lignocellulose, as the most abundant natural organic carbon on earth, plays a key role in regulating the global carbon cycle, but there have been only few studies in marine ecosystems. Little information is available about the extant lignin-degrading bacteria in coastal wetlands, limiting our understanding of their ecological roles and traits in lignocellulose degradation. We utilized in situ lignocellulose enrichment experiments coupled with 16S rRNA amplicon and shotgun metagenomics sequencing to identify and characterize bacterial consortia attributed to different lignin/lignocellulosic substrates in the southern-east intertidal zone of East China Sea. We found the consortia enriched on woody lignocellulose showed higher diversity than those on herbaceous substrate. This also revealed substrate-dependent taxonomic groups. A time-dissimilarity pattern with increased alpha diversity over time was observed. Additionally, this study identified a comprehensive set of genes associated with lignin degradation potential, containing 23 gene families involved in lignin depolymerization, and 371 gene families involved in aerobic/anaerobic lignin-derived aromatic compound pathways, challenging the traditional view of lignin recalcitrance within marine ecosystems. In contrast to similar cellulase genes among the lignocellulose substrates, significantly different ligninolytic gene groups were observed between consortia under woody and herbaceous substrates. Importantly, we not only observed synergistic degradation of lignin and hemi-/cellulose, but also pinpointed the potential biological actors at the levels of taxa and functional genes, which indicated that the alternation of aerobic and anaerobic catabolism could facilitate lignocellulose degradation. Our study advances the understanding of coastal bacterial community assembly and metabolic potential for lignocellulose substrates. IMPORTANCE It is essential for the global carbon cycle that microorganisms drive lignocellulose transformation, due to its high abundance. Previous studies were primarily constrained to terrestrial ecosystems, with limited information about the role of microbes in marine ecosystems. Through in situ lignocellulose enrichment experiment coupled with high-throughput sequencing, this study demonstrated different impacts that substrates and exposure times had on long-term bacterial community assembly and pinpointed comprehensive, yet versatile, potential decomposers at the levels of taxa and functional genes in response to different lignocellulose substrates. Moreover, the links between ligninolytic functional traits and taxonomic groups of substrate-specific populations were revealed. It showed that the synergistic effect of lignin and hemi-/cellulose degradation could enhance lignocellulose degradation under alternation of aerobic and anaerobic conditions. This study provides valuable taxonomic and genomic insights into coastal bacterial consortia for lignocellulose degradation.

Bacteria rather than fungi mediate the chemodiversity of dissolved organic matter in a mudflat intertidal zone.

Dissolved organic matter (DOM) in natural ecosystems is intimately associated with microbial communities. However, it remains unclear whether the diversity patterns followed by microbes can be transmitted to DOM compounds. Considering the structural properties of DOM compounds and the roles of microbes in ecosystems, we hypothesized that bacteria tended to be more closely associated with DOM compounds than fungi. To test the above hypothesis and bridge this knowledge gap, the diversity patterns and ecological processes for the DOM compounds, and the bacterial and fungal communities in a mudflat intertidal zone were comparatively investigated. As a result, spatial scaling patterns followed by microbes, including the diversity-area and distance-decay relationships, were also observed for DOM compounds. Lipid-like and aliphatic-like molecules comprised the major DOM compounds associated with environmental factors. Both the alpha- and beta-chemodiversity of DOM compounds were significantly associated with the diversity of bacterial communities, but not fungal communities. Co-occurrence ecological network analysis suggested that DOM compounds were more frequently associated with bacteria than fungi. Further, consistent community assembly patterns were observed for DOM and bacterial communities, but not fungal communities. Integrating multiple lines of evidence, this study demonstrated that bacteria rather than fungi mediated the chemodiversity of DOM in the mudflat intertidal zone. This study elucidates the spatial patterns of complex dissolved organic matter (DOM) pools in the intertidal ecosystem, shedding light on the intricate relationship between DOM compounds and bacterial communities.

Environmental heterogeneity and dispersal limitation simultaneously determine the spatial scaling of different microbial functional groups.

Uncovering the mechanisms driving patterns of diversity across space and through time is of critical importance in microbial community ecology. Previous studies suggest that microorganisms also follow the same spatial scaling patterns as macro-organisms. However, it remains unclear whether different microbial functional groups differ in spatial scaling and how different ecological processes may contribute to such differences. In this study, two typical spatial scaling patterns, taxa-area (TAR) and distance-decay relationships (DDR), were investigated for the whole prokaryotic community and seven microbial functional groups using marker genes, including amoA (AOA), amoA (AOB), aprA, dsrB, mcrA, nifH and nirS. Different microbial functional groups harbored different spatial scaling patterns. Microbial functional groups had weaker TAR slope coefficients than the whole prokaryotic community. The archaeal ammonia-oxidizing group, however, displayed a stronger DDR pattern than the bacterial ammonia-oxidizing group. For both TAR and DDR, rare subcommunities were mainly responsible for the observed microbial spatial scaling patterns. Significant associations between environmental heterogeneity and spatial scaling metrics were observed for multiple microbial functional groups. Dispersal limitation, which positively correlated with phylogenetic breadth, was also strongly associated with the strength of microbial spatial scaling. The results demonstrated that environmental heterogeneity and dispersal limitation simultaneously contributed to microbial spatial scaling patterns. This study links microbial spatial scaling patterns with ecological processes, providing mechanistic insights into the typical diversity patterns followed by microbes.

Vertically stratified methane, nitrogen and sulphur cycling and coupling mechanisms in mangrove sediment microbiomes.

BACKGROUND: Mangrove ecosystems are considered as hot spots of biogeochemical cycling, yet the diversity, function and coupling mechanism of microbially driven biogeochemical cycling along the sediment depth of mangrove wetlands remain elusive. Here we investigated the vertical profile of methane (CH4), nitrogen (N) and sulphur (S) cycling genes/pathways and their potential coupling mechanisms using metagenome sequencing approaches. RESULTS: Our results showed that the metabolic pathways involved in CH4, N and S cycling were mainly shaped by pH and acid volatile sulphide (AVS) along a sediment depth, and AVS was a critical electron donor impacting mangrove sediment S oxidation and denitrification. Gene families involved in S oxidation and denitrification significantly (P < 0.05) decreased along the sediment depth and could be coupled by S-driven denitrifiers, such as Burkholderiaceae and Sulfurifustis in the surface sediment (0-15 cm). Interestingly, all S-driven denitrifier metagenome-assembled genomes (MAGs) appeared to be incomplete denitrifiers with nitrate/nitrite/nitric oxide reductases (Nar/Nir/Nor) but without nitrous oxide reductase (Nos), suggesting such sulphide-utilizing groups might be an important contributor to N2O production in the surface mangrove sediment. Gene families involved in methanogenesis and S reduction significantly (P < 0.05) increased along the sediment depth. Based on both network and MAG analyses, sulphate-reducing bacteria (SRB) might develop syntrophic relationships with anaerobic CH4 oxidizers (ANMEs) by direct electron transfer or zero-valent sulphur, which would pull forward the co-existence of methanogens and SRB in the middle and deep layer sediments. CONCLUSIONS: In addition to offering a perspective on the vertical distribution of microbially driven CH4, N and S cycling genes/pathways, this study emphasizes the important role of S-driven denitrifiers on N2O emissions and various possible coupling mechanisms of ANMEs and SRB along the mangrove sediment depth. The exploration of potential coupling mechanisms provides novel insights into future synthetic microbial community construction and analysis. This study also has important implications for predicting ecosystem functions within the context of environmental and global change. Video Abstract.

Distinct Ecological Processes Mediate Domain-Level Differentiation in Microbial Spatial Scaling.

The spatial scaling of biodiversity, such as the taxa-area relationship (TAR) and distance-decay relationship (DDR), is a typical ecological pattern that is followed by both microbes and macrobes in natural ecosystems. Previous studies focusing on microbes mainly aimed to address whether and how different types of microbial taxa differ in spatial scaling patterns, leaving the underlying mechanisms largely untouched. In this study, the spatial scaling of different microbial domains and their associated ecological processes in an intertidal zone were comparatively investigated. The significant spatial scaling of biodiversity could be observed across all microbial domains, including archaea, bacteria, fungi, and protists. Among them, archaea and fungi were found with much stronger DDR slopes than those observed in bacteria and protists. For both TAR and DDR, rare subcommunities were mainly responsible for the observed spatial scaling patterns, except for the DDR of protists and bacteria. This was also evidenced by extending the TAR and DDR diversity metrics to Hill numbers. Further statistical analyses demonstrated that different microbial domains were influenced by different environmental factors and harbored distinct local community assembly processes. Of these, drift was mainly responsible for the compositional variations of bacteria and protists. Archaea were shaped by strong homogeneous selection, whereas fungi were more affected by dispersal limitation. Such differing ecological processes resulted in the domain-level differentiation of microbial spatial scaling. This study links ecological processes with microbial spatial scaling and provides novel mechanistic insights into the diversity patterns of microbes that belong to different trophic levels. IMPORTANCE As the most diverse and numerous life form on Earth, microorganisms play indispensable roles in natural ecological processes. Revealing their diversity patterns across space and through time is of essential importance to better understand the underlying ecological mechanisms controlling the distribution and assembly of microbial communities. However, the diversity patterns and their underlying ecological mechanisms for different microbial domains and/or trophic levels require further exploration. In this study, the spatial scaling of different microbial domains and their associated ecological processes in a mudflat intertidal zone were investigated. The results showed different spatial scaling patterns for different microbial domains. Different ecological processes underlie the domain-level differentiation of microbial spatial scaling. This study links ecological processes with microbial spatial scaling to provide novel mechanistic insights into the diversity patterns of microorganisms that belong to different trophic levels.

To assemble or not to assemble: metagenomic profiling of microbially mediated biogeochemical pathways in complex communities.

High-throughput profiling of microbial functional traits involved in various biogeochemical cycling pathways using shotgun metagenomic sequencing has been routinely applied in microbial ecology and environmental science. Multiple bioinformatics data processing approaches are available, including assembly-based (single-sample assembly and multi-sample assembly) and read-based (merged reads and raw data). However, it remains not clear how these different approaches may differ in data analyses and affect result interpretation. In this study, using two typical shotgun metagenome datasets recovered from geographically distant coastal sediments, the performance of different data processing approaches was comparatively investigated from both technical and biological/ecological perspectives. Microbially mediated biogeochemical cycling pathways, including nitrogen cycling, sulfur cycling and B12 biosynthesis, were analyzed. As a result, multi-sample assembly provided the most amount of usable information for targeted functional traits, at a high cost of computational resources and running time. Single-sample assembly and read-based analysis were comparable in obtaining usable information, but the former was much more time- and resource-consuming. Critically, different approaches introduced much stronger variations in microbial profiles than biological differences. However, community-level differences between the two sampling sites could be consistently observed despite the approaches being used. In choosing an appropriate approach, researchers shall balance the trade-offs between multiple factors, including the scientific question, the amount of usable information, computational resources and time cost. This study is expected to provide valuable technical insights and guidelines for the various approaches used for metagenomic data analysis.

Effective data filtering is prerequisite for robust microbial association network construction.

Microorganisms do not exist as individual population in the environment. Rather, they form complex assemblages that perform essential ecosystem functions and maintain ecosystem stability. Besides the diversity and composition of microbial communities, deciphering their potential interactions in the form of association networks has attracted many microbiologists and ecologists. Much effort has been made toward the methodological development for constructing microbial association networks. However, microbial profiles suffer dramatically from zero values, which hamper accurate association network construction. In this study, we investigated the effects of zero-value issues associated with microbial association network construction. Using the TARA Oceans microbial profile as an example, different zero-value-treatment approaches were comparatively investigated using different correlation methods. The results suggested dramatic variations of correlation coefficient values for differently treated microbial profiles. Most specifically, correlation coefficients among less frequent microbial taxa were more affected, whichever method was used. Negative correlation coefficients were more problematic and sensitive to network construction, as many of them were inferred from low-overlapped microbial taxa. Consequently, microbial association networks were greatly differed. Among various approaches, we recommend sequential calculation of correlation coefficients for microbial taxa pairs by excluding paired zero values. Filling missing values with pseudo-values is not recommended. As microbial association network analyses have become a widely used technique in the field of microbial ecology and environmental science, we urge cautions be made to critically consider the zero-value issues in microbial data.

Random sampling associated with microbial profiling leads to overestimated stochasticity inference in community assembly.

Revealing the mechanisms governing the complex community assembly over space and time is a central issue in ecology. Null models have been developed to quantitatively disentangle the relative importance of deterministic vs. stochastic processes in structuring the compositional variations of biological communities. Similar approaches have been recently extended to the field of microbial ecology. However, the profiling of highly diverse biological communities (e.g., microbial communities) is severely influenced by random sampling issues, leading to undersampled community profiles and overestimated ß-diversity, which may further affect stochasticity inference in community assembly. By implementing simulated datasets, this study demonstrate that microbial stochasticity inference is also affected due to random sampling issues associated with microbial profiling. The effects on microbial stochasticity inference for the whole community and the abundant subcommunities were different using different randomization methods in generating null communities. The stochasticity of rare subcommunities, however, was persistently overestimated irrespective of which randomization method was used. Comparatively, the stochastic ratio approach was more sensitive to random sampling issues, whereas the Raup-Crick metric was more affected by randomization methods. As more studies begin to focus on the mechanisms governing abundant and rare subcommunities, we urge cautions be taken for microbial stochasticity inference based on ß-diversity, especially for rare subcommunities. Randomization methods to generate null communities shall also be carefully selected. When necessary, the cutoff used for judging the relative importance of deterministic vs. stochastic processes shall be redefined.

Contrasting archaeal and bacterial community assembly processes and the importance of rare taxa along a depth gradient in shallow coastal sediments.

Marine microbial communities assemble along a sediment depth gradient and are responsible for processing organic matter. Composition of the microbial community along the depth is affected by various biotic and abiotic factors, e.g., the change of redox gradient, the availability of organic matter, and the interactions of different taxa. The community structure is also subjected to some random changes caused by stochastic processes of birth, death, immigration and emigration. However, the high-resolution shifts of microbial community and mechanisms of the vertical assembly processes in marine sediments remain poorly described. Archaeal and bacterial communities were analyzed based on 16S rRNA gene amplicon sequencing and metagenomes in the Bohai Sea sediment samples. The archaeal community was dominated by Thaumarchaeota with increased alpha diversity along depth. Proteobacteria was the dominant bacterial group with decreased alpha diversity as depth increased. Sampling sites and depths collectively affected the beta-diversity for both archaeal and bacterial communities. The dominant mechanism determining archaeal community assembly was determinism, which was mostly contributed by homogeneous selection, i.e., consistent selection pressures in different locations or depths. In contrast, bacterial community assembly was dominated by stochasticity. Co-occurrence networks among different taxa and key functional genes revealed a tight community with low modularity in the bottom sediment, and disproportionately more interactions among low abundant ASVs. This suggests a significant contribution to community stabilization by rare taxa, and suggests that the bottom layer, rather than surface sediments may represent a hotspot for benthic microbial interactions.

PCycDB: a comprehensive and accurate database for fast analysis of phosphorus cycling genes.

BACKGROUND: Phosphorus (P) is one of the most essential macronutrients on the planet, and microorganisms (including bacteria and archaea) play a key role in P cycling in all living things and ecosystems. However, our comprehensive understanding of key P cycling genes (PCGs) and microorganisms (PCMs) as well as their ecological functions remains elusive even with the rapid advancement of metagenome sequencing technologies. One of major challenges is a lack of a comprehensive and accurately annotated P cycling functional gene database. RESULTS: In this study, we constructed a well-curated P cycling database (PCycDB) covering 139 gene families and 10 P metabolic processes, including several previously ignored PCGs such as pafA encoding phosphate-insensitive phosphatase, ptxABCD (phosphite-related genes), and novel aepXVWPS genes for 2-aminoethylphosphonate transporters. We achieved an annotation accuracy, positive predictive value (PPV), sensitivity, specificity, and negative predictive value (NPV) of 99.8%, 96.1%, 99.9%, 99.8%, and 99.9%, respectively, for simulated gene datasets. Compared to other orthology databases, PCycDB is more accurate, more comprehensive, and faster to profile the PCGs. We used PCycDB to analyze P cycling microbial communities from representative natural and engineered environments and showed that PCycDB could apply to different environments. CONCLUSIONS: We demonstrate that PCycDB is a powerful tool for advancing our understanding of microbially driven P cycling in the environment with high coverage, high accuracy, and rapid analysis of metagenome sequencing data. The PCycDB is available at https://github.com/ZengJiaxiong/Phosphorus-cycling-database . Video Abstract.

Functional Traits Resolve Mechanisms Governing the Assembly and Distribution of Nitrogen-Cycling Microbial Communities in the Global Ocean.

Microorganisms drive much of the marine nitrogen (N) cycle, which jointly controls the primary production in the global ocean. However, our understanding of the microbial communities driving the global ocean N cycle remains fragmented. Focusing on "who is doing what, where, and how?", this study draws a clear picture describing the global biogeography of marine N-cycling microbial communities by utilizing the Tara Oceans shotgun metagenomes. The marine N-cycling communities are highly variable taxonomically but relatively even at the functional trait level, showing clear functional redundancy properties. The functional traits and taxonomic groups are shaped by the same set of geo-environmental factors, among which, depth is the major factor impacting marine N-cycling communities, differentiating mesopelagic from epipelagic communities. Latitudinal diversity gradients and distance-decay relationships are observed for taxonomic groups, but rarely or weakly for functional traits. The composition of functional traits is strongly deterministic as revealed by null model analysis, while a higher degree of stochasticity is observed for taxonomic composition. Integrating multiple lines of evidence, in addition to drawing a biogeographic picture of marine N-cycling communities, this study also demonstrated an essential microbial ecological theory-determinism governs the assembly of microbial communities performing essential biogeochemical processes; the environment selects functional traits rather than taxonomic groups; functional redundancy underlies stochastic taxonomic community assembly. IMPORTANCE A critical question in microbial ecology is how the complex microbial communities are formed in natural ecosystems with the existence of thousands different species, thereby performing essential ecosystem functions and maintaining ecosystem stability. Previous studies disentangling the community assembly mechanisms mainly focus on microbial taxa, ignoring the functional traits they carry. By anchoring microbial functional traits and their carrying taxonomic groups involved in nitrogen cycling processes, this study demonstrated an important mechanism associated with the complex microbial community assembly. Evidence shows that the environment selects functional traits rather than taxonomic groups, and functional redundancy underlies stochastic taxonomic community assembly. This study is expected to provide valuable mechanistic insights into the complex microbial community assembly in both natural and artificial ecosystems.

Determinism governs the succession of disturbed bacterioplankton communities in a coastal maricultural ecosystem.

Bacterioplankton community is the major engine that drives the biogeochemical cycling of various nutrient and essential elements in the coastal ecosystem. Unraveling the mechanisms governing the succession of such complex bacterioplankton communities in dynamic environment is a challenging issue in environmental science. In this study, we investigated the diversity patterns and succession mechanisms of both free-living and particle-attached bacterioplankton communities that have been exposed to low oxygen and typhoon Lekima. The community evenness was the lowest in August when the temperature was high and dissolved oxygen was low. Similar patterns in community succession were observed for free-living and particle-attached bacterioplankton community after the passing through of typhoon Lekima. Both the free-living and particle-attached bacterioplankton communities in the surface and bottom water columns were strongly affected by geo-environmental factors, among which temperature was the common factor, suggesting that the metabolic theory of ecology also underlie the dynamic patterns of bacterioplankton communities. Although the surface and bottom bacterioplankton compositions were initially different taxonomically and shaped by different environmental conditions, they followed a similar succession pattern over the sampling months. Temperature, dissolved inorganic carbon and dissolved inorganic phosphorus were the major factors associated with the variations of surface bacterioplankton ASVs. Time-decay relationship, in which community similarity decreases with increasing time interval, was clearly observed. Such pattern shall be attributed to the combined effects of time and the changing environmental factors over the sampling months, rather than time alone. Integrating multiple lines of evidences, we demonstrated that determinism governed the succession of both free-living and particle-attached bacterioplankton communities in the coastal maricultural ecosystem, with higher stochastic ratio in habitable months (i.e. fall). This study is expected to provide valuable mechanistic insights into the succession of disturbed complex bacterioplankton communities.

Metagenomic evidence for the microbial transformation of carboxyl-rich alicyclic molecules: A long-term macrocosm experiment.

Carboxyl-rich alicyclic molecules (CRAMs) widely exist in the ocean and constitute the central part of the refractory dissolved organic matter (RDOM) pool. Although a consensus has been reached that microbial activity forms CRAMs, the detailed molecular mechanisms remain largely unexplored. To better understand the underlying genetic mechanisms driving the microbial transformation of CRAM, a long-term macrocosm experiment spanning 220 days was conducted in the Aquatron Tower Tank at Dalhousie University, Halifax, Canada, with the supply of diatom-derived DOM as a carbon source. The DOM composition, community structure, and metabolic pathways were characterised using multi-omics approaches. The addition of diatom lysate introduced a mass of labile DOM into the incubation seawater, which led to a low degradation index (IDEG) and refractory molecular lability boundary (RMLB) on days 1 and 18. The molecular compositions of the DOM molecules in the later incubation period (from day 120 to day 220) were more similar in composition to those on day 0, suggesting a rapid turnover of phytoplankton debris by microbial communities. Taxonomically, while Alpha proteobacteria dominated during the entire incubation period, Gamma proteobacteria became more sensitive and abundant than the other bacterial groups on days 1 and 18. Recalcitrant measurements such as IDEG and RMLB were closely related to the DOM molecules, bacterial community, and Kyoto encyclopaedia of Genes and Genomes (KEGG) modules, suggesting close associations between RDOM accumulation and microbial metabolism. KEGG modules that showed strong positive correlation with CRAMs were identified using a microbial ecological network approach. The identified KEGG modules produced the substrates, such as the acetyl-CoA or 3­hydroxy-3-methylglutaryl-CoA, which could participate in the mevalonate pathway to generate the precursor of CRAM analogues, isopentenyl-PP, suggesting a potential generation pathway of CRAM analogues in bacteria and archaea. This study revealed the potential genetic and molecular processes involved in the microbial origin of CRAM analogues, and thus indicated a vital ecological role of bacteria and archaea in RDOM production. This study also offered new perspectives on the carbon sequestration in the ocean.

MCycDB: A curated database for comprehensively profiling methane cycling processes of environmental microbiomes.

Methane is a critical greenhouse gas with significant impacts on environmental and global change. However, CH4 cycling processes and coupling mechanisms with the biogeochemical cycling of carbon, nitrogen, sulfur and metals in the environment remain elusive. To fill such knowledge gaps, we constructed a manually curated methane cycling database (MCycDB) for comprehensive and accurate analysis of methane cycling microbial communities. MCycDB contains 298 methane cycling gene families covering 10 methane metabolism pathways with 610,208 representative sequences, and associated reference sequences from the NCBI RefSeq database with 48 phyla and 2,197 genera, and five phyla and 100 genera for bacteria and archaea, respectively. Also, homologous groups from public orthology databases were identified and included in MCycDB to reduce false positive assignments. We applied MCycDB to profile methane cycling gene families and associated taxonomic groups from various environments. Gene families involved in methanogenesis were abundant in hot spring sediment and less abundant in freshwater, whereas the ones involved in aerobic oxidation of methane were abundant in permafrost and peatland. This study demonstrates that MCycDB is a useful tool for studying microbially-driven methane cycling processes with high specificity, coverage and accuracy.

Eco-engineering approaches for ocean negative carbon emission.

The goal of achieving carbon neutrality in the next 30-40 years is approaching worldwide consensus and requires coordinated efforts to combat the increasing threat of climate change. Two main sets of actions have been proposed to address this grand goal. One is to reduce anthropogenic CO2 emissions to the atmosphere, and the other is to increase carbon sinks or negative emissions, i.e., removing CO2 from the atmosphere. Here we advocate eco-engineering approaches for ocean negative carbon emission (ONCE), aiming to enhance carbon sinks in the marine environment. An international program is being established to promote coordinated efforts in developing ONCE-relevant strategies and methodologies, taking into consideration ecological/biogeochemical processes and mechanisms related to different forms of carbon (inorganic/organic, biotic/abiotic, particulate/dissolved) for sequestration. We focus on marine ecosystem-based approaches and pay special attention to mechanisms that require transformative research, including those elucidating interactions between the biological pump (BP), the microbial carbon pump (MCP), and microbially induced carbonate precipitation (MICP). Eutrophic estuaries, hypoxic and anoxic waters, coral reef ecosystems, as well as aquaculture areas are particularly considered in the context of efforts to increase their capacity as carbon sinks. ONCE approaches are thus expected to be beneficial for both carbon sequestration and alleviation of environmental stresses.

VB12Path for Accurate Metagenomic Profiling of Microbially Driven Cobalamin Synthesis Pathways.

Cobalamin (vitamin B12; VB12) is an indispensable nutrient for all living entities in the Earth's biosphere and plays a vital role in both natural and host environments. Currently in the metagenomic era, gene families of interest are extracted and analyzed based on functional profiles by searching shotgun metagenomes against public databases. However, critical issues exist in applying public databases for specific processes such as VB12 biosynthesis pathways. We developed a curated functional gene database termed VB12Path for accurate metagenomic profiling of VB12 biosynthesis gene families of microbial communities in complex environments. VB12Path contains a total of 60 VB12 synthesis gene families, 287,731 sequences, and 21,154 homology groups, and it aims to provide accurate functional and taxonomic profiles of VB12 synthesis pathways for shotgun metagenomes and minimize false-positive assignments. VB12Path was applied to characterize cobalamin biosynthesis gene families in human intestines and marine environments. The results demonstrated that ocean and human intestine had dramatically different VB12 synthesis processes and that gene families belonging to salvage and remodeling pathway dominated human intestine but were lowest in the ocean ecosystem. VB12Path is expected to be a useful tool to study cobalamin biosynthesis processes via shotgun metagenome sequencing in both environmental and human microbiome research. IMPORTANCE Vitamin B12 (VB12) is an indispensable nutrient for all living entities in the world but can only be synthesized by a small subset of prokaryotes. Therefore, this small subset of prokaryotes controls ecosystem stability and host health to some extent. However, critical accuracy and comprehensiveness issues exist in applying public databases to profile VB12 synthetic gene families and taxonomic groups in complex metagenomes. In this study, we developed a curated functional gene database termed VB12Path for accurate metagenomic profiling of VB12 communities in complex environments. VB12Path is expected to serve as a valuable tool to uncover the hidden microbial communities producing this precious nutrient on Earth.