Comparative Analysis of Root Microbiomes of Rice Cultivars with High and Low Methane Emissions Reveals Differences in Abundance of Methanogenic Archaea and Putative Upstream Fermenters.

Rice cultivation worldwide accounts for ∼7 to 17% of global methane emissions. Methane cycling in rice paddies is a microbial process not only involving methane producers (methanogens) and methane metabolizers (methanotrophs) but also other microbial taxa that affect upstream processes related to methane metabolism. Rice cultivars vary in their rates of methane emissions, but the influence of rice genotypes on methane cycling microbiota has been poorly characterized. Here, we profiled the rhizosphere, rhizoplane, and endosphere microbiomes of a high-methane-emitting cultivar (Sabine) and a low-methane-emitting cultivar (CLXL745) throughout the growing season to identify variations in the archaeal and bacterial communities relating to methane emissions. The rhizosphere of the high-emitting cultivar was enriched in methanogens compared to that in the low emitter, whereas the relative abundances of methanotrophs between the cultivars were not significantly different. Further analysis of cultivar-sensitive taxa identified families enriched in the high emitter that are associated with methanogenesis-related processes. The high emitter had greater relative abundances of sulfate-reducing and iron-reducing taxa which peak earlier in the season than methanogens and are necessary to lower soil oxidation reduction potential before methanogenesis can occur. The high emitter also had a greater abundance of fermentative taxa which produce methanogenesis precursors (acetate, CO2, and H2). Furthermore, the high emitter was enriched in taxa related to acetogenesis which compete with methanogens for CO2 and H2 These taxa were enriched in a spatio-specific manner and reveal a complex network of microbial interactions on which plant genotype-dependent factors can act to affect methanogenesis and methane emissions.IMPORTANCE Rice cultivation is a major source of anthropogenic emissions of methane, a greenhouse gas with a potentially severe impact on climate change. Emission variation between rice cultivars suggests the feasibility of breeding low-emission rice, but there is a limited understanding of how genotypes affect the microbiota involved in methane cycling. Here, we show that the root microbiome of the high-emitting cultivar is enriched both in methanogens and in taxa associated with fermentation, iron, and sulfate reduction and acetogenesis, processes that support methanogenesis. Understanding how cultivars affect microbes with methanogenesis-related functions is vital for understanding the genetic basis for methane emission in rice and can aid in the development of breeding programs that reduce the environmental impact of rice cultivation

Soil microbial communities in restored and unrestored coastal dune ecosystems in California

Most restoration projects involving invasive plant eradication tend to focus on plant removal with little consideration given to how these invasives change soil microbial communities. However, soil microorganisms can determine invasibility of habitats and, in turn, be altered by invasives once established, potentially inhibiting native plant establishment. We studied soil microbial communities in coastal dunes with varying invasion intensity and different restoration approaches (herbicide, mechanical excavation) at Point Reyes National Seashore. Overall, we found evidence of a strong link between bacterial and fungal soil communities and the presence of invasives and restoration approach. Heavily invaded sites were characterized by a lower abundance of putatively identified nitrifiers, fermentative bacteria, fungal parasites, and fungal dung saprotrophs and a higher abundance of cellulolytic bacteria and a class of arbuscular mycorrhizal fungi (Archaeosporomycetes). Changes in soil microbiota did not fully dissipate following removal of invasives using herbicide, with exception of reductions in cellulolytic bacteria and Archaeosporomycetes abundance. Mechanical restoration effectively removed both invasives and soil legacy effects by inverting or “flipping” rhizome-contaminated surface soils with soils from below and may have inadvertently induced other adverse effects on soils that impeded reestablishment of native dune plants. Land managers should consider additional measures to counteract lingering legacy effects and/or focus restoration efforts in areas where legacy effects are less pronounced

Can We Use Functional Annotation of Prokaryotic Taxa (FAPROTAX) to Assign the Ecological Functions of Soil Bacteria?

FAPROTAX is a promising tool for predicting ecological relevant functions of bacterial and archaeal taxa derived from 16S rRNA amplicon sequencing. The database was initially developed to predict the function of marine species using standard microbiological references. This study, however, has attempted to access the application of FAPROTAX in soil environments. We hypothesized that FAPROTAX was compatible with terrestrial ecosystems. The potential use of FAPROTAX to assign ecological functions of soil bacteria was investigated using meta-analysis and our newly designed experiments. Soil samples from two major terrestrial ecosystems, including agricultural land and forest, were collected. Bacterial taxonomy was analyzed using Illumina sequencing of the 16S rRNA gene and ecological functions of the soil bacteria were assigned by FAPROTAX. The presence of all functionally assigned OTUs (Operation Taxonomic Units) in soil were manually checked using peer-reviewed articles as well as standard microbiology books. Overall, we showed that sample source was not a predominant factor that limited the application of FAPROTAX, but poor taxonomic identification was. The proportion of assigned taxa between aquatic and non-aquatic ecosystems was not significantly different (p > 0.05). There were strong and significant correlations (σ = 0.90–0.95, p < 0.01) between the number of OTUs assigned to genus or order level and the number of functionally assigned OTUs. After manual verification, we found that more than 97% of the FAPROTAX assigned OTUs have previously been detected and potentially performed functions in agricultural and forest soils. We further provided information regarding taxa capable of N-fixation, P and K solubilization, which are three main important elements in soil systems and can be integrated with FAPROTAX to increase the proportion of functionally assigned OTUs. Consequently, we concluded that FAPROTAX can be used for a fast-functional screening or grouping of 16S derived bacterial data from terrestrial ecosystems and its performance could be enhanced through improving the taxonomic and functional reference databases

MICROPHERRET: MICRObial PHEnotypic tRait ClassifieR using Machine lEarning Techniques

Background: In recent years, there has been a rapid increase in the number of microbial genomes reconstructed through shotgun sequencing, and obtained by newly developed approaches including metagenomic binning and single-cell sequencing. However, our ability to functionally characterize these genomes by experimental assays is orders of magnitude less efficient. Consequently, there is a pressing need for the development of swift and automated strategies for the functional classification of microbial genomes. Results: The present work leverages a suite of supervised machine learning algorithms to establish a range of 86 metabolic and other ecological functions, such as methanotrophy and plastic degradation, starting from widely obtainable microbial genome annotations. Tests performed on independent datasets demonstrated robust performance across complete, fragmented, and incomplete genomes above a 70% completeness level for most of the considered functions. Application of the algorithms to the Biogas Microbiome database yielded predictions broadly consistent with current biological knowledge and correctly detecting functionally-related nuances of archaeal genomes. Finally, a case study focused on acetoclastic methanogenesis demonstrated how the developed machine learning models can be refined or expanded with models describing novel functions of interest. Conclusions: The resulting tool, MICROPHERRET, incorporates a total of 86 models, one for each tested functional class, and can be applied to high-quality microbial genomes as well as to low-quality genomes derived from metagenomics and single-cell sequencing. MICROPHERRET can thus aid in understanding the functional role of newly generated genomes within their micro-ecological context

Environmental drivers of soil and plant microbiomes in agricultural and grassland ecosystems

Soils and plant microbial communities are intricately linked to ecosystem functioning as they play important roles in nutrients dynamics as decomposers and feedback to plant communities as mutualists and pathogens. Numerous soil physicochemical factors as well as the land use management are shaping the composition and dynamics of microbial community. In addition, global warming and climate change are the most prominent of all environmental factors that influence all kinds of the living organisms including microbes associated to the plant soil systems. A better understanding of the environmental drivers shaping these microbial communities especially under future climate will help to understand and predict the expected changes of ecosystems functions and accordingly of the services they provide. In addition, such knowledge will help to detect potential ways on how soil microorganisms can be harnessed to help mitigating the negative consequences of climate change.The Global Change Experimental Facility (GCEF) is settled in the field research station of the Helmholtz Centre for Environmental Research (UFZ) in Bad Lauchstädt, Saxony-Anhalt, Germany (51_22’60 N, 11_50’60 E, 118 m a.s.l.). This facility has been designed to investigate the consequences of a predicted future climate scenario expected in 50-70 years in Central Germany on ecosystem processes under different land-use regimes applied on large field plots in comparison to similar sets of plots under the ambient climate. We performed our study using this research facility, with the aim to analyze the impact of future climate conditions, soil physicochemical factors, and/or land use type and intensity on microbial communities in different habitats (rhizosphere soil, plant endosphere, and plant residues) in grassland and cropland ecosystems. To assess the microbial communities, we used the highly sensitive and powerful highthroughput next generation sequencing, Illumina Miseq.This thesis constitutes the first assessment of microbial communities in the GCEF experimental facility. The samples were collected in 2015 for manuscript 4, while for manuscripts 1, 2, 3, 5, 6, the samples were collected in 2018-2019. Manuscript 1: (Sansupa, Wahdan, Hossen et al., 2021; Applied Science 2021, 11, 688) “Can we use functional annotation of prokaryotic taxa (FAPROTAX) to assign the ecological functions of investigated the potential use of FAPROTAX for bacterial functional annotation in non-aquatic ecosystems, specifically in soil. For this study, we used microbial datasets of soil systems including rhizosphere soil of Trifolium pratense from the extensively used meadow plots in the GCEF. We hypothesized that FAPROTAX can be used in terrestrial ecosystems. Our survey revealed that FAPROTAX tool can be used for screening or grouping of 16S derived bacterial data from terrestrial ecosystems and its performance could be enhanced through improving the taxonomic and functional reference databases. Manuscript 2: (Wahdan et al., 2021; Frontiers in Microbiology 12:629169) “Targeting the active rhizosphere microbiome of Trifolium pratense in grassland evidences a stronger-than-expected belowground biodiversity-ecosystem functioning link”. In this study, we used the bromodeoxyuridine (BrdU) immunocapture technique combined with pair-end Illumina sequencing to differentiate between total and active microbiomes (including both bacteria and fungi) in the rhizosphere of T. pratense. In the same rhizosphere soil samples, we also measured the activities of three microbial extracellular hydrolytic enzymes, (ß-glucosidase, N-acetylglucosaminidase, and acid phosphatase), which play central roles in the C, N, and P acquisition. We investigated the proportion of active and total rhizosphere microbiomes, and their responses to the manipulated future climate in the GCEF. In addition, we identified the possible links between total and active microbiomes and the soil ecosystem function (extracellular enzyme production). Our results revealed that the active microbes of the rhizosphere represented 42.8 and 32.1% of the total bacterial and fungal operational taxonomic units (OTUs), respectively. Active and total microbial fractions were taxonomically and functionally diverse and displayed different responses to variations of soil physicochemical factors. We also showed that the richness of overall and specific functional groups of active microbes in rhizosphere soil significantly correlated with the measured enzyme activities, while total microbial richness did not. Manuscript 3: (Wahdan et al., 2021; Microbiology Open 10:e1217) “Deciphering Trifolium pratense L. (red clover) holobiont reveals a resistant microbial community assembly to future climate changes predicted for the next 50–70 years”. We investigated the microbial communities of bacteria and fungi associated with four plant parts of T. pratense (the rhizosphere and the endopheres of the roots, whole shoot system (leaves and stems), and of the flower) and evaluated their potential ecological and metabolic functions in response to future climate conditions. This study was performed on the GCEF extensively managed grassland plots. Our analyses indicated that plant tissue/compartments differentiation enables the formation of a unique ecological niches that harbor specific microbial communities. Except for the fungal communities of the aboveground compartments, T. pratense microbiome diversity and community composition showed a resistance against the future climate changes. We also analyzed the predicted bacterial metabolic functional genes of red clover. Thereby, we detected microbial genes involved in plant growth processes, such as biofertilisation (nitrogen fixation, phosphorus solubilisation, and siderophore biosynthesis) and biostimulation (phytohormone and auxin production), which were not influenced by the future climate. Manuscript 4: (Wahdan et al., 2021; Environmental Microbiology) “Organic agricultural practice enhances arbuscular mycorrhizal symbiosis in correspondence to soil warming and altered precipitation patterns”. This study was performed on the conventional and organic farming plots under both ambient and future climate conditions. We evaluated the effect of climate (ambient vs. future), agricultural practice (conventional vs. organic farming) and their interaction on Arbuscular Mycorrhizal Fungi (AMF) community composition and richness inside wheat roots. In addition, we evaluated the relationship between molecular richness of indigenous root AMF and wheat yield parameters. Future climate altered the total AMF community composition and a sub-community of Glomeraceae. Further, application of different agricultural practices altered both total AMF and Glomeraceae community, whereby organic farming appeared to enhance total AMF and Diversisporaceae richness. Under the future climate scenario, organic farming enhanced total AMF and Gigasporaceae richness in comparison with conventional farming. Our results revealed a positive correlation between AMF richness and wheat nutrient contents not only in organic farming system but also under conventionally managed fields. Manuscript 5: (Wahdan et al., 2020; Microorganisms 8, 908) “Future climate significantly alters fungal plant pathogen dynamics during the early phase of wheat litter decomposition”. This study was performed on the conventional farming plots. We investigated the structure and ecological functions of fungal communities colonizing wheat during the early phase of decomposition (0, 30, and 60 days) under current and future climate conditions. We found that plant pathogenic fungi dominated (~87% of the total sequences) within the wheat residue mycobiome. Destructive wheat fungal pathogens such as Fusarium graminearum, Fusarium tricinctum, and Zymoseptoria tritci were detected under ambient and future climates. Additionally, the future climate brought new pathogens to the system. Manuscript 6: (Wahdan et al., 2021; Microbial Ecology 10.1007/s00248-021-01840-6) “Life in the wheat litter: effects of future climate on microbiome and function during the early phase of decomposition”. This study was performed on the conventional farming plots. We assessed the effects of climate change on microbial richness, community compositions, interactions and their functions (production of extracellular enzymes) in decomposing residues of wheat. In addition, we investigated the effects of climate change on litter residues physicochemical factors as well as on mass loss during the early phase of decomposition. Future climate significantly accelerated litter mass loss as compared with ambient one. Our results indicated that future climate significantly increased fungal richness and altered fungal communities over time, while bacterial communities were more resistant in wheat residues. Fungi corresponded to different physicochemical elements of litter under ambient (C, Ca2+ and pH) and future (C/N, N, P, K+, Ca2+ and pH) climate conditions. Also, a highly correlative interactions between richness of bacteria and fungi were detected under future climate. Activities of microbial β-glucosidase and N-acetylglucosaminidase in wheat straw were significantly higher under future climate. Such high enzymatic activities were coupled with a significant positive correlation between microbial (both bacteria and fungi) richness and community compositions with these two enzymatic activities only under future climate.:CONTENTS BIBLIOGRAPHIC DESCRIPTION……………………………………………….......III ZUSAMMENFASSUNG………………………………………………………...........V SUMMARY……………………………………………………………………………..X GENERAL INTRODUCTION…………………………………………………………………...............1 I-1 Ecosystem functions carried out by soil and plant microbiomes…………………..2 I-2 Biodiversity and functional diversity and maintenance of ecosystem functions……………..3 I-3 Total vs. active microbial diversity for assessing ecosystem functions……………4 I-4 Factors influencing soil and plant microbiota…………………………………..……6 I-4.1 Elements of climate changes……………………………………………................7 I-4.2 Climate changes influence microbes in an interacting, complex manner………8 I-4.3 Environmental factors controlling the response of microorganisms to climate changes………………………………………………………………………………….....10 I-5 Interplay between climate and land use intensity in agroecosystems……………11 I-6 Study site, and overall objectives………………………………………………....…12 I-7 Methods used for the taxonomic and functional characterization of the microbiomes……...15 I-8 Presentation of aims and hypotheses of the publications/manuscripts in different chapters.................................................................................................................16 I-9References.........................................................................................................20 CHAPTER 1 Can we use functional annotation of prokaryotic taxa (FAPROTAX) to assign the ecological functions of soil bacteria? .....................................................................29 Publication…………………………………………………………………………...........31 Supplementary materials…………………………………………………………….......42 CHAPTER 2 Targeting the active rhizosphere microbiome of Trifolium pratense in grassland evidences a stronger-than-expected belowground biodiversity-ecosystem functioning link………………..........................................................................…49 Publication………………………………………………………………………………51 Supplementary materials……………………………………………………………..67 CHAPTER 3 Deciphering Trifolium pratense L. holobiont reveals a microbiome resilient to future climate changes……………………………………………….…………………………..89 Publication………………………………………………………………………………….91 Supplementary materials……………………………………………………………….111 CHAPTER 4 Organic agricultural practice enhances arbuscular mycorrhizal symbiosis in correspondence to soil warming and altered precipitation patterns………………125 Publication……………………………………………………………………………….127 Supplementary materials………………………………………………………….......140 CHAPTER 5 Future climate significantly alters fungal plant pathogen dynamics during the early phase of wheat litter decomposition…...................………………….……………..156 Publication………………………………………………...…………….….…………...158 Supplementary materials………………………………………………….…....……..175 CHAPTER 6 Life in the wheat litter: effects of future climate on microbiome and function during the early phase of decomposition…………………………………….....……....…….181 Publication…………………………………..…………………………………….....…...183 Supplementary materials………………………………………………………………..199 GENERAL DISCUSSION…………………………………………………………….......210 D-I Approaches and main findings of the result chapters………………………..…211 D-2 Conclusion and implications of the study findings…………………………...…215 D-3 Technical limitation of the study……………………………………………......…217 D-4 Future prospects of the study field ...……………………………………………217 D-5 References…………………………………………………………………………..219 DATA AVAILABILITY……………………………………………………………………...223 ACKNOWLEDGEMENTS……………………………………………………………......224 CURRICULUM VITAE……………………………………………………………….....…225 LIST OF PUBLICATIONS………………………………………………………….........226 CONFERENCE PROCEEDINGS…………………………………………………….....227 STATUTORY DECLARATION………………………………………………................228 VERIFICATION OF AUTHOR PARTS……………………………………………........22

Elucidate microbial characteristics in a fullscale treatment plant for offshore oil produced wastewater

Oil-produced wastewater treatment plants, especially those involving biological treatment processes, harbor rich and diverse microbes. However, knowledge of microbial ecology and microbial interactions determining the efficiency of plants for oil-produced wastewater is limited. Here, we performed 16S rDNA amplicon sequencing to elucidate the microbial composition and potential microbial functions in a full-scale well-worked offshore oil-produced wastewater treatment plant. Results showed that microbes that inhabited the plant were diverse and originated from oil and marine associated environments. The upstream physical and chemical treatments resulted in low microbial diversity. Organic pollutants were digested in the anaerobic baffled reactor (ABR) dominantly through fermentation combined with sulfur compounds respiration. Three aerobic parallel reactors (APRs) harbored different microbial groups that performed similar potential functions, such as hydrocarbon degradation, acidogenesis, photosynthetic assimilation, and nitrogen removal. Microbial characteristics were important to the performance of oil-produced wastewater treatment plants with biological processes

Characterization of the core bacteriobiome in the rhizosphere of greenhouse vegetables: taxonomic diversity and putative functions

The aim of the study. The aim was to profile 16S rRNA gene diversity and to assess functional potential of bacterial assemblages in the rhizosphere of some unconventional vegetables grown in protected greenhouse conditions in West Siberia. Location and time of the study. Novosibirsk, Russia, 2016. Methodology. At the end of the growing season in the middle of September the rhizosphere soil was collected from the plants of wax gourd (Benincasa hispida), bitter melon (Momordica charantia), kiwano (Cucumis metuliferus) and cowpea (Vigna unguiculata) grown on peat-based substrate in a polyethylene-protected greenhouse that has been in operation for more than 40 years. The metagenomic DNA was extracted and amplified with V3-V4 primers for 16S rRNA genes, and the amplicons sequenced with Illumina MiSeq. The obtained OTUs tables were used to predict putative functions by running through the FAPROTAX database. Main results. The rhizosphere bacteriobiome was dominated by Proteobacteria (32±11% of the total number of sequence reads), Acidobacteria (23±7%) and Actinobacteria (18±3%) phyla, together accounting for about three quarters of the rhizosphere bacteriobiome. In total 20 bacterial phyla were found. The rhizosphere bacteriobiome was surprisingly diverse with Shannon index ranging 7.0–7.5. The number of the observed operational taxonomic units (OTUs) per sample was very high, ranging 4,500–4,900, and the potential number of OTUs estimated as 5,100–5,700; all those OTUs were evenly and equitably represented in the bacteriobiome, and dominance indices (Simpson dominance and Berger-Parker) were very low. The main dominant OTU represented Bradyrhizobiaceae family and accounted for just 1% on average. Overall the study identified 27 OTUs belonging to the Bradyrhizobiaceae family, but only four of them were ascribed to nitrogen fixation by FAPROTAX. Function prediction by FAPROTAX also suggested that bacteriobiome had a marked potential for the carbon cycle, denitrification, aromatic compound and plant polymer degradation, but no plant pathogens. The biggest difference in rhizosphere bacteriobiome diversity was observed between the bitter melon and the other three vegetable crops: bitter melon had much increased abundance of Arthrobacter and Sphingomonas as compared with wax gourd, kiwano and cowpea, and increased number of bacterial species associated with aromatic compounds degradation. Conclusion. Based on the finding that the studied rhizosphere bacteriobiomes were very diverse, we conclude that the crops were able to recruit diverse microbiota from the peat-based soil substrate, which, in its turn, means that diverse soil substrate microbiota has been sustained over several decades of the greenhouse operation. All crops apparently shaped distinct bacteriobiomes in their rhizosphere, which ideally should be included into studies of plant-associated bacterial diversity profiles for breeding and sustainable production.The aim of the study. The aim was to profile 16S rRNA gene diversity and to assess functional potential of bacterial assemblages in the rhizosphere of some unconventional vegetables grown in protected greenhouse conditions in West Siberia. Location and time of the study. Novosibirsk, Russia, 2016. Methodology. At the end of the growing season in the middle of September the rhizosphere soil was collected from the plants of wax gourd (Benincasa hispida), bitter melon (Momordica charantia), kiwano (Cucumis metuliferus) and cowpea (Vigna unguiculata) grown on peat-based substrate in a polyethylene-protected greenhouse that has been in operation for more than 40 years. The metagenomic DNA was extracted and amplified with V3-V4 primers for 16S rRNA genes, and the amplicons sequenced with Illumina MiSeq. The obtained OTUs tables were used to predict putative functions by running through the FAPROTAX database. Main results. The rhizosphere bacteriobiome was dominated by Proteobacteria (32±11% of the total number of sequence reads), Acidobacteria (23±7%) and Actinobacteria (18±3%) phyla, together accounting for about three quarters of the rhizosphere bacteriobiome. In total 20 bacterial phyla were found. The rhizosphere bacteriobiome was surprisingly diverse with Shannon index ranging 7.0–7.5. The number of the observed operational taxonomic units (OTUs) per sample was very high, ranging 4,500–4,900, and the potential number of OTUs estimated as 5,100–5,700; all those OTUs were evenly and equitably represented in the bacteriobiome, and dominance indices (Simpson dominance and Berger-Parker) were very low. The main dominant OTU represented Bradyrhizobiaceae family and accounted for just 1% on average. Overall the study identified 27 OTUs belonging to the Bradyrhizobiaceae family, but only four of them were ascribed to nitrogen fixation by FAPROTAX. Function prediction by FAPROTAX also suggested that bacteriobiome had a marked potential for the carbon cycle, denitrification, aromatic compound and plant polymer degradation, but no plant pathogens. The biggest difference in rhizosphere bacteriobiome diversity was observed between the bitter melon and the other three vegetable crops: bitter melon had much increased abundance of Arthrobacter and Sphingomonas as compared with wax gourd, kiwano and cowpea, and increased number of bacterial species associated with aromatic compounds degradation. Conclusion. Based on the finding that the studied rhizosphere bacteriobiomes were very diverse, we conclude that the crops were able to recruit diverse microbiota from the peat-based soil substrate, which, in its turn, means that diverse soil substrate microbiota has been sustained over several decades of the greenhouse operation. All crops apparently shaped distinct bacteriobiomes in their rhizosphere, which ideally should be included into studies of plant-associated bacterial diversity profiles for breeding and sustainable production

Effect of proton pump inhibitor on microbial community, function, and kinetics in anaerobic digestion with ammonia stress

The proton pump is a convincing mechanism for ammonia inhibition in anaerobic digestion, which explained how the ammonia accumulated intercellularly due to diffusion of free ammonia. Proton pump inhibitor (PPI) was dosed for mitigating the accumulation in anaerobic digestion with ammonia stress, with respect to kinetics. Results show PPI inhibited beta-oxidation of fatty acids by targeting ATPase in anaerobic digestion with ammonia stress. Alternatively, PPI stimulated syntrophic acetate oxidization. Random forest located key genera as syntrophic consortia. Methane increased 18.72 +/- 7.39% with 20 mg/L PPI at the first peak, consistent with microbial results. The deterministic Gompertz kinetics and stochastic Gaussian processes contributed 97.63 +/- 8.93% and 2.37 +/- 8.93% in accumulated methane production, respectively. Thus, the use of PPI for anaerobic digestion allowed mitigate ammonia inhibition based on the mechanism of proton pump, facilitate intercellularly ammonia accumulation, stimulate syntrophic consortia, and eliminate uncertainty of process failure, which resulted in efficient methane production under ammonia stress

Classification Methods for 16S rRNA Based Functional Annotation

Microbial communities play an essential role in Earth’s ecosystems. The goal of this study was to investigate whether the functional potential of microorganisms forming these diverse communities can be directly identified using a 16S rRNA marker gene with supervised learning methods. The recently developed FAPROTAX database has been used along with the SILVA database to produce a training set where 16S rRNA sequences are linked to a number of metabolic functions. Since gene sequences cannot be explicitly used as feature vectors by most classification algorithms, the present research aimed to investigate possible feature engineering approaches for 16S rRNA. Techniques based on Multiple Sequence Alignment (MSA) and N-grams are proposed and tested. The results showed that the feature representation based on the Ngrams outperformed MSA, especially when implemented with large and diverse functional groups. This suggests that a clustering-like alignment procedure results in a biased feature representation of the marker gene. Since classifiers trained using Random Forest and Support Vector Machines techniques were able to accurately detect a range of functional groups it is concluded that the 16S rRNA gene provides substantial information for the direct identification of functional capabilities

Subcuticular and biofilm microbiomes in Holothuria tubulosa and their potential for denitrification

Holothurians, as benthic invertebrates inhabiting marine ecosystems, have a crucial function in that they actively process organic detritus in the sediments. Previous works have provided evidence of the capability of holothurians to reduce nitrate and ammonium concentrations in aquaculture tanks. However, the mechanisms underlying this nitrogen decrease still need to be elucidated and might be related to bacterial symbionts in the holothurians. Here we characterize the community of bacterial symbionts in the biofilm and subcuticle of Holothuria tubulosa and explore the presence of nitrification and denitrification genes. To characterize these bacterial symbionts, we extracted DNA and amplified the V3-V4 hypervariable region of the 16S rRNA gene. We obtained a notable contribution of Bacteroidota, Alphaproteobacteria (mostly Rhodobacterales), and Gammaproteobacteria (mostly Pseudomonadales) both within the biofilm and subcuticle of H. tubulosa. Subsequently, we tested the presence of specific genes encoding enzymes involved in nitrification (i.e. archaeal amoA and bacterial amoA) and denitrification (i.e. nirS and nosZ). Our results confirm the presence of denitrification genes in the holothurian biofilms. These findings indicate that the holothurians house a diverse community of bacterial symbionts, which includes species with the potential for nitrogen removal. Therefore, holothurian holobionts may play a multifaceted ecological role, both processing organic detritus and reducing nitrogen levels in coastal areas. These roles could be extended to sustainable aquaculture, making them valuable ecosystem engineers with significant implications for ecosystem and aquaculture health.Project PID2022-137865OB-I00 funded by MICIU/AEI/10.13039/501100011033/ERDF, EUHOLOSYSTEMS Project from the Junta de Andalucía, Consejería de Transformación Económica, Indus - tria, Conocimiento y Universidades (grant no. P20.00705)Agencia Estatal de Investigación (PTA2018-016205-I)Ministerio de Ciencia, Innovación y Uni - versidades of Spain (CRONOS project, RTI2018-098849-BI00) at the University of GranadaMarie Skłodowska-Curie postdoctoral fellowship (HORIZON-291 MSCA-2021-PF-01, project number: 101066750) by the European Commission at Princeton Universit