Determination of carbon-fixing potential of Bathyarchaeota in marine sediment by DNA stable isotope probing analysis

Bathyarchaeota is believed to play a crucial role in the global carbon cycle due to its vast biomass,broad distribution,and diverse habitat.However,its physiological and metabolic features are hard to determine without pure culture.While metagenomic analyses have shown that Bathyarchaeota has a complete inorganic carbon fixation(Wood-Ljungdahl,WL)pathway,no direct functional confirmation has been reported.To explore the inorganic carbon fixation ability of Bathyarchaeota,we used lignin and sodium bicarbonate-^(13)C(NaH^(13)CO_(3))in the long-term incubation of marine sediment samples.We found that Bathyarchaeota grew continuously in the cultivation system with lignin,and its abundance increased up to 15.3 times after10 months,increasing its fraction of all archaea from 30%to 80%.We monitored theδ^(13)C of total organic carbon to identify microbial carbon fixation in the cultivation systems,finding that it increased in the first month while NaH^(13)CO_(3)was present but only increased continuously afterward when lignin was also present.Furthermore,ultracentrifugation was performed on DNA extracted from samples at different cultivation stages to separate DNA of different buoyant densities,and bathyarchaeotal and bacterial 16S ribosomal RNA(r RNA)gene abundance were quantified using qPCR.Compared to bacteria,bathyarchaeotal 16S rRNA tended to be concentrated in heavy layers after 4 months of incubation with lignin and NaH^(13)CO_(3),indicating that Bathyarchaeota DNA contained^(13)C through proliferation based on lignin utilization and NaH^(13)CO_(3)assimilation,proving the carbon fixation capacity of Bathyarchaeota.

Elevated and atmospheric-level methane consumption by soil methanotrophs of three grasslands in China

Background:Methane(CH4)oxidation driven by soil aerobic methanotrophs demonstrates the capacity of grassland as a CH4 sink.Methods:In this study,we compared the oxidation characteristics of atmospheric-level and elevated concentration(10%)CH4 in a typical grassland(steppe)on the Loess Plateau,an alpine meadow(meadow)on the Qinghai-Tibet Plateau,and an inland arid-area artificial grassland(pasture)in northwest China and investigated the communities of active methanotrophs and their contribution to CH4 oxidation using DNA-based stable-isotope probing and Illumina Miseq sequencing.Results:The results showed that the oxidation of atmospheric CH4 only occurred in steppe and meadow soils where the USCγgroup of methanotrophs was numerically dominant in the methanotroph community.Pasture soils,with their very low relative abundance of USCγ,did not show atmospheric CH4 oxidation.However,a DNA-stable isotope probing experiment with 10%CH4 indicated that conventional CH4 oxidizers(Methylocaldum and Methylocystis)rather than USCγcommunities assimilated significant amounts of 13CH4 for growth.Conclusions:The CH4 oxidation mechanisms in the three experimental grassland soils varied significantly.The USCγgroup may be obligate oligotrophic microorganisms or their growth requires specific unknown conditions.

重庆紫色水稻土中“全程”和“半程”氨氧化微生物的垂直分异

【目的】系统评估全程氨氧化细菌(complete ammonia oxidizing bacteria, Comammox bacteria)、半程氨氧化细菌(AOB)和古菌(AOA)在典型水稻土剖面的垂直分异规律。2015年发现的"全程"氨氧化细菌(Comammox Nitrospira)可将氨分子一步氧化为硝酸盐,实现硝化作用。而经典的"半程"氨氧化细菌(AOB)或古菌(AOA)将氨分子氧化为亚硝酸盐后,再由系统发育完全不同的硝化细菌将其氧化为硝酸盐。全程氨氧化细菌实现了一步硝化全过程,根本改变了学术界对2类微生物分步硝化的经典认知,但相关研究仍处于初步阶段。【方法】选择重庆北碚地区2017年典型水稻土并采集5、10、20和40 cm不同深度土壤(剖面采样点的上下误差不超过1cm),提取水稻土总DNA后,利用标靶功能基因amoA,通过实时荧光定量PCR技术分析全程氨氧化细菌(Comammox)、半程氨氧化细菌(AOB)和古菌(AOA)在水稻土不同深度的数量变异规律。【结果】半程氨氧化细菌AOB和古菌AOA均随土壤深度增加呈显著下降趋势。然而,全程氨氧化细菌的两大类微生物则表现出相反的规律,Comammox Clade A的丰度随着土壤剖面的加深而显著增加(P<0.05),但Clade B并未有类似规律。Clade A在水稻土不同层次的土层中均比Clade B高出1个数量级,在5 cm和40 cm处的最低和最高值分别为3.42×10~7、8.46×10~7 copies/g。AOA与AOB的丰度大致相当,5cm剖面处数量最高分别为1.23×10~7、1.83×10~5copies/g,但其平均丰度远低于全程氨氧化细菌,Comammox与AOA、AOB amoA功能基因拷贝数之比为10–2000。【结论】全程氨氧化细菌(Comammox bacteria)广泛分布于水稻土不同土层中,且数量远高于"半程"氨氧化细菌和古菌,意味着Comammox可能在水稻土硝化作用中起重要作用。

典型草地土壤好氧甲烷氧化的微生物生态过程

【目的】针对我国甘肃三个典型生态区草地土壤(玛曲MQ、临泽LZ和环县HX),研究其甲烷氧化潜力、甲烷氧化菌(methane-oxidizingbacteria,MOB)丰度及可能存在的群落分异规律。【方法】通过原位分析、室内高浓度甲烷模拟培养三种典型土壤及实时荧光定量、高通量测序的方法研究甲烷氧化菌标靶基因pmoA序列的组成及其丰度变化规律。【结果】三种典型草地土壤的原位甲烷氧化菌的丰度存在显著差异,表现为MQ>HX>LZ,其数量范围为为0.18–6.86×10^7g/d.w.s.;甲烷氧化潜力也表现出类似规律,其通量为109–169mg/(m^2·h);甲烷氧化潜力与原位土壤中甲烷氧化菌丰度有正相关。三种草地土壤甲烷氧化菌存在明显的空间异质性,采用高通量测序的方法,发现三种草地原位土壤中的优势类群为USCγ(Upland Soil Cluster gamma,USCγ);然而,室内高浓度甲烷氧化过程中,传统的甲烷氧化菌均发生明显增加,MQ土壤中TypeⅡ的Methylocystis为优势类群,而LZ和HX土壤的优势类群均为TypeⅠ型Methylosarcina。【结论】这些研究结果表明,我国甘肃典型草地土壤中也存在难培养的大气甲烷氧化菌和经典的可培养甲烷氧化菌,这些微生物极可能氧化极低浓度的大气甲烷,也可能利用闭蓄于土壤中的高浓度甲烷生长。未来应采用先进技术原位观测大气甲烷氧化过程并分离相应微生物类群,研究草地土壤甲烷氧化菌地理分异规律及其环境驱动机制。

Soil biodiversity in a rapidly changing world

Microbes are simple single-cell organisms,but have an enormous practical significance for human kind.Soil microbes make our planet habitable,and the planet's rapidly changing environments in turn have a profound impact on the soil microbial communities,both positively and negatively.It is thus crucial to better understand how abiotic and biotic factors interact to assemble microbiomes under the given environmental conditions,and how they modulate the intrinsic link between microbial diversity and ecosystem function.From a functional viewpoint.

Soil microbial community assemblage and its seasonal variability in alpine treeline ecotone on the eastern Qinghai-Tibet Plateau

The alpine treeline ecotone is characterized as the upper limit of the forest in the high-mountain ecosystem.Due to the freeze-thaw cycles,the soil organism community,such as microbial communities are expected to change between seasons.However,there are limited microbialcommunity studies focused on the high altitude alpine ecosystem.We conducted a study in the alpine treeline ecotone on the eastern Qinghai-Tibet Plateau,China,and investigated the seasonal variability of the soil microbial community.We collected all soil samples within the alpine treeline ecotone,between the treeline and timberline in the high-mountain region.The 16S rRNA genes of the microbial communities(bacterial and archaeal)were analyzed by highthroughput sequencing to the genus level.The results showed that soil microbial community in the alpine treeline ecotone was consistently dominated by eight phyla which consisted of 95% of the total microbial community,including Proteobacteria,Actinobacteria,Acidobacteria,Firmicutes,Planctomycetes,Chloroflexi,Bacteroidetes,and Verrucomicrobia.The overall diversity and evenness of the community were relatively stable,with an average of 0.5% difference between seasons.The highest seasonal variability occurred at the upper boundary of the alpine treeline ecotone,and few or almost no seasonal change was observed at lower elevations,indicating dense forest cover and litter deposition might have created a local microclimate that reduced seasonal variation among the surrounding environmental conditions.Our study was one of the first group that documented the microbial community assemblage in the treeline ecotone on the Qinghai-Tibet Plateau.

OPPORTUNITIES AND APPROACHES FOR MANIPULATING SOIL-PLANT MICROBIOMES FOR EFFECTIVE CROP NITROGEN USE IN AGROECOSYSTEMS

Soil microbiomes drive the biogeochemical cycling of nitrogen and regulate soil N supply and loss,thus,pivotal nitrogen use efficiency(NUE).Meanwhile,there is an increasing awareness that plant associated microbiomes and soil food web interactions is vital for modulating crop productivity and N uptake.The rapid advances in modern omics-based techniques and biotechnologies make it possible to manipulate soil-plant microbiomes for improving NUE and reducing N environmental impacts.This paper summarizes current progress in research on regulating soil microbial N cycle processes for NUE improvement,plant-microbe interactions benefiting plant N uptake,and the importance of soil microbiomes in promoting soil health and crop productivity.We also proposes a potential holistic(rhizosphere-root-phyllosphere)microbe-based approach to improve NUE and reduce dependence on mineral N fertilizer in agroecosystems,toward nature-based solution for nutrient management in intensive cropping systems.

Stable-isotope probing of bacterial community for dissolved inorganic carbon utilization in Microcystis aeruginosa-dominated eutrophic water

Dissolved inorganic carbon(DIC) is an important source of carbon in aquatic ecosystems,especially under conditions of increased frequency of cyanobacterial bloom. However, the importance of bacteria in direct or indirect utilization of DIC has been widely overlooked in eutrophic freshwater. To identify the functional bacteria that can actively utilize DIC in eutrophic freshwater during cyanobacterial bloom, stable-isotope probing(SIP) experiments were conducted on eutrophic river water with or without inoculation with cyanobacteria(Microcystis aeruginosa). Our 16 S rRNA sequencing results revealed the significance of Betaproteobacteria, with similar relative abundance as Alphaproteobacteria, in the active assimilation of H^(13)CO^(3-) into their DNA directly or indirectly, which include autotrophic genera Betaproteobacterial ammonia-oxidizing bacteria. Other bacterial groups containing autotrophic members, e.g. Planctomycetes and Nitrospira, also presented higher abundance among free-living bacteria in water without cyanobacteria. Microcystis aggregates showed a preference for some specific bacterial members that may utilize H^(13)CO^(3-) metabolized by Microcystis as organic matter, e.g. Bacteroidetes(Cytophagales, Sphingobacteriales), and microcystindegrading bacteria Betaproteobacteria(Paucibacter/Burkholderiaceae). This study provides some valuable information regarding the functional bacteria that can actively utilize DIC in eutrophic freshwater.

Methanotrophy-driven accumulation of organic carbon in four paddy soils of Bangladesh

Biological methane oxidation is a crucial process in the global carbon cycle that reduces methane emissions from paddy fields and natural wetlands into the atmosphere.However,soil organic carbon accumulation associated with microbial methane oxidation is poorly understood.Therefore,to investigate methane-derived carbon incorporation into soil organic matter,paddy soils originated from different parent materials(Inceptisol,Entisol,and Alfisol) were collected after rice harvesting from four major rice-producing regions in Bangladesh.Following microcosm incubation with 5%(volume/volume)^(13) CH_(4),soil^(13) C-atom abundances significantly increased from background level of 1.08% to 1.88%–2.78%,leading to a net methane-derived accumulation of soil organic carbon ranging from 120 to 307 mg kg^(-1).Approximately 23.6%–60.0% of the methane consumed was converted to soil organic carbon during microbial methane oxidation.The phylogeny of^(13) C-labeled pmoA(enconding the alpha subunit of the particulate methane monooxygenase) and 16 S rRNA genes further revealed that canonical α(type II) and γ(type I) Proteobacteria were active methane oxidizers.Members within the Methylobacter-and Methylosarcina-affiliated type Ia lineages dominated active methane-oxidizing communities that were responsible for the majority of methane-derived carbon accumulation in all three paddy soils,while Methylocystis-affiliated type IIa lineage was the key contributor in one paddy soil of Inceptisol origin.These results suggest that methanotroph-mediated synthesis of biomass plays an important role in soil organic matter accumulation.This study thus supports the concept that methanotrophs not only consume the greenhouse gas methane but also serve as a key biotic factor in maintaining soil fertility.

Heterotrophy-coordinated diazotrophy is associated with significant changes of rare taxa in soil microbiome

Soil heterotrophic respiration during decomposition of carbon(C)-rich organic matter plays a vital role in sustaining soil fertility.However,it remains poorly understood whether dinitrogen(N_(2))fixation occurs in support of soil heterotrophic respiration.In this study,^(15)N_(2)-tracing indicated that strong N_(2)fixation occurred during heterotrophic respiration of carbon-rich glucose.Soil organic ^(15)N increased from 0.37 atom%to 2.50 atom%under aerobic conditions and to 4.23 atom%under anaerobic conditions,while the concomitant CO_(2)flux increased by 12.0-fold under aerobic conditions and 5.18-fold under anaerobic conditions.Soil N_(2)fixation was completely absent in soils replete with inorganic N,although soil N bioavailability did not alter soil respiration.High-throughput sequencing of the 16S rRNA gene further indicated that:i)under aerobic conditions,only 15.2%of soil microbiome responded positively to glucose addition,and these responses were significantly associated with soil respiration and N_(2)fixation and ii)under anaerobic conditions,the percentage of responses was even lower at 5.70%.Intriguingly,more than 95%of these responses were originally rare with<0.5%relative abundance in background soils,including typical N_(2)-fixing heterotrophs such as Azotobacter and Clostridium and well-recognized non-N_(2)-fixing heterotrophs such as Sporosarcina,Agromyces,and Sedimentibacter.These results suggest that only a small portion of the soil microbiome could respond quickly to the amendment of readily accessible organic C in a fluvo-aquic soil and highlighted that rare phylotypes might have played more important roles than previously appreciated in catalyzing soil C and nitrogen turnovers.Our study indicates that N_(2)fixation could be closely associated with microbial turnover of soil organic C when available in excess.

Evidence for niche differentiation of nitrifying communities in grassland soils after 44 years of different field fertilization scenarios

Long-term nitrogen(N)fertilization imposes strong selection on nitrifying communities in agricultural soil,but how a progressively changing niche affects potentially active nitrifiers in the field remains poorly understood.Using a 44-year grassland fertilization experiment,we investigated community shifts of active nitrifiers by DNA-based stable isotope probing(SIP)of field soils that received no fertilization(CK),high levels of organic cattle manure(HC),and chemical N fertilization(CF).Incubation of DNA-SIP microcosms showed significant nitrification activities in CF and HC soils,whereas no activity occurred in CK soils.The 44 years of inorganic N fertilization selected only 13C-ammonia-oxidizing bacteria(AOB),whereas cattle slurry applications created a niche in which both ammonia-oxidizing archaea(AOA)and AOB could be actively13C-labeled.Phylogenetic analysis indicated that Nitrosospira sp.62-like AOB dominated inorganically fertilized CF soils,while Nitrosospira sp.41-like AOB were abundant in organically fertilized HC soils.The 13C-AOA in HC soils were affiliated with the 29i4 lineage.The 13C-nitrite-oxidizing bacteria(NOB)were dominated by both Nitrospira-and Nitrobacter-like communities in CF soils,and the latter was overwhelmingly abundant in HC soils.The 13C-labeled nitrifying communities in SIP microcosms of CF and HC soils were largely similar to those predominant under field conditions.These results provide direct evidence for a strong selection of distinctly active nitrifiers after 44 years of different fertilization regimes in the field.Our findings imply that niche differentiation of nitrifying communities could be assessed as a net result of microbial adaption over 44 years to inorganic and organic N fertilization in the field,where distinct nitrifiers have been shaped by intensified anthropogenic N input.

In Memoriam:David D.Myrold(1955–2021)

David D.Myrold,65,Professor of Soil Microbiology in the Department of Crop and Soil Science at Oregon State University,passed away in Corvallis,Oregon,USA on July 15,2021.Dave was well known to many soil scientists for his work on soil nitrogen cycling,a career of research work that was summarized in his latest 2021 publication—Transformation of Nitrogen.