Characterization of the first rice paddy cluster I isolate, Methyloterricola oryzae gen. nov., sp. nov. and amended description of Methylomagnum ishizawai.

Three gammaproteobacterial methanotrophic strains (73aT, 175 and 114) were isolated from stems of rice plants. All strains are Gram-negative, motile and grow on methane or methanol as sole carbon sources. They oxidize methane using the particulate methane monooxygenase. Strains 114 and 175 possess additionally a soluble methane monooxygenase. All strains contain significant amounts of the cellular fatty acids C16 : 0, C16 : 1ω6c and C16 : 1ω7c, typical for type Ib methanotrophs. Characteristic for strains 114 and 175 are high amounts of C14 : 0 and C16 : 1ω6c , while strain 73aT contains high quantities of C16 : 1ω5c. 16S rRNA gene sequence analyses showed that strains 114 and 175 are most closely related to Methylomagnum ishizawai (≥99.6 % sequence identity). Strain 73aT is representing a new genus within the family Methylococcaceae, most closely related to Methylococcus capsulatus (94.3 % sequence identity). Phylogenetic analysis of the PmoA sequence indicates that strain 73aT represents rice paddy cluster I (RPCI), which has almost exclusively been detected in rice ecosystems. The G+C content of strain 73aT is 61.0 mol%, while strains 114 and 175 have a G+C content of 63.3 mol%. Strain 73aT (=LMG 29185T, =VKM B-2986T) represents the type strain of a novel species and genus, for which the name Methyloterricola oryzae gen. nov., sp. nov. is proposed and a description is provided. Strains 175 (=LMG 28717, VKM B-2989) and 114 are members of the species Methylomagnum ishizawai. This genus was so far only represented by one isolate, so an amended description of the species is given.

Effects of light and autochthonous carbon additions on microbial turnover of allochthonous organic carbon and community composition.

The fate of allochthonous dissolved organic carbon (DOC) in aquatic systems is primarily controlled by the turnover of heterotrophic bacteria. However, the roles that abiotic and biotic factors such as light and DOC release by aquatic primary producers play in the microbial decomposition of allochthonous DOC is not well understood. We therefore tested if light and autochthonous DOC additions would increase allochthonous DOC decomposition rates and change bacterial growth efficiencies and community composition (BCC). We established continuous growth cultures with different inocula of natural bacterial communities and alder leaf leachates (DOCleaf) with and without light exposure before amendment. Furthermore, we incubated DOCleaf together with autochthonous DOC from lysed phytoplankton cultures (DOCphyto). Our results revealed that pretreatments of DOCleaf with light resulted in a doubling of bacterial growth efficiency (BGE), whereas additions of DOCphyto or combined additions of DOCphyto and light had no effect on BGE. The change in BGE was not accompanied by shifts in the phylogenetic structure of the BCC, but BCC was influenced by the DOC source. Our results highlight that a doubling of BGE is not necessarily accompanied by a shift in BCC and that BCC is more strongly affected by resource properties.

Microbial methane production in oxygenated water column of an oligotrophic lake.

The prevailing paradigm in aquatic science is that microbial methanogenesis happens primarily in anoxic environments. Here, we used multiple complementary approaches to show that microbial methane production could and did occur in the well-oxygenated water column of an oligotrophic lake (Lake Stechlin, Germany). Oversaturation of methane was repeatedly recorded in the well-oxygenated upper 10 m of the water column, and the methane maxima coincided with oxygen oversaturation at 6 m. Laboratory incubations of unamended epilimnetic lake water and inoculations of photoautotrophs with a lake-enrichment culture both led to methane production even in the presence of oxygen, and the production was not affected by the addition of inorganic phosphate or methylated compounds. Methane production was also detected by in-lake incubations of lake water, and the highest production rate was 1.8-2.4 nM⋅h(-1) at 6 m, which could explain 33-44% of the observed ambient methane accumulation in the same month. Temporal and spatial uncoupling between methanogenesis and methanotrophy was supported by field and laboratory measurements, which also helped explain the oversaturation of methane in the upper water column. Potentially methanogenic Archaea were detected in situ in the oxygenated, methane-rich epilimnion, and their attachment to photoautotrophs might allow for anaerobic growth and direct transfer of substrates for methane production. Specific PCR on mRNA of the methyl coenzyme M reductase A gene revealed active methanogenesis. Microbial methane production in oxygenated water represents a hitherto overlooked source of methane and can be important for carbon cycling in the aquatic environments and water to air methane flux.

Differential Impact of Plant Secondary Metabolites on the Soil Microbiota.

Plant metabolites can shape the microbial community composition in the soil. Two indole metabolites, benzoxazolinone (BOA) and gramine, produced by different Gramineae species, and quercetin, a flavonoid synthesized by many dicot species, were studied for their impacts on the community structure of field soil bacteria. The three plant metabolites were directly added to agricultural soil over a period of 28 days. Alterations in bacterial composition were monitored by next generation sequencing of 16S rRNA gene PCR products and phospholipid fatty acid analysis. Treatment of the soil with the plant metabolites altered the community composition from phylum to amplicon sequence variant (ASV) level. Alpha diversity was significantly reduced by BOA or quercetin, but not by gramine. BOA treatment caused a decrease of the relative abundance of 11 ASVs, while only 10 ASVs were increased. Gramine or quercetin treatment resulted in the increase in relative abundance of many more ASVs (33 or 38, respectively), most of them belonging to the Proteobacteria. Isolation and characterization of cultivable bacteria indicated an enrichment in Pseudarthrobacter or Pseudomonas strains under BOA/quercetin or BOA/gramine treatments, respectively. Therefore, the effects of the treatments on soil bacteria were characteristic for each metabolite, with BOA exerting a predominantly inhibitory effect, with only few genera being able to proliferate, while gramine and quercetin caused the proliferation of many potentially beneficial strains. As a consequence, BOA or gramine biosynthesis, which have evolved in different barley species, is accompanied with the association of distinct bacterial communities in the soil, presumably after mutual adaptation during evolution.

Development of a Distinct Microbial Community Upon First Season Crop Change in Soils of Long-Term Managed Maize and Rice Fields.

The introduction of crop rotation regimes in paddy soils, for example, rice in combination with maize, implements the establishment of new paddy fields to compensate for reduced rice production on existing fields. To study responses of the soil and rhizosphere microbiota upon introduction of a new crop species into continuous cropping agroecosystems, we conducted experiments with soils from adjacent fields where rice and maize were grown successively for more than 30 years. In microcosm experiments, rice and maize plants were cultivated in both soils under the respective plant-required management regime, i.e., rice cultivation under flooded conditions and maize under non-flooded conditions. 16S rRNA gene and fungal ITS region amplicon analysis showed that the soil and rhizosphere microbiota was clearly distinct between soils after long-term rice/maize management. Upon change of the management regime, the bulk soil microbiota became different to both, the former microbial community in the soil and the community being characteristic for the respective type of long-term cropping. Nevertheless, the influence of the soil management history remained clearly visible besides the impact of the new management regime. Similar results were observed for the rhizosphere, though the combined effect of plant species and altered management was even more effective in this compartment compared to the bulk soil. The newly introduced crop plant did not take over characteristic members of the rhizosphere microbiota of the previously cultivated crop; instead, some previously rare taxa became enriched. Thus, the formerly grown crop species did not directly affect the recruitment of microorganisms in the rhizosphere of the following crop species. Further, the results show that the rhizosphere and bulk soil microbiota do not develop straight toward the specific microbiota that is characteristic for a continuous cropping system, but reach a distinct stage upon introduction of a new crop species and new management practices.

Evidence for signatures of ancient microbial life in paleosols.

Loess-paleosol sequences are terrestrial archives of past climate change. They may host traces of ancient microbial life, but little information is available on the recovery of microbial biomarkers from such deposits. We hypothesized that microbial communities in soil horizons up to an age of 127 kyr carry information related to past environments. We extracted DNA from a loess-paleosol sequence near Toshan, Northern Iran, with 26 m thick deposits showing different degrees of soil development, performed quantitative PCR and 16S rRNA gene amplicon sequencing. Periods of soil formation archived within the loess sediment led to higher diversity and bacterial abundance in the paleosol horizons. Community composition fluctuated over the loess-paleosol sequence and was mainly correlated with age and depth, (ADONIS R2 < 0.14, P ≤ 0.002), while responses to paleosol soil traits were weaker. Phyla like Bacteriodetes, Proteobacteria or Acidobacteria were more prevalent in paleosol horizons characterized by intense soil formation, while weakly developed paleosols or loess horizons hosted a higher percentage and diversity of Actinobacteria. Taken together, our findings indicate that the microbial community in loess-paleosol sequences carries signatures of earlier environmental conditions that are preserved until today.

Salinity-independent dissipation of antibiotics from flooded tropical soil: a microcosm study.

River deltas are frequently facing salinity intrusion, thus challenging agricultural production in these areas. One adaption strategy to increasing salinity is shrimp production, which however, heavily relies on antibiotic usage. This study was performed to evaluate the effect of increasing salinity on the dissipation rates of antibiotics in tropical flooded soil systems. For this purpose, paddy top soil from a coastal Vietnamese delta was spiked with selected frequently used antibiotics (sulfadiazine, sulfamethazine, sulfamethoxazole, trimethoprim) and incubated with flood water of different salt concentrations (0, 10, 20 g L-1). Antibiotic concentrations were monitored in water and soil phases over a period of 112 days using liquid chromatography and tandem mass spectrometry. We found that sulfamethazine was the most persistent antibiotic in the flooded soil system (DT50 = 77 days), followed by sulfadiazine (DT50 = 53 days), trimethoprim (DT50 = 3 days) and sulfamethoxazole (DT50 = 1 days). With the exception of sulfamethoxazole, the apparent distribution coefficient increased significantly (p < 0.05) for all antibiotics in course of the incubation, which indicates an accumulation of antibiotics in soil. On a whole system basis, including soil and water into the assessment, there was no overall salinity effect on the dissipation rates of antibiotics, suggesting that common e-fate models remain valid under varying salinity.

Crop Rotation and Straw Application Impact Microbial Communities in Italian and Philippine Soils and the Rhizosphere of Zea mays.

Rice is one of the most important nourishments and its cultivation binds large agricultural areas in the world. Its cultivation leads to huge water consumption and high methane emissions. To diminish these problems, crop rotation between paddy rice and maize is introduced in Asia, but can lead to losses of carbon and water by the formation of desiccation cracks. To counteract these problems rice straw can be applied. We analyzed soil microbial responses to different crop rotation systems [rice-rice (RR), maize-maize (MM), maize-rice (MR)] and to rice straw application in the soil and rhizosphere of maize. Zea mays was grown in microcosms using soils from different field locations, each including different crop rotation regimes. The bacterial and fungal community composition was analyzed by 16S rRNA gene and ITS based amplicon sequencing in the bulk soil and rhizosphere. The microbiota was clearly different in soils from the different field locations (analysis of similarity, ANOSIM: R = 0.516 for the bacterial community; R = 0.817 for the fungal community). Within the field locations, crop rotation contributed differently to the variation in microbial community composition. Strong differences were observed in communities inhabiting soils under monosuccession (RR vs. MM) (ANOSIM: R = 0.923 for the bacterial and R = 0.714 for the fungal community), while the communities in soils undergoing MR crop rotation were more similar to those of the corresponding RR soils (ANOSIM: R = 0.111-0.175). The observed differences could be explained by altered oxygen availabilities in RR and MR soils, resulting in an enrichment of anaerobic bacteria in the soils, and the presence of the different crops, leading to the enrichment of host-plant specific microbial communities. The responses of the microbial communities to the application of rice straw in the microcosms were rather weak compared to the other factors. The taxa responding in bulk soil and rhizosphere were mostly distinct. In conclusion, this study revealed that the different agricultural management practices affect microbial community composition to different extent, not only in the bulk soil but also in the rhizosphere, and that the microbial responses in bulk soil and rhizosphere are distinct.

Shifts among Eukaryota, Bacteria, and Archaea define the vertical organization of a lake sediment.

BACKGROUND: Lake sediments harbor diverse microbial communities that cycle carbon and nutrients while being constantly colonized and potentially buried by organic matter sinking from the water column. The interaction of activity and burial remained largely unexplored in aquatic sediments. We aimed to relate taxonomic composition to sediment biogeochemical parameters, test whether community turnover with depth resulted from taxonomic replacement or from richness effects, and to provide a basic model for the vertical community structure in sediments. METHODS: We analyzed four replicate sediment cores taken from 30-m depth in oligo-mesotrophic Lake Stechlin in northern Germany. Each 30-cm core spanned ca. 170 years of sediment accumulation according to 137Cs dating and was sectioned into layers 1-4 cm thick. We examined a full suite of biogeochemical parameters and used DNA metabarcoding to examine community composition of microbial Archaea, Bacteria, and Eukaryota. RESULTS: Community ß-diversity indicated nearly complete turnover within the uppermost 30 cm. We observed a pronounced shift from Eukaryota- and Bacteria-dominated upper layers (<5 cm) to Bacteria-dominated intermediate layers (5-14 cm) and to deep layers (>14 cm) dominated by enigmatic Archaea that typically occur in deep-sea sediments. Taxonomic replacement was the prevalent mechanism in structuring the community composition and was linked to parameters indicative of microbial activity (e.g., CO2 and CH4 concentration, bacterial protein production). Richness loss played a lesser role but was linked to conservative parameters (e.g., C, N, P) indicative of past conditions. CONCLUSIONS: By including all three domains, we were able to directly link the exponential decay of eukaryotes with the active sediment microbial community. The dominance of Archaea in deeper layers confirms earlier findings from marine systems and establishes freshwater sediments as a potential low-energy environment, similar to deep sea sediments. We propose a general model of sediment structure and function based on microbial characteristics and burial processes. An upper "replacement horizon" is dominated by rapid taxonomic turnover with depth, high microbial activity, and biotic interactions. A lower "depauperate horizon" is characterized by low taxonomic richness, more stable "low-energy" conditions, and a dominance of enigmatic Archaea.

Draft Genome Sequences of Two Gammaproteobacterial Methanotrophs Isolated from Rice Ecosystems.

The genomes of the aerobic methanotrophs "Methyloterricola oryzae" strain 73aT and Methylomagnum ishizawai strain 175 were sequenced. Both strains were isolated from rice plants. Methyloterricola oryzae strain 73aT represents the first isolate of rice paddy cluster I, and strain 175 is the second representative of the recently described genus Methylomagnum.

Redox stability regulates community structure of active microbes at the sediment-water interface.

Changes in redox conditions occur in a wide range of microbial habitats, in particular at the sediment-water interface (SWI) of aquatic systems. A mesocosm study using intact sediment cores from Lake Stechlin (Germany) was performed to investigate the impact of redox changes on microbial communities at the SWI. The SWI was exposed to permanent oxic (OX) or anoxic (ANOX) or to variable (VR) redox conditions, and for molecular analysis sediment samples were taken at the start and after seven days of the treatment. We performed 16S rRNA amplicon sequencing to identify redox-specific changes in the composition of metabolically active microbes. Generally, the community of active microbes in the VR cores was similar to in the OX cores, but differed significantly from the ANOX cores. Interestingly, VR conditions resulted in a high fraction of a Crenothrix-like microorganism increasing in read abundance from 4 to 5% initially, up to 69% over the experimental period. This implies that periodic redox fluctuations select for specific bacteria in environments such as seiches-affected sediments of stratified lakes. In Lake Stechlin sediment cores, these redox fluctuations lead to increased activities of specific microorganisms and high organic matter turnover rates with profound implications for aquatic organic matter cycling.

Effects of Elevated Tropospheric Ozone Concentration on the Bacterial Community in the Phyllosphere and Rhizoplane of Rice.

Microbes constitute a vital part of the plant holobiont. They establish plant-microbe or microbe-microbe associations, forming a unique microbiota with each plant species and under different environmental conditions. These microbial communities have to adapt to diverse environmental conditions, such as geographical location, climate conditions and soil types, and are subjected to changes in their surrounding environment. Elevated ozone concentration is one of the most important aspects of global change, but its effect on microbial communities living on plant surfaces has barely been investigated. In the current study, we aimed at elucidating the potential effect of elevated ozone concentrations on the phyllosphere (aerial part of the plant) and rhizoplane (surface of the root) microbiota by adopting next-generation 16S rRNA amplicon sequencing. A standard japonica rice cultivar Nipponbare and an ozone-tolerant breeding line L81 (Nipponbare background) were pre-grown in a greenhouse for 10 weeks and then exposed to ozone at 85 ppb for 7 h daily for 30 days in open top chambers. Microbial cells were collected from the phyllosphere and rhizoplane separately. The treatment or different genotypes did not affect various diversity indices. On the other hand, the relative abundance of some bacterial taxa were significantly affected in the rhizoplane community of ozone-treated plants. A significant effect of ozone was detected by homogeneity of molecular variance analysis in the phyllosphere, meaning that the community from ozone-treated phyllosphere samples was more variable than those from control plants. In addition, a weak treatment effect was observed by clustering samples based on the Yue and Clayton and weighted UniFrac distance matrices among samples. We therefore conclude that the elevated ozone concentrations affected the bacterial community structure of the phyllosphere and the rhizosplane as a whole, even though this effect was rather weak and did not lead to changes of the function of the communities.

Microbial Response to Experimentally Controlled Redox Transitions at the Sediment Water Interface.

The sediment-water interface of freshwater lakes is characterized by sharp chemical gradients, shaped by the interplay between physical, chemical and microbial processes. As dissolved oxygen is depleted in the uppermost sediment, the availability of alternative electron acceptors, e.g. nitrate and sulfate, becomes the limiting factor. We performed a time series experiment in a mesocosm to simulate the transition from aerobic to anaerobic conditions at the sediment-water interface. Our goal was to identify changes in the microbial activity due to redox transitions induced by successive depletion of available electron acceptors. Monitoring critical hydrochemical parameters in the overlying water in conjunction with a new sampling strategy for sediment bacteria enabled us to correlate redox changes in the water to shifts in the active microbial community and the expression of functional genes representing specific redox-dependent microbial processes. Our results show that during several transitions from oxic-heterotrophic condition to sulfate-reducing condition, nitrate-availability and the on-set of sulfate reduction strongly affected the corresponding functional gene expression. There was evidence of anaerobic methane oxidation with NOx. DGGE analysis revealed redox-related changes in microbial activity and expression of functional genes involved in sulfate and nitrite reduction, whereas methanogenesis and methanotrophy showed only minor changes during redox transitions. The combination of high-frequency chemical measurements and molecular methods provide new insights into the temporal dynamics of the interplay between microbial activity and specific redox transitions at the sediment-water interface.

Measuring bacterial activity and community composition at high hydrostatic pressure using a novel experimental approach: a pilot study.

In this pilot study, we describe a high-pressure incubation system allowing multiple subsampling of a pressurized culture without decompression. The system was tested using one piezophilic (Photobacterium profundum), one piezotolerant (Colwellia maris) bacterial strain and a decompressed sample from the Mediterranean deep sea (3044 m) determining bacterial community composition, protein production (BPP) and cell multiplication rates (BCM) up to 27 MPa. The results showed elevation of BPP at high pressure was by a factor of 1.5 ± 1.4 and 3.9 ± 2.3 for P. profundum and C. maris, respectively, compared to ambient-pressure treatments and by a factor of 6.9 ± 3.8 fold in the field samples. In P. profundum and C. maris, BCM at high pressure was elevated (3.1 ± 1.5 and 2.9 ± 1.7 fold, respectively) compared to the ambient-pressure treatments. After 3 days of incubation at 27 MPa, the natural bacterial deep-sea community was dominated by one phylum of the genus Exiguobacterium, indicating the rapid selection of piezotolerant bacteria. In future studies, our novel incubation system could be part of an isopiestic pressure chain, allowing more accurate measurement of bacterial activity rates which is important both for modeling and for predicting the efficiency of the oceanic carbon pump.