Ridge with no-tillage facilitates microbial N2 fixation associated with methane oxidation in rice soil.

Aerobic methane-oxidizing bacteria (MOB) play a crucial role in mitigating the greenhouse gas methane emission, particularly prevalent in flooded wetlands. The implementation of ridge with no-tillage practices within a rice-rape rotation system proves effective in overcoming the restrictive redox conditions associated with waterlogging. This approach enhances capillary water availability from furrows, especially during periods of low rainfall, thereby supporting plant growth on the ridges. However, the microbe-mediated accumulation of soil organic carbon and nitrogen remains insufficiently understood under this agricultural practice, particularly concerning methane oxidation, which holds ecological and agricultural significance in the rice fields. In this study, the ridge and ditch soils from a 28-year-old ridge with no-tillage rice field experiment were utilized for incubation with 13C-CH4 and 15NN2 to estimate the methane-oxidizing and N2-fixing potentials. Our findings reveal a significantly higher net production of fresh soil organic carbon in the ridge compared to the ditch soil during methane oxidation, with values of 626 and 543 µg 13C g-1 dry weight soil, respectively. Additionally, the fixed 15N exhibited a twofold increase in the ridge soil (14.1 µg 15N g-1 dry weight soil) compared to the ditch soil. Interestingly, the result of DNA-based stable isotope probing indicated no significant differences in active MOB and N2 fixers between ridge and ditch soils. Both Methylocystis-like type II and Methylosarcina/Methylomonas-like type I MOB catalyzed methane into organic biomass carbon pools. Soil N2-fixing activity was associated with the 15N-labeling of methane oxidizers and non-MOB, such as methanol oxidizers (Hyphomicrobium) and conventional N2 fixers (Burkholderia). Methane oxidation also fostered microbial interactions, as evidenced by co-occurrence patterns. These results underscore the dual role of microbial methane oxidation - not only as a recognized sink for the potent greenhouse gas methane but also as a source of soil organic carbon and bioavailable nitrogen. This emphasizes the pivotal role of microbial methane metabolism in contributing to soil carbon and nitrogen accumulation in ridge with no-tillage systems.

Active Methanotrophs in Suboxic Alpine Swamp Soils of the Qinghai-Tibetan Plateau.

Methanotrophs are the only biofilters for reducing the flux of global methane (CH4) emissions in water-logged wetlands. However, adaptation of aerobic methanotrophs to low concentrations of oxygen and nitrogen in typical swamps, such as that of the Qinghai-Tibetan Plateau, is poorly understood. In this study, we show that Methylobacter-like methanotrophs dominate methane oxidation and nitrogen fixation under suboxic conditions in alpine swamp soils. Following incubation with 13C-CH4 and 15N-N2 for 90 days under suboxic conditions with repeated flushing using an inert gas (i.e., argon), microbial carbon and nitrogen turnover was measured in swamp soils at different depths: 0-20 cm (top), 40-60 cm (intermediate), and 60-80 cm (deep). Results show detectable methane oxidation and nitrogen fixation in all three soil depths. In particular, labeled carbon was found in CO2 enrichment (13C-CO2), and soil organic carbon (13C-SOC), whereas labeled nitrogen (15N) was detected in soil organic nitrogen (SON). The highest values of labeled isotopes were found at intermediate soil depths. High-throughput amplicon sequencing and Sanger sequencing indicated the dominance of Methylobacter-like methanotrophs in swamp soils, which comprised 21.3-24.0% of the total bacterial sequences, as measured by 13C-DNA at day 90. These results demonstrate that aerobic methanotroph Methylobacter is the key player in suboxic methane oxidation and likely catalyzes nitrogen fixation in swamp wetland soils in the Qinghai-Tibetan Plateau.

Response of a methane-driven interaction network to stressor intensification.

Microorganisms may reciprocally select for specific interacting partners, forming a network with interdependent relationships. The methanotrophic interaction network, comprising methanotrophs and non-methanotrophs, is thought to modulate methane oxidation and give rise to emergent properties beneficial for the methanotrophs. Therefore, microbial interaction may become relevant for community functioning under stress. However, empirical validation of the role and stressor-induced response of the interaction network remains scarce. Here, we determined the response of a complex methane-driven interaction network to a stepwise increase in NH4Cl-induced stress (0.5-4.75 g L-1, in 0.25-0.5 g L-1 increments) using enrichment of a naturally occurring complex community derived from a paddy soil in laboratory-scale incubations. Although ammonium and intermediates of ammonium oxidation are known to inhibit methane oxidation, methanotrophic activity was unexpectedly detected even in incubations with high ammonium levels, albeit rates were significantly reduced. Sequencing analysis of the 16S rRNA and pmoA genes consistently revealed divergent communities in the reference and stressed incubations. The 16S rRNA-based co-occurrence network analysis revealed that NH4Cl-induced stress intensification resulted in a less complex and modular network, likely driven by less stable interaction. Interestingly, the non-methanotrophs formed the key nodes, and appear to be relevant members of the community. Overall, stressor intensification unravels the interaction network, with adverse consequences for community functioning.

Toxic responses of metabolites, organelles and gut microorganisms of Eisenia fetida in a soil with chromium contamination.

The toxic sensitivity in different physiological levels of chromium (Cr) contaminated soils with environmentally equivalent concentrations (EEC) was fully unknown. The earthworm Eisenia fetida was exposed to a Cr-contaminated soil at the EEC level (referred to as Cr-CS) to characterize the induced toxicity at the whole body, organ, tissue, subcellular structure and metabolic levels. The results showed that the survival rate, weight and biodiversity of the gut microorganisms (organ) had no significant difference (p > 0.05) between control and Cr-CS groups. Qualitative histopathological and subcellular evaluations from morphology showed earthworms obvious injuries. The organelle injuries combined with the metabolic changes provided additional evidence that the Cr-CS damaged the nucleus and probably disturbed the nucleic acid metabolism of earthworms. 2-hexyl-5-ethyl-3-furansulfonate, dimethylglycine, betaine and scyllo-inositol were sensitive and relatively quantitative metabolites that were recommended as potential biomarkers for Cr-CS based on their significant weights in the multivariate analysis model. In addition, the relative abundance of Burkholderiaceae, Enterobacteriaceae and Microscillaceae of the earthworm guts in the Cr-CS group significantly increased, particularly for Burkholderiaceae (increased by 13.1%), while that of Aeromonadaceae significantly decreased by 5.6% in contrast with the control group. These results provided new insights into our understanding of the toxic effects of the EEC level of Cr contaminated soil from different physiological levels of earthworms and extend our knowledge on the composition and sensitivity of the earthworm gut microbiota in Cr contaminated soil ecosystems. Furthermore, these toxic responses from gut microorganisms to metabolites of earthworms provided important data to improve the adverse outcome pathway and toxic mechanism of the Cr-CS if the earthworm genomics and proteomics would be also gained in the future.

Reduction in soil N2O emissions by pH manipulation and enhanced nosZ gene transcription under different water regimes.

Several studies have been carried out to examine nitrous oxide (N2O) emissions from agricultural soils in the past. However, the emissions of N2O particularly during amelioration of acidic soils have been rarely studied. We carried out the present study using a rice-rapeseed rotation soil (pH 5.44) that was amended with dolomite (0, 1 and 2 g kg-1 soil) under 60% water filled pore space (WFPS) and flooding. N2O emissions and several soil properties (pH, NH4+N, NO3--N, and nosZ gene transcripts) were measured throughout the study. The increase in soil pH with dolomite application triggered soil N transformation and transcripts of nosZ gene controlling N2O emissions under both water regimes (60% WFPS and flooding). The 60% WFPS produced higher soil N2O emissions than that of flooding, and dolomite largely reduced N2O emissions at higher pH under both water regimes through enhanced transcription of nosZ gene. The results suggest that ameliorating soil acidity with dolomite can substantially mitigate N2O emissions through promoting nosZ gene transcription.

[Effects of culture supernatant of human amnion mesenchymal stem cells on biological characteristics of human fibroblasts].

OBJECTIVE: To investigate the effects of culture supernatant of human amnion mesenchymal stem cells (hAMSCs-CS) on biological characteristics of human fibroblasts. METHODS: (1) hAMSCs were isolated from deprecated human fresh amnion tissue of placenta and then sub-cultured. The morphology of hAMSCs on culture day 3 and hAMSCs of the third passage were observed with inverted phase contrast microscope. (2) Two batches of hAMSCs of the third passage were obtained, then the expression of vimentin of cells was observed with immunofluorescence method, and the expression of cell surface marker CD90, CD73, CD105, and CD45 was detected by flow cytometer. (3) hAMSCs-CS of the third passage at culture hour 72 were collected, and the content of insulin-like growth factor Ⅰ (IGF-Ⅰ), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), and basic fibroblast growth factor (bFGF) were detected by enzyme-linked immunosorbent assay. (4) Human fibroblasts were isolated from deprecated human fresh prepuce tissue of circumcision and then sub-cultured. Human fibroblasts of the third passage were used in the following experiments. Cells were divided into blank control group and 10%, 30%, 50%, and 70% hAMSCs-CS groups according to the random number table (the same grouping method below), with 48 wells in each group. Cells in blank control group were cultured with DMEM/F12 medium containing 2% fetal bovine serum (FBS), while cells in the latter 4 groups were cultured with DMEM/F12 medium containing corresponding volume fraction of hAMSCs-CS and 2% FBS. The proliferation activity of cells was detected by cell counting kit 8 and microplate reader at culture hour 12, 24, 48, and 72, respectively, and corresponding volume fraction of hAMSCs-CS which causing the best proliferation activity of human fibroblasts was used in the following experiments. (5) Human fibroblasts were divided into blank control group and 50% hAMSCs-CS group and treated as in (4), with 4 wells in each group, at post scratch hour (PSH) 0 (immediately after scratch), 12, 24, 48, and 72, the migration distance of cells was observed and measured with inverted phase contrast microscope. (6) Human fibroblasts were grouped and treated as in (5), with 3 battles in each group, and apoptosis rate of cells was detected by flow cytometer. Data were processed with analysis of variance of factorial design, analysis of variance for repeated measurement, one-way analysis of variance, LSD test, and t test. RESULTS: (1) On culture day 3, most hAMSCs were in large form, and spindle-shaped with much prominences like fibroblasts or in flat polygonal shape. hAMSCs of the third passage were spindle-shaped. The expression of vimentin of hAMSCs of the third passage was strongly positive, and the expressions of surface markers CD90, CD73, and CD105 of the cells were positive, while the expression of CD45 of the cells was negative. (2) The content of IGF-Ⅰ, VEGF, EGF, and bFGF in hAMSCs-CS were respectively (11.7±1.0), (316±68), (6.1±0.4), and (1.49±0.05) pg/mL. (3) At culture hour 12-72, the proliferation activity of human fibroblasts in each hAMSCs-CS group was significantly higher than that in blank control group (with P values below 0.01), and the proliferation activity of human fibroblasts in 50% hAMSCs-CS group was the highest. (4) The width of scratch in two groups was nearly the same at PSH 0. The migration distance of cells in 50% hAMSCs-CS group was significantly longer than that in blank control group at PSH 12-72 (with P values below 0.01). (5) The apoptosis rate of human fibroblasts in blank control group was (16.2±2.4)%, which was significantly higher than that in 50% hAMSCs-CS group [(7.4±3.6)%, t=6.710, P<0.01]. CONCLUSIONS: hAMSCs-CS can promote proliferation and migration of human fibroblasts and inhibit the apoptosis of human fibroblasts.

Effects of dicyandiamide and dolomite application on N2O emission from an acidic soil.

Soil acidification is a major problem for sustainable agriculture since it limits productivity of several crops. Liming is usually adopted to ameliorate soil acidity that can trigger soil processes such as nitrification, denitrification, and loss of nitrogen (N) as nitrous oxide (N2O) emissions. The loss of N following liming of acidic soils can be controlled by nitrification inhibitors (such as dicyandiamide). However, effects of nitrification inhibitors following liming of acidic soils are not well understood so far. Here, we conducted a laboratory study using an acidic soil to examine the effects of dolomite and dicyandiamide (DCD) application on N2O emissions. Three levels of DCD (0, 10, and 20 mg kg(-1); DCD0, DCD10, and DCD20, respectively) were applied to the acidic soil under two levels of dolomite (0 and 1 g kg(-1)) which were further treated with two levels of N fertilizer (0 and 200 mg N kg(-1)). Results showed that N2O emissions were highest at low soil pH levels in fertilizer-treated soil without application of DCD and dolomite. Application of DCD and dolomite significantly (P ≤ 0.001) reduced N2O emissions through decreasing rates of NH4 (+)-N oxidation and increasing soil pH, respectively. Total N2O emissions were reduced by 44 and 13% in DCD20 and dolomite alone treatments, respectively, while DCD20 + dolomite reduced N2O emissions by 54% when compared with DCD0 treatment. The present study suggests that application of DCD and dolomite to acidic soils can mitigate N2O emissions.