Microbial functioning refers to microbial activity because only the active microorganisms drive biogeochemical processes. Despite the importance of active microorganisms, most methods focus on estimating total microbial biomass and fail to evaluate its active fraction. At first, we have described the differences among the active, potentially active, and dormant microbial states in soil and suggested threshold values of parameters for their identification. Secondly, we critically reviewed the ability of a broad range of approaches to estimate and characterize the active and the potentially active microorganisms in soil. Following approaches were evaluated: plate count and microbial cultures; direct microscopy combined with cell staining; ATP, PLFA, DNA and RNA content; microarray analyses; PCR-based approaches; stable isotope probing; soil proteomics, enzymes activity; and various approaches based on respiration and substrate utilization. The “static” approaches, mainly based on the single-stage determination of cell components (ATP, DNA, RNA, and molecular biomarkers), detect well the presence of microorganisms and total biomass, but they fail to evaluate the active part and consequently the functions. In contrast, the dynamic approaches, estimating the changes of these parameters during microbial growth and based on process rates: substrate utilization and product formation, e.g., respiration, help to evaluate active microbial biomass and relate it to specific process rates. Based on a comparison of all approaches for their universality (possibility to analyze active, potentially active and dormant microorganisms), we concluded that 1) direct microscopy with complementary stains, 2) a combination of RNA-based FISH with staining of total microbial biomass, and 3) approaches based on microbial growth were the most advantageous and allowed simultaneous quantitative estimation of active, potentially active, and dormant microorganisms in soil. The active microorganisms compose only about 0.1–2% of the total microbial biomass and very seldom exceed 5% in soils without input of easily available substrates. Nonetheless, the fraction of potentially active microorganisms (ready to start utilization of available substrates within few hours) is much higher, contributing between 10 and 40% (up to 60%) of the total microbial biomass. Therefore, we emphasize the role of potentially active microorganisms with quick response to fluctuating substrate input in soil microhabitats and hotspots. The transition from the potentially active to the active state occurs in minutes to hours, but the shift from dormant to active state takes anywhere from hours to days. Despite very fast activation, the reverse process – fading to the potentially active and dormant stage – requires a much longer period and is very different for individual criteria: ATP, DNA, RNA, enzyme production, respiration rates. This leads to further difficulties in the estimation of the active part of microbial community by methods based on these parameters. Consequently, the standardization, further elaboration, and broad application of approaches focused on the portion of active microorganisms in soil and their functions are urgently needed. We conclude that because active microorganisms are the solely microbial drivers of main biogeochemical processes, analyses of the active and potentially active fractions are necessary in studies focused on soil functions.
Two peatland micro-relief forms (microforms) – hummocks and hollows – differ by their hydrological characteristics (water table level, i.e. oxic-anoxic conditions) and vegetation communities. We studied the CH4 and CO2 production potential and the localization of methanogenic pathways in both hummocks and hollows at depths of 15, 50, 100, 150 and 200cm in a laboratory incubation experiment. For this purpose, we measured CH4 and CO2 production rates, peat elemental composition, as well as δ13C values of gases and solids; the specific inhibitor of methanogenesis BES (2-bromo-ethane sulfonate, 1mM) was aimed to preferentially block the acetoclastic pathway. The cumulative CH4 production of all depths was almost one fold higher in hollows than in hummocks, with no differences in CO2. With depth, CO2 and CH4 production decreased, and the relative contribution of the hydrogenotrophic pathway of methanogenesis increased. The highest methanogenic activity among all depths and both microforms was measured at 15cm of hollows (91%) at which the highest relative contribution of acetoclastic vs. hydrogenotrophic pathway (92 and 8%, respectively) was detected. For hummocks, the CH4 production was the highest at 50cm (82%), where relative contribution of acetoclastic methanogenesis comprised 89%. The addition of 1mM BES was not selective and inhibited both methanogenic pathways in the soil. Thus, BES was less efficient in partitioning the pathways compared with the δ13C signature. We conclude that the peat microforms – dry hummocks and wet hollows – play an important role for CH4 but not for CO2 production when the effects of living vegetation are excluded.
Bacterial communities play key roles in maintaining ecosystem multifunctionality. With increasing land use intensity, soil biogeochemical and microbial characteristics change significantly and may affect the multifunctionality of ecosystems. The relationship between soil microbial communities and resistance of multiple ecosystem functions under land-use change has not previously been assessed in the karst regions. Soils from four karst ecosystems (primary forest, secondary forest, abandoned land and cultivated land) were analyzed for microbial communities as predictor of multifunctional resistance to land use change by using high-throughput sequencing, structural equation modeling and random forest modeling. We evaluated the multifunctional resistance of soil ecosystems by measuring indicators related to soil carbon, nitrogen and phosphorus cycling. The resistance of Proteobacteria was the highest in the secondary forest, and that of Verrucomicrobia was the highest in the abandoned and cultivated lands. With increasing land-use intensity, C-cycling functional resistance decreased by 77% and nitrogen functional resistance increased by 17% in the abandoned land, compared with those in the secondary forest. Bacterial communities had the largest direct positive effect on multifunctional resistance and N-related functional resistance. Among bacterial communities, Verrucomicrobia and Chloroflexi were the two most important phyla that affected soil multifunctional resistance. Armatimonadetes_unclassified, Chloroflexia and OPB35_soil_group were the best predictor of total organic carbon, total nitrogen and total phosphorus content, respectively. Our results suggested strong links between microbial community composition and multifunctional resistance in various karst ecosystems, and provided insights into the importance of microbial community composition in the recovery of ecosystems following human intervention.
Nitrogen (N) is a crucial nutrient for the growth and activity of rhizosphere microorganisms, particularly during drought conditions. Plant root-secreted mucilage contains N that could potentially nourish rhizosphere microbial communities. However, there remains a significant gap in understanding mucilage N content, its source, and its utilization by microorganisms under drought stress. In this study, we investigated the impact of four maize varieties (DH02 and DH04 from Kenya, and Kentos and Keops from Germany) on the secretion rates of mucilage from aerial roots and explored the origin of mucilage N supporting microbial life in the rhizosphere. We found that DH02 exhibited a 96% higher mucilage secretion rate compared to Kentos, while Keops showed 114% and 89% higher secretion rates compared to Kentos and DH04, respectively. On average, the four maize varieties released 4 μg N per root tip per day, representing 2% of total mucilage secretion. Notably, the natural abundance of 15 N isotopes increased (higher δ 15 N signature) with mucilage N release. This indicates a potential dilution of the isotopic signal from biological fixation of atmospheric N by mucilage-inhabiting bacteria as mucilage secretion rates increase. We proposed a model linking mucilage secretion to a mixture of isotopic signatures and estimated that biological N fixation may contribute to 45 - 75% of mucilage N per root tip. The N content of mucilage from a single maize root tip can support a bacterial population ranging from 10 7 to 10 10 cells per day. In conclusion, mucilage serves as a significant N-rich resource for microbial communities in the rhizosphere during drought conditions.
Photosynthesis of higher plants drives carbon (C) allocation below-ground and controls the supply of assimilates to roots and to rhizosphere microorganisms. To investigate the effect of limited photosynthesis on C allocation, redistribution and reutilization in plant and soil microorganisms, perennial grass Lolium perenne and legume Medicago sativa were clipped or shaded. Plants were labelled with three 14C pulses to trace allocation and reutilization of C assimilated before clipping or shading. Five days after the last 14C pulse, plants were clipped or shaded and the total CO2 and 14CO2 efflux from the soil was measured. 14C in above- and below-ground plant biomass and bulk soil, rhizosphere soil and microorganisms was determined 10 days after clipping or shading. After clipping, 2% of the total assimilated 14C originating mainly from root reserves were detected in the newly grown shoots. This corresponded to a translocation of 5 and 8% of total 14C from reserve organs to new shoots of L. perenne and M. sativa, respectively. The total CO2 efflux from soil decreased after shading of both plant species, whereas after clipping, this was only true for L. perenne. The 14CO2 efflux from soil did not change after clipping of both species. An increased 14CO2 efflux from soil under shading for both plants indicated that lower assimilation was compensated by higher utilization of the reserve C for root and rhizomicrobial respiration. We conclude that C stored in roots is an important factor for plant recovery after limiting photosynthesis. This stored C is important for shoot regrowth after clipping, whereas after shading, it is utilized mainly for maintenance of root respiration. Based on these results as well as on a review of several studies on C reutilization for regrowth after clipping, we conclude that because of the high energy demand for nitrogen fixation, legumes use a higher portion (9–10%) of stored C for regrowth compared to grasses (5–7%). The effects of limited photosynthesis were of minor importance for the exudation of the reserve C and thus, have no effect on the uptake of this C by microorganisms.
Novel biochar-based fertilizers, produced by combining biochar particles with chemical fertilizers, have strong potential to enrich soil with carbon (C) and nitrogen (N), and to increase plant productivity. Application of biochar-based fertilizers modifies soil microbial community compositions, thereby influencing greenhouse gas emissions, including methane (CH4) fluxes. Due to the improved aeration in soils amended with biochar particles, we hypothesized that biochar-based urea would decrease CH4 production and increase CH4 oxidation, leading to increase of total CH4 uptake by soil. A three-year field experiment was conducted in a subtropical Moso bamboo forest to compare the impacts of biochar-based urea and traditional urea on seasonal CH4 uptake by soil, as well as on soil physicochemical and microbial attributes. Urea application lowered the annual soil CH4 uptake by 6 % and 16 % at 100 and 300 kg N/ha, respectively, within the first year. Biochar-based urea application at 300 kg N/ha increased the annual CH4 uptake by 12 % in both the first and second years, whereas the effects weakened over time. Soil CH4 uptake was positively correlated with CH4 oxidation rate but negatively with CH4 production rate. The urea-induced decrease in CH4 uptake was attributed to the increased NH4+ and NO3− contents and mcrA gene abundance as well as decreased pmoA/mcrA ratio, thereby increasing CH4 production rate. The biochar-based urea increased CH4 uptake by enhanced soil aeration and labile C supply, which created favorable conditions for methanotrophs. This resulted in increased pmoA gene abundance and the pmoA/mcrA ratio, thereby accelerating CH4 oxidation. Overall, these findings underscore the potential of biochar-based fertilizers in augmenting CH4 uptake within subtropical forest soils. Notably, the transition from traditional urea to biochar-based urea in China Moso bamboo forests alone has the potential to lift annual soil CH4 uptake by an estimated 4450 t.
