Microbial Extracellular Enzyme Activity Research Articles

Release of intracellular enzymes increases the total extracellular activities of carbohydrate processing enzymes in marine environment.

Microbial extracellular enzymatic activities (EEAs) produced by microbes to degrade biopolymers are the 'gatekeeper' of carbon cycle in the marine ecosystem. It is usually assumed that these extracellular enzymes are actively secreted by microbes. But biopolymers degrading enzymes also exist in the intracellular space. Cell lysis will passively release these enzymes into the environments and contribute to the total EEAs. However, to what extent the cell lysis can contribute to the total EEAs are still unclear. Here, using extreme cell lysis method, we evaluated the maximum contribution of cell lysis to total EEAs in culturable marine bacteria and coastal seawater. For carbohydrate processing enzymes (β-glucosidase, alginate lyase and chitinase), the release of intracellular enzymes could contribute positively (up to 56.1% increase for β-glucosidase in seawater) to the total EEAs. For protease and leucine aminopeptidase, the cell lysis did not increase and even decreased the total EEAs. For alkaline phosphatase, the intracellular enzymes generally had no contribution to the total EEAs. These results showed that passively released intracellular enzymes could substantially increase the total extracellular activities of carbohydrate processing enzymes, which should be considered in building the link between the EEAs and organic carbon cycle in the ocean.

Smooth vetch covering alters soil aggregate microbial metabolic limitations in citrus orchards

Green manure covering alters the supply-demand relationship between soil resources and microorganisms by improving soil structure and increasing carbon inputs. However, it remain unclear how soil microorganism respond to the imbalance between resources and demands at the aggregate scale. The present study analyzed the stoichiometric ratios of organic carbon, nitrogen, and phosphorus nutrients, microbial extracellular enzyme activities, and biomass in large macroaggregates (LMA, 2–8 mm), small macroaggregates (SMA, 0.25–2 mm), and microaggregates (MIA, < 0.25 mm) under smooth vetch covering. The relationship between microbial carbon use efficiency and nutrient restriction in soil aggregates was elucidated and the alteration was revealed in microbial metabolic pathways and carbon sequestration functions within soil aggregates under green manure covering. The results showed that: (1) Smooth vetch covering significantly increased the C: N resource imbalance (ln(MBC: MBN)/ln(SOC: TN)) in soil aggregates from 0.50 to 0.69, and thus increased the nitrogen requirement of soil aggregates. However, the N:P resource imbalance (ln(MBN: MBP)/ln(TN: TP)) decreased from 2.25 to 1.78, especially in microaggregates. (2) To mitigate the limitation imposed by nitrogen availability at the aggregate level, microorganisms have ramped up the activity of C- (46.72 %–98.64 %), N- (2.32 %–121.00 %), and P- acquiring enzymes (119.11 %–187.78 %). Nevertheless, the spatial heterogeneity inherent in soil aggregates, characterized by varying particle sizes, has led to a pivotal shift in microbial nutrient limitation. As soil aggregate size diminishes, the primary constraint on microbial activity transitions from nitrogen limitation to phosphorus limitation, reflecting the dynamic interplay between soil structure and nutrient availability. (3) In addition, smooth vetch covering increased the microbial carbon use efficiency of soil aggregates, and it increased with the increase of the particle size of soil aggregates: LMA (178.80 %) > SMA (147.23 %) > MIA (9.99 %). Microorganisms allocated more energy to generate biomass instead of obtaining limiting nutrient elements. These results indicate that smooth vetch covering could increase the organic carbon content of soil aggregates. In addition, in order to adapt to nutrient imbalance, soil aggregates microorganisms choose a “egoistic” metabolic pathway, which alleviates nutrient limitation and assimilated more energy and nutrients into their own biomass and reduces the release of organic carbon. It is evident that integrating Vicia villosa Roth var. glabresens Koch as a ‘green manure’ in orchards serves a dual purpose: it mitigates the environmental impact of chemical fertilizers while simultaneously promoting soil carbon sequestration. This approach offers a robust theoretical framework for achieving the harmonious integration of ecological sustainability and economic viability in economic forestry, such as citrus orchards.

Effects of Land-Use Type and Salinity on Soil Carbon Mineralization in Coastal Areas of Northern Jiangsu Province

Sea level rise due to glacier melting caused by climate warming is a major global challenge, but the mechanism of the effect of salinity on soil carbon (C) mineralization in different land types is not clear. The pathways by which salinity indirectly affects soil carbon mineralization rates need to be investigated. Whether or not the response mode is consistent among different land-use types, as well as the intrinsic links and interactions between soil microbial resource limitation, environmental stress, microbial extracellular enzyme activity, and soil carbon mineralization, remain to be demonstrated. In this paper, three typical land-use types (wetland, forest, and agroforestry) were selected, and different salinity levels (0‰, 3‰, 6‰, and 32‰) were designed to conduct a 125-day laboratory incubation experiment to determine the soil CO2 release rate, soil physicochemical properties, and soil enzyme activities, and to correlate C mineralization with biotic and abiotic factors. A correlation analysis of soil physical and chemical properties, extracellular enzyme activities, and carbon mineralization rates was conducted to investigate their intrinsic linkages, and a multiple linear regression of C mineralization at different sites was performed to explore the variability of mineralization among different site types. Structural equation models were established in the pre- and post-incubation stages to study the pathways of soil C mineralization at different incubation times, and the mechanism of mineralization was further verified by enzyme stoichiometry. The results showed that, at the end of 125 days of incubation, the 32‰ salinity addition reduced the cumulative mineralization of forest and agroforestry types by 28.41% and 34.35%, respectively, compared to the 0‰ salinity addition. Soil C mineralization in the three different land-use types was highly correlated with the active C fractions of readily oxidizable C (ROC), dissolved organic C, and microbial biomass C (MBC) in the soil, with the standardized coefficients of multivariate linear regression reaching 0.67 for MBC in the wetland and −0.843 for ROC in the forest. Under long-term salinity additions, increased salinity would reduce the microbial respiratory quotient value by inhibiting β-glucosidase activity, thus indirectly affecting the rate of CO2 release. With added salinity, the mineralization of non-saline soil was more susceptible to the inhibitory effect of salinity, whereas the mineralization of salinized soil was more controlled by soil C pools.

Fast labile carbon and litter exhaustion under no-tillage after 5-year soil warming

No-tillage continuously contribute organic matter and increase carbon sequestration in the soil. However, global warming may stimulate microbial activity and accelerate soil organic matter (SOM) decomposition, also especially in the topsoil. Understanding the responses of microbial utilization of SOM accumulated under no-tillage to higher temperatures is necessary to develop management strategies to sustain soil fertility. Determine the response of microbial extracellular enzyme activities and SOM decomposition to long-term warming by incorporation of 14C-labelled maize litter to in situ warmed soil from no-tillage and till systems. We compared decomposability of litter and SOM in soils from a 5-year in situ experiment with two tillage systems (Till and No-till) and under two temperature levels: ambient temperature and continuous soil warming (+1.6 °C at 5 cm soil depth). We hypothesized that decomposition of crop litter (14C-labeled maize) at increasing temperature (15, 21, and 27 °C for 59 days) will be more intensive in warmed soil vs. ambient and the effects should be stronger under No-till vs. Till system. As expected, soil from field warming had always higher total CO2 efflux (from 5.5 to 12%) than the non-warmed counterpart in Till and No-till systems. Five-year warming increased temperature sensitivity (Q10) of CO2 efflux for 2-folds under No-tillage vs. Till. Litter decomposition (measured as 14CO2) in No-till with warming was 7.9% greater than in No-tillage without warming. Three extracellular enzymes (β-glucosidase, chitinase, and sulfatase) had higher activities under warming in No-till but not under Till system. Our results demonstrated that the 5-year in situ warming accelerated preferential microbial utilization of labile SOM fractions in No-till notably, and from stable SOM in Till system. Consequently, warming will accelerate SOM decomposition in future and this acceleration will be especially pronounced on the labile SOM under No-tillage systems.

Root distribution and properties of a young alley-cropping system: effects on soil carbon storage and microbial activity

In agroforestry systems, the root distributions and properties of the annual and perennial vegetation are poorly known, although they are recognized for fostering soil carbon (C) stocks through annual root-derived C inputs and by altering microbial activity. This study aimed to evaluate the potential contribution of roots from the understory vegetation strip (UVS) and the crop to top- and subsoil C stocks (0–100 cm) to a 3-year-old agroforestry system of the alley-cropping type. Root biomass, chemical composition, functional traits and anatomical structure were assessed in parallel to a characterization of soil physicochemical and microbiological properties, including enzymatic activity, at different depths and locations perpendicular to the tree line. The root biomass and properties were heterogeneous in the young alley-cropping system due to the presence of different plant communities and the heterogeneity of the soil mineral N content according to the location perpendicular to the tree line. The soil microbiological properties and organic C stocks did not vary horizontally at this stage of agroforestry but should be monitored through multiple-time samples to confirm a differentiation in subsequent years suggested by the tight link between root stoichiometry and microbial extracellular enzymatic activities that we found. Altogether, our results suggested that increasing the root biomass in topsoil in agroforestry systems positively contributes to increasing soil organic C stocks, but in deeper soil layers, an increase in litter inputs with a high C:N ratio might accentuate microbial N limitations and limit soil C storage.

Experimental increases in pH and P availability exert long-term impacts on decomposition in forests

Forests are an important sink for atmospheric carbon (C), but global change phenomena threaten to alter their C storage capacity. One such global change driver is acid deposition, which can alter rates of decomposition by changing the composition of microbial communities as well as the quality of litter inputs, and ultimately nutrient cycling. The impacts of acid deposition in forests can potentially be ameliorated by increasing soil pH and addressing concurrent nutrient deficiencies. Here, we explore the capacity for experimental acid deposition reversal-induced shifts in the soil microbial community and litter quality to drive changes in rates of C cycling relative to ambient, acidic conditions. Specifically, we conducted a laboratory-based microcosm incubation experiment using soil and litter collected from a 7-year field-based acid deposition reversal experiment (through addition of Hi-Ca limestone to raise pH and triple super phosphate to increase phosphorus [P] availability) to explore the impacts of long-term acid deposition on rates of litter decomposition. We combined litter and soil from untreated (ambient, acidic) and treated (elevated pH + P) plots in a reciprocal design and incubated them in the lab, measuring rates of CO2 evolution and microbial extracellular enzyme activity during litter decomposition. Decomposition was generally depressed on soils from plots with elevated pH and P availability relative to soils from ambient conditions early in the experiment, while decomposition was greater on litter from elevated pH and P plots late in the experiment. There were no effects of treatment on cumulative CO2 evolution over the course of the experiment. Greater P content in litter from elevated pH + P plots led to reduced phosphatase activity in treated plots. We also saw greater β-glucosidase activity in elevated pH + P plots, likely due to greater nutrient availability in these plots, allowing microbes to direct foraging toward more labile forms of C. These results suggest that acid deposition reversal can depress decomposition by altering microbial community functioning, relative to ambient, acidic conditions where we saw accelerated rates of decomposition. Our results emphasize the importance of considering the potential for shifts in microbial communities under acidified conditions to alter C storage and ecosystem functioning.

Effects of Spatial Variability and Drainage on Extracellular Enzyme Activity in Coastal Freshwater Forested Wetlands of Eastern North Carolina, USA

Drainage of freshwater wetlands is common in coastal regions, although the effects on microbial extracellular enzyme activity (a key mediator of soil organic matter decomposition) in relation to spatial variability (microtopography and soil depth) are poorly understood. Soils were collected from organic (Oi, Oe, Oa) and mineral (A, AB, B) horizons from a natural and drained coastal forested wetland in North Carolina, USA. Activity of seven enzymes were measured: α-glucosidase (AG), β-glucosidase (BG), cellobiohydrolase (CBH), xylosidase (XYL), phenol oxidase (POX), peroxidase (PER) and N-acetyl glucosamide (NAG). Enzyme activity rates were normalized by soil weight, soil organic C (SOC), and microbial biomass C (MBC). Specific enzyme activity (per SOC or MBC) was more sensitive to drainage and soil depth compared to normalization by soil weight. In Oi and Oa horizons, specific enzyme activity (per MBC) (AG, BG, XYL, POX, PER) was higher in the natural compared to drained wetland but lower (AG, CBH, XYL, POX, PER, NAG) in the AB or B mineral soils. Results from this study indicate that organic soil horizons of natural freshwater wetlands contain a highly active microbial community driven by inputs of plant-derived C, while deeper soils of the drained wetland exhibit higher microbial metabolic activity, which likely plays a role in SOC storage of these systems.

Effects of different forms of nitrogen addition on microbial extracellular enzyme activity in temperate grassland soil

BackgroundNitrogen (N) deposition alters litter decomposition and soil carbon (C) sequestration by influencing the microbial community and its enzyme activity. Natural atmospheric N deposition comprises of inorganic N (IN) and organic N (ON) compounds. However, most studies have focused on IN and its effect on soil C cycling, whereas the effect of ON on microbial enzyme activity is poorly understood. Here we studied the effects of different forms of externally supplied N on soil enzyme activities related to decomposition in a temperate steppe. Ammonium nitrate was chosen as IN source, whereas urea and glycine were chosen as ON sources. Different ratios of IN to ON (Control, 10:0, 7:3, 5:5, 3:7, and 0:10) were mixed with equal total amounts of N and then used to fertilize the grassland soils for 6 years.ResultsOur results show that IN deposition inhibited lignin-degrading enzyme activity, such as phenol oxidase (POX) and peroxidase (PER), which may restrain decomposition and thus induce accumulation of recalcitrant organic C in grassland soils. By contrast, deposition of ON and mixed ON and IN enhanced most of the C-degrading enzyme activities, which may promote the organic matter decomposition in grassland soils. In addition, the β-N-acetyl-glucosaminidase (NAG) activity was remarkably stimulated by fertilization with both IN and ON, maybe because of the elevated N availability and the lack of N limitation after long-term N fertilization at the grassland site. Meanwhile, differences in soil pH, soil dissolved organic carbon (DOC), and microbial biomass partially explained the differential effects on soil enzyme activity under different forms of N treatments.ConclusionsOur results emphasize the importance of organic N deposition in controlling soil processes, which are regulated by microbial enzyme activities, and may consequently change the ecological effect of N deposition. Thus, more ON deposition may promote the decomposition of soil organic matter thus converting C sequestration in grassland soils into a C source.

Microbial extracellular enzyme activity with simulated climate change

It is critical to understand the consequences of environmental change for the microbial regulation of carbon and nutrient cycling. Specifically, understanding microbial community traits, such as extracellular enzyme activity, can help inform nutrient cycling models and address knowledge gaps. We analyzed data on extracellular enzyme activities and litter decomposition from an 18-month experiment in which microbial communities were reciprocally transplanted along a climate gradient in Southern California. Communities were from desert, scrubland, grassland, pine–oak, and subalpine ecosystems. We aimed to test how enzyme activities responded to climate change following transplantation and how those responses related to decomposition rates. We hypothesized that microbial communities would specialize on their native climate conditions, resulting in higher enzyme activities when transplanted back into their native climate. We investigated the relationship between extracellular enzyme Vmax values, substrate mass loss, and microbial biomass as well as variation in these variables across the climate gradient. We found little evidence for climate specialization, and there was rarely a reduction in enzyme functioning after microbial communities were transplanted into new climate conditions. Moreover, observed differences in decomposition were not related to changes in extracellular enzyme potential, although there were significant differences in enzyme activities and decomposition rates across sites. These results suggest that direct, physiological impacts of climate are likely to be important for enzyme-mediated decomposition, but climate specialization will not constrain the microbial response to climate change in our system.

Effects of COVID-19 lockdown on water quality, microbial extracellular enzyme activity, and sediment-P release in the Ganga River, India.

This study investigates possible improvement in water quality and ecosystem functions in the Ganga River as influenced by COVID-19 lockdown in India. A total of 132 samples were collected during summer-2020 low flow (coinciding COVID-19 lockdown) for water (sub-surface and sediment-water interface) and 132 samples separately for sediment (river bottom and land-water interface) considering 518-km main river stem including three-point sources (one releases urban sewage and the other two add metal-rich industrial effluents) and a pollution-impacted tributary. Parameters such as dissolved oxygen deficit and the concentrations of carbon, nutrients (N and P), and heavy metals were measured in water. Sediment P-release was measured in bottom sediment whereas extracellular enzymes (EE; alkaline phosphatase, FDAase, protease, and β-D-glucosidase) and CO2 emission were measured at land-water interface to evaluate changes in water quality and ecosystem functions. The data comparisons were made with preceding year (2019) measurements. Sediment-P release and the concentrations of carbon, nutrients, and heavy metals declined significantly (p<0.05) in 2020 compared to those recorded in 2019. Unlike the preceding year, we did not observe benthic hypoxia (DO <2.0 mg L−1) in 2020 even at the most polluted site. The EE activities, which declined sharply in the year 2019, showed improvement during the 2020. The stability coefficient and correlative evidences also showed a large improvement in the water quality and functional variables. Positive changes in functional attributes indicated a transient recovery when human perturbations withdrawn. The study suggests that timing the ecosystem recovery windows, as observed here, may help taking management decision to design mitigation actions for rivers to recover from anthropogenic perturbations.

Warming and Labile Substrate Addition Alter Enzyme Activities and Composition of Soil Organic Carbon

Warming can increase the efflux of carbon dioxide (CO2) from soils and can potentially feedback to climate change. In addition to warming, the input of labile carbon can enhance the microbial activity by stimulating the co-metabolism of recalcitrant soil organic matter (SOM). This is particularly true with SOM under invaded ecosystems where elevated CO2and warming may increase the biomass of invasive species resulting in higher addition of labile substrates. We hypothesized that the input of labile carbon would instigate a greater soil organic carbon (SOC) loss with warming compared to the ambient temperature. We investigated this by incubating soils collected from a native pine (Pinus taeda) forest to which labile carbon from the invasive species kudzu (Pueraria lobata) was added. We evaluated the microbial extracellular enzyme activity, molecular composition of SOC and the temperature sensitivity of soil CO2efflux under warming and labile carbon addition. After 14 months of soil incubation, the addition of labile C through kudzu extract increased the activity of β-1,4-glucosidase compared with the control. However, the activity of N-acetyl-β-D-glucosaminidase and fungal biomass (ergosterol) decreased with labile carbon addition. The activity of peroxidase increased with warming after 14 months of soil incubation. Although the carbon content of incubated soils did not vary with substrate and temperature treatments, the molecular composition of SOC indicated a general decrease in biopolymers such as cutin, suberin, long-chain fatty acids, and phytosterol with warming and an increasing trend of microbial-derived compounds with labile substrate addition. In soils that received an addition of labile C, the macro-aggregate stability was higher while the temperature sensitivity of soil C efflux was lower compared with the control. The increase in aggregate stability could enhance the physical protection of SOC from microbial decomposition potentially contributing to the observed pattern of temperature sensitivity. Our results suggest that warming could preferentially accelerate the decomposition of recalcitrant compounds while the addition of labile substrates could enhance microbial-derived compounds that are relatively resistant to further decomposition. Our study further emphasizes that global change factors such as plant invasion and climate change can differentially alter soil microbial activity and the composition of SOC.

Plant removal across an elevational gradient marginally reduces rates, substantially reduces variation in mineralization.

The loss of aboveground plant diversity alters belowground ecosystem function; yet, the mechanisms underpinning this relationship and the degree to which plant community structure and climate mediate the effects of plant species loss remain unclear. Here, we explored how plant species loss through experimental removal shaped belowground function in ecosystems characterized by different climatic regimes and edaphic properties. We measured plant community composition as well as potential carbon (C) and nitrogen (N) mineralization and microbial extracellular enzyme activity in soils collected from four unique plant removal experiments located along an elevational gradient in Colorado, USA. We found that, regardless of the identity of the removed species or the climate at each site, plant removal decreased the absolute variation in potential N mineralization rates and marginally reduced the magnitude of N mineralization rates. While plant species removal also marginally reduced C mineralization rates, C mineralization, unlike N mineralization, displayed sensitivity to the climatic and edaphic differences among sites, where C mineralization was greatest at the high elevation site that receives the most precipitation annually and contains the largest soil total C pool. Plant removal had little impact on soil enzyme activity. Removal effects were not contingent on the amount of biomass removed annually, and shifts in mineralization rates occurred despite only marginal shifts in plant community structure following plant species removal. Our results present a surprisingly simple and consistent pattern of belowground response to the loss of dominant plant species across an elevational gradient with different climatic and edaphic properties, suggesting a common response of belowground ecosystem function to plant species loss regardless of which plant species are lost or the broader climatic context.

Enhanced plant leaf P and unchanged soil P stocks after a quarter century of warming in the arctic tundra

AbstractPhosphorus (P) limits or co‐limits plant and microbial life in multiple ecosystems, including the arctic tundra. Although current global carbon (C) models focus on the coupling between soil nitrogen (N) and C, ecosystem P response to climate warming may also influence the global C cycle. Permafrost soils may see enhanced or reduced P availability under climate warming through multiple mechanisms including changing litter inputs through plant community change, changing plant–microbial dynamics, altered rates of mineralization of soil organic P through increased microbial activity, and newly exposed mineral‐bound P via deeper thaw. We investigated the effect of long‐term warming on plant leaf, multiple soil and microbial C, N, and P pools, and microbial extracellular enzyme activities, in Alaskan tundra plots underlain by permafrost. Here, we show that 25 yr of experimental summer warming increases community‐level plant leaf P through changing community composition to favour relatively P‐rich plant species. However, despite associated increases in P‐rich litter inputs, we found only a few responses in the belowground pools of P available for plant and microbial uptake, including a weak positive response for citric acid–extractable PO4 in the surface soil, a decrease in microbial biomass P, and no change in soil P (or C or N) stocks. This weak, neutral, or negative belowground P response to warming despite enhanced litter P inputs is consistent with a growing number of studies in the arctic tundra that find no long‐term response of soil C and N stocks to warming.

Large Mammalian Herbivores and the Paradox of Soil Carbon in Grazing Ecosystems: Role of Microbial Decomposers and Their Enzymes

Grazing is the dominant land use across the world, and large mammalian herbivores exert strong influence over biogeochemical cycles. Grazing ecosystems feature C-rich soils, even though herbivores consume a major fraction of plant production to reduce detrital input to soil. Yet, counter-intuitively, moderate grazing can promote net soil-C storage in many ecosystems compared to grazer-exclusion. We address this enigmatic influence of grazers on soil-C and test their indirect effect on proximate drivers of decomposition: microbial extracellular enzyme activity. We used a replicated long-term grazer-exclusion experiment to measure responses in above- and belowground plant biomass, soil-C stock, microbial biomass, labile/recalcitrant C pools and three enzymes relevant to the C-cycle: peroxidase—which initiates decomposition of recalcitrant matter, alongside beta-glucosidase and cellobiohydrolase—which act further downstream on more labile fractions. Consistent with other ecosystems, upto 12 years of herbivore exclusion did not increase soil-C in the fenced plots despite higher plant biomass and higher potential detrital C-inputs. Grazer-exclusion did not alter microbial biomass; peroxidase increased threefold and beta-glucosidase was doubled; cellobiohydrolase was unaffected. Grazer-exclusion also led to twofold increase in recalcitrant-C and in microbial respiration, but it did not influence labile-C. Structural equation models supported the hypothesis that grazing favours soil-C via its indirect effect on peroxidase, but they did not support that the effects can run in the opposite direction where soil-C affects enzymes. Grazer-mediated shifts in how microbes deploy enzymes emerge as a plausible mechanism that affects soil-C. These linkages may be important to maintain soil-C sequestration in drylands which support large mammalian herbivores.

Saltwater and phosphorus drive unique soil biogeochemical processes in freshwater and brackish wetland mesocosms

AbstractCoastal ecosystems are exposed to saltwater intrusion but differential effects on biogeochemical cycling are uncertain. We tested how elevated salinity and phosphorus (P) individually and interactively affect microbial activities and biogeochemical cycling in freshwater and brackish wetland soils. In experimental mesocosms, we added crossed gradients of elevated concentrations of soluble reactive P (SRP) (0, 20, 40, 60, 80 μg/L) and salinity (0, 4, 7, 12, 16 ppt) to freshwater and brackish peat soils (10, 14, 17, 22, 26 ppt) for 35 d. We quantified changes in water chemistry [dissolved organic carbon (DOC), ammonium (), nitrate + nitrite (N + N), SRP concentrations], soil microbial extracellular enzyme activities, respiration rates, microbial biomass C, and soil chemistry (%C, %N, %P, C:N, C:P, N:P). DOC, , and SRP increased in freshwater but decreased in brackish mesocosms with elevated salinity. DOC similarly decreased in brackish mesocosms with added P, and N + N decreased with elevated salinity in both freshwater and brackish mesocosms. In freshwater soils, water column P uptake occurred only in the absence of elevated salinity and when P was above 40 µg/L. Freshwater microbial EEAs, respiration rates, and microbial biomass C were consistently higher compared to those from brackish soils, and soil phosphatase activities and microbial respiration rates in freshwater soils decreased with elevated salinity. Elevated salinity increased arylsulfatase activities and microbial biomass C in brackish soils, and elevated P increased microbial respiration rates in brackish soils. Freshwater soil %C, %N, %P decreased and C:P and N:P increased with elevated salinity. Elevated P increased %C and C:N in freshwater soils and increased %P but decreased C:P and N:P in brackish soils. Freshwater soils released more C and nutrients than brackish soils when exposed to elevated salinity, and both soils were less responsive to elevated P than expected. Freshwater soils became more nutrient‐depleted with elevated salinity, whereas brackish soils were unaffected by salinity but increased P uptake. Microbial activities in freshwater soils were inhibited by elevated salinity and unaffected by added P, but brackish soil microbial activities slightly increased with elevated salinity and P.

Seasonal changes in soil properties, microbial biomass and enzyme activities across the soil profile in two alpine ecosystems

Microbial biomass and extracellular enzyme activities control the rate of soil organic carbon decomposition, thereby affecting soil carbon pool. However, seasonal dynamics of soil microbial properties at different depths of the soil profile remain unclear. In this study, we sampled soils in the early, middle and late growing season at different soil depths (0–100 cm) in two alpine ecosystems (meadow and shrubland) on the Tibetan Plateau. We measured plant belowground biomass, soil properties, microbial biomass and extracellular enzyme activities. We found that soil properties changed significantly with sampling time and soil depth. Specifically, most of soil properties consistently decreased with increasing soil depth, but inconsistently varied with sampling time. Moreover, root biomass and microbial biomass decreased with increasing soil depth and increased with sampling time during the growing season. However, microbial extracellular enzyme activities and their vector properties all changed with depth, but did not vary significantly with time. Taken together, these results show that soil properties, microbial biomass and extracellular enzyme activities mostly decline with increasing depth of the soil profile, and soil properties and microbial biomass are generally more variable during the growing season than extracellular enzyme activities across the soil profile in these alpine ecosystems. Further studies are needed to investigate the changes in soil microbial community composition and function at different soil depths over the growing season, which can enhance our mechanistic understanding of whole-profile soil carbon dynamics of alpine ecosystems under climate change.

Influence of water availability and temperature on estimates of microbial extracellular enzyme activity.

Soils are highly heterogeneous and support highly diverse microbial communities. Microbial extracellular enzymes breakdown complex polymers into small assimilable molecules representing the limiting step of soil organic matter mineralization. This process occurs on to soil particles although currently it is typically estimated in laboratory aqueous solutions. Herein, estimates of microbial extracellular enzyme activity were obtained over a broad range of temperatures and water availabilities frequently observed at soil upper layers. A Pseudomonas strain presented optimum extracellular enzyme activities at high water activity whereas a desiccation resistant bacterium (Deinococcus) and a soil thermophilic isolate (Parageobacillus) showed optimum extracellular enzyme activity under dried (i.e., water activities ranging 0.5–0.8) rather that wet conditions. Different unamended soils presented a distinctive response of extracellular enzyme activity as a function of temperature and water availability. This study presents a procedure to obtain realistic estimates of microbial extracellular enzyme activity under natural soil conditions of extreme water availability and temperature. Improving estimates of microbial extracellular enzyme activity contribute to better understand the role of microorganisms in soils.

Soil C, N and P cycling enzyme responses to nutrient limitation under elevated CO2

Elevated CO2 (eCO2) can stimulate plant productivity and increase carbon (C) input to soils, but nutrient limitation restricts productivity. Despite phosphorus (P)-limited ecosystems increasing globally, it is unknown how nutrient cycling, particularly soil microbial extra cellular enzyme activity (EEA), will respond to eCO2 in such ecosystems. Long-term nutrient manipulation plots from adjacent P-limited acidic and limestone grasslands were exposed to eCO2 (600 ppm) provided by a mini-Free Air CO2 Enrichment system. P-limitation was alleviated (35 kg-P ha−1 y−1 (P35)), exacerbated (35 kg-N ha−1 y−1 (N35), 140 kg-N ha−1 y−1 (N140)), or maintained (control (P0N0)) for > 20 years. We measured EEAs of C-, N- and P-cycling enzymes (1,4-β-glucosidase, cellobiohydrolase, N-acetyl β-D-glucosaminidase, leucine aminopeptidase, and acid phosphatase) and compared C:N:P cycling enzyme ratios using a vector analysis. Potential acid phosphatase activity doubled under N additions relative to P0N0 and P35 treatments. Vector analysis revealed reduced C-cycling investment and increased P-cycling investment under eCO2. Vector angle significantly increased with P-limitation (P35 < P0N0 < N35 < N140) indicating relatively greater investment in P-cycling enzymes. The limestone grassland was more C limited than the acidic grassland, characterised by increased vector length, C:N and C:P enzyme ratios. The absence of interactions between grassland type and eCO2 or nutrient treatment for all enzyme indicators signaled consistent responses to changing P-limitation and eCO2 in both grasslands. Our findings suggest that eCO2 reduces C limitation, allowing increased investment in P- and N-cycle enzymes with implications for rates of nutrient cycling, potentially alleviating nutrient limitation of ecosystem productivity under eCO2.Graphic abstract

Persistence of microbial extracellular enzymes in soils under different temperatures and water availabilities.

Microbial extracellular enzyme activity (EEA) is critical for the decomposition of organic matter in soils. Generally, EEA represents the limiting step governing soil organic matter mineralization. The high complexity of soil microbial communities and the heterogeneity of soils suggest potentially complex interactions between microorganisms (and their extracellular enzymes), organic matter, and physicochemical factors. Previous studies have reported the existence of maximum soil EEA at high temperatures although microorganisms thriving at high temperature represent a minority of soil microbial communities. To solve this paradox, we attempt to evaluate if soil extracellular enzymes from thermophiles could accumulate in soils. Methodology at this respect is scarce and an adapted protocol is proposed. Herein, the approach is to analyze the persistence of soil microbial extracellular enzymes at different temperatures and under a broad range of water availability. Results suggest that soil high‐temperature EEA presented longer persistence than enzymes with optimum activity at moderate temperature. Water availability influenced enzyme persistence, generally preserving for longer time the extracellular enzymes. These results suggest that high‐temperature extracellular enzymes could be naturally accumulated in soils. Thus, soils could contain a reservoir of enzymes allowing a quick response by soil microorganisms to changing conditions. This study suggests the existence of novel mechanisms of interaction among microorganisms, their enzymes and the soil environment with relevance at local and global levels.

Environmental factors affect the response of microbial extracellular enzyme activity in soils when determined as a function of water availability and temperature.

Microorganisms govern soil carbon cycling with critical effects at local and global scales. The activity of microbial extracellular enzymes is generally the limiting step for soil organic matter mineralization. Nevertheless, the influence of soil characteristics and climate parameters on microbial extracellular enzyme activity (EEA) performance at different water availabilities and temperatures remains to be detailed. Different soils from the Iberian Peninsula presenting distinctive climatic scenarios were sampled for these analyses. Results showed that microbial EEA in the mesophilic temperature range presents optimal rates under wet conditions (high water availability) while activity at the thermophilic temperature range (60°C) could present maximum EEA rates under dry conditions if the soil is frequently exposed to high temperatures. Optimum water availability conditions for maximum soil microbial EEA were influenced mainly by soil texture. Soil properties and climatic parameters are major environmental components ruling soil water availability and temperature which were decisive factors regulating soil microbial EEA. This study contributes decisively to the understanding of environmental factors on the microbial EEA in soils, specifically on the decisive influence of water availability and temperature on EEA. Unlike previous belief, optimum EEA in high temperature exposed soil upper layers can occur at low water availability (i.e., dryness) and high temperatures. This study shows the potential for a significant response by soil microbial EEA under conditions of high temperature and dryness due to a progressive environmental warming which will influence organic carbon decomposition at local and global scenarios.