Leucine Aminopeptidase Activity Research Articles

Keep oxygen in check: An improved in-situ zymography approach for mapping anoxic hydrolytic enzyme activities in a paddy soil

Paddy soils regularly experience redox oscillations during the wetting and draining stages, yet the effects of short-term presence of oxygen (O2) on in-situ microbial hotspots and enzyme activities in anoxic ecosystems remain unclear. To fill this knowledge gap, we applied soil zymography to localize hotspots and activities of phosphomonoesterase (PME), β-glucosidase (BG), and leucine aminopeptidase (LAP) in three compartments of rice-planted rhizoboxes (top bulk, rooted, and bottom bulk paddy soil) under oxic (+O2) and anoxic (O2) conditions. Short-term (35 min) aeration decreased PME activity by 13–49 %, BG by 4–52 %, and LAP by 12–61 % as compared with O2 in three soil compartments. The percentage of hotspot area was higher by 3–110 % for PME, by 10–60 % for BG, and by 12–158 % for LAP under +O2 vs. O2 conditions depending on a rice growth stage. Irrespective of the aeration conditions, the rhizosphere extent of rice plants for three enzymes was generally greater under higher moisture conditions and at earlier growth stage. Higher O2 sensitivity for the tested enzymes at bottom bulk soil versus other compartments suggested that short-term aeration during conventional zymography may lead to underestimation of nutrient mobilization in subsoil compared to top bulk soil. The intolerance of anaerobic microorganisms against the toxicity of O2 in the cells and the shift of microbial metabolic pathways may explain such a short-term suppression by O2. Our findings, therefore, show that anoxic conditions and soil moisture should be kept during zymography and probably other in-situ soil imaging methods when studying anoxic systems.

Ectomycorrhizal and non-mycorrhizal rhizosphere fungi increase root-derived C input to soil and modify enzyme activities: A 14 C pulse labelling of Picea abies seedlings.

Consequences of interactions between ectomycorrhizal fungi (EcMF) and non-mycorrhizal rhizosphere fungi (NMRF) for plant carbon (C) allocation belowground and nutrient cycling in soil remain unknown. To address this topic, we performed a mesocosm study with Norway spruce seedlings [Picea abies (L.) H. Karst] inoculated with EcMF, NMRF, or a mixture of both (MIX). 14 CO2 pulse labelling of spruce was applied to trace and visualize the 14 C incorporation into roots, rhizohyphosphere and hyphosphere. Activities and localization of enzymes involved in the C, nitrogen (N) and phosphorus (P) cycling were visualized using zymography. Spruce seedlings inoculated with EcMF and NMRF allocated more C to soils (EcMF: 10.7%; NMRF: 3.5% of total recovered C) compared to uninoculated control seedlings. The 14 C activity in the hyphosphere was highest for EcMF and lowest for NMRF. In the presence of both, NMRF and EcMF (MIX), the 14 C activity was 64% lower compared with EcMF inoculation alone. This suggests a suppressed C allocation via EcMF likely due to the competition between EcMF and NMRF for N and P. Furthermore, we observed 57% and 49% higher chitinase and leucine-aminopeptidase activities in the rhizohyphosphere of EcMF compared to the uninoculated control, respectively. In contrast, β-glucosidase activity (14.3 nmol cm-2 h-1 ) was highest in NMRF likely because NMRF consumed rhizodeposits efficiently. This was further supported by that enzyme stoichiometry in soil with EcMF shifted to a higher investment of nutrient acquisition enzymes (e.g., chitinase, leucine-aminopeptidase, acid phosphatase) compared to NMRF inoculation, where investment in β-glucosidase increased. In conclusion, the alleviation of EcMF from C limitation promotes higher activities of enzymes involved in the N and P cycle to cover the nutrient demand of EcMF and host seedlings. In contrast, C limitation of NMRF probably led to a shift in investment towards higher activities of enzymes involved in the C cycle.

Effects of Phytoremediation on Microbial Biomass, Composition, and Function in a Sulphide-Rich Tailing From a Metal-Contaminated Region

Mining activities lead to serious land deterioration and large scale mine waste generation. Reclamation has been carried out on several technogenic materials to encourage the development of soils. To date no detailed studies have been conducted to assess if soil developed in reclaimed tailings can be suitable for microbial community sustainability and associated plant population. This study investigated if 1) soil metal contamination affects microbial biomass and composition in sulphide tailings and 2) phytoremediation of tailing increases microbial abundance, diversity, and function. Microbial biomass was assessed using Phospholipid fatty acid analysis (PLFA). Soil bacterial and fungal microbiota was determined by high throughput sequencing of 16S rRNA gene for bacteria and internal transcribed spacer region for fungi using the Illumina platform. Total copper, nickel, iron, and titium were higher in unreclaimed sites compared to vegetated areas but the total microbial biomass was significantly higher in reclaimed sites compared to reference areas. More importantly, the levels of microbial biomass were not impacted by metals since the bioavailable Cu, Ni, and Ti were low in all the sites. Site-specific bacterial and fungal genera were identified. Proteobacteria was the most dominant bacterial phylum while Ascomicota was the predominant fungal phylum. Interestinlgy, Acidiferrobacter, an acidophilic, thermotolerant and facultatively anaerobic was the most predominant genus in unreclaimed site that is characterized by extreme acidity (pH = 2.8). Analysis of microbial diversity revealed higher Chao 1, # of OTUs, Shannon index, and species richness in bacterial and fungal populations from reclaimed sites compared to controls. The levels of β-glucosidase (BG), cellobiohydrolase (CBH), β-N-acetylglucosaminidase (NAGase), aryl sulfatase (AS), acid phosphatase (AP), alkaline phosphatase (AlP), glycine aminopeptidase (GAP), and leucine aminopeptidase (LAP) activities were significantly higher in vegetated sites compared to reference areas. Strong positive correlation coefficients were observed between soil organic matter and total microbial biomass (r = 0.99). These two factors were positively correlated with enzymatic activities and bacterial population diversity. Overall, newly developed soils can sustain diverse microbial communities and associated vegetations.

Replacement of fishmeal with plant protein in the diets of juvenile lumpfish (Cyclopterus lumpus, L. 1758): Effects on digestive enzymes and microscopic structure of the digestive tract

This study investigated the effects of plant protein concentrates on the activity of digestive enzymes and microscopic structure of the intestine of juvenile lumpfish. The experiment was carried out using triplicate groups of lumpfish of 7 g average initial body weight, to 40 g average final weight, after 54 days. Four experimental diets were used; a fishmeal (FM) based control diet and three test diets with the plant protein concentrates; soy protein concentrate (SPC) and pea protein concentrate (PPC) (1:1 ratio) replacing FM in proportions of 25% (PP25), 50% (PP50) and 75% (PP75), respectively. Nested ANOVA showed several significant treatment differences in histomorphometry. Overall, fish fed test diets exhibited several changes such as shorter mucosal fold height (MFH) in the anterior intestine (AI), increased number of goblet cells (GCs) and width of lamina propria (WLP) in distal intestine (DI), compared to control. Principal component analysis (PCA) on histological indices showed that all three treatment groups (i.e. PP25, PP50 and PP75) had significantly altered overall intestinal architecture, compared to the control group. The observed enteritis was negatively related with the condition factor (CF). Regarding enzyme activities fewer changes were observed across diets. Fish fed the PP50 diet exhibited an increased activity of leucine aminopeptidase (LAP) in the pyloric caeca and a decrease on chymotrypsin (CHY) in the mid intestine (MI) compared to the control. Nevertheless, the overall changes captured by PCA on enzymes were associated only with condition factor (CF) and not with the diets. Overall, histological evaluation confirmed that lumpfish intestinal morphometry was significantly altered by plant protein ingredients at 25%, 50% and 75% levels, but replacing of FM up to 50% did not affected the growth and the enzyme activities up to 75% inclusion level.

Effects of Plastic Film Mulching on Soil Enzyme Activities and Stoichiometry in Dryland Agroecosystems.

Soil extracellular enzymes are pivotal for microbial nutrient cycling in the ecosystem. In order to study the effects of different nitrogen application rates under plastic film mulching on soil extracellular enzyme activities and stoichiometry, five nitrogen application levels (i.e., 0, 90, 150, 225 and 300 kg·hm−2) were set based on two treatments: plastic film mulching (PM) and no film mulching (LD). We measured the soil extracellular enzyme activities (EEAs) and stoichiometry (EES) of four enzymes (i.e., β-1,4-glucosidase (βG), leucine aminopeptidase (LAP), β-1,4-N-acetylaminoglucosidase (NAG) and alkaline phosphatase (AP)) involved in the C, N and P cycles of soil microorganisms in surface soil at five maize growth stages (seedling stage, jointing stage, trumpet stage, grout stage and harvest stage). The results showed that there were significant differences in soil EEA at different maize growth stages. The soil nutrient content and soil EEA were significantly improved under PM, and the stoichiometric ratio of extracellular enzymes (EC:N:P) was closer to 1:1:1, which indicated that PM was beneficial to the balance of soil nutrients and the activity of microorganisms. At each stage, with the increase in nitrogen application levels, the soil EEA showed a trend of increasing first and then decreasing (or remained unchanged), and both LD and PM treatments reached their highest activity at the 225 kg·hm−2 nitrogen application rate. When the nitrogen application level was less than 225 kg·hm−2, the soil enzyme activity was mainly limited by the N nutrient, and when the nitrogen application level reached 300 kg·hm−2, it was mainly limited by the P nutrient. RDA and correlation analysis showed that the soil C:P, C:N, N:P and pH had significant effects on soil βG, NAG + LAP and AP activities as well as EC:N, EC:P and EN:P.

Carbon, Nitrogen, Phosphorus, and Extracellular Soil Enzyme Responses to Different Land Use

Extracellular soil enzymes play a key role in soil organic matter decomposition and nutrient cycling. However, it is not fully understood how these enzymes respond to different land use. Long-term research studies were used to evaluate how diversified management practices affect extracellular enzymes driving C cycling [phenol oxidases (PO), peroxidases (PP), α-glucosidase (AG), β-glucosidase (BG), cellobiohydrolase (CB), β-1,-4-Nacetylglucosaminidase (NAG)], N cycling [leucine aminopeptidase (LAP)], and P cycling [phosphomonoesterase (PME)]. The soil pH, contents of total organic C, total N, mehlich-3 P, soil respiration and soil nitrogen supply capacity were also measured. Different land use included tillage frequency, tillage regimes, mineral N fertilization, crop rotations and liquid dairy manure. Compared to medium or high tillage frequency, low tillage frequency increased total organic C and total N and soil respiration as well as NAG and PME activities, whereas it decreased soil nitrogen supply, mehlich-3 P, and soil pH, as well as PO, PP, AG, BG, CB, and LAP activities. Non till was associated with lower PP and PO activities than moldboard plow. Nitrogen fertilization decreased soil pH and PO activity but increased PME activity. Barley (Hordeum vulgare) in rotation with forage increased total organic C, total N, soil nitrogen supply and soil respiration by 31, 21, 44, and 33%, respectively, in comparison with barley in monoculture. The application of liquid dairy manure increased soil pH, total N and soil nitrogen supply and soil enzyme activities (AG, BG, NAG) in comparison to the mineral N fertilizer. When principal component analysis was performed, soil pH, PO, PP, CB, LAP, and PME were grouped in the first component, which explained the highest variance. This is the core group controlling the C, N, and P cycling. The activities of C, N, and P acquiring enzymes, soil nitrogen supply and soil respiration were related to changes in soil total C and N, and extractable P contents across a broad range of management practices. Increased PO and PP activities reflect total C decline.

Reduction in root active zones under drought stress controls spatial distribution and catalytic efficiency of enzyme activities in rhizosphere of wheat

Adaptation of plants and microbes to drought stress affects various biochemical processes such as enzyme activities in the rhizosphere. Nonetheless, it is largely unknown how this adaptation changes the spatial distribution of enzyme activities and catalytic properties of enzymes in the rhizosphere. Also, it is very important to know about the spatial pattern of enzyme activities in seminal and lateral roots regions to clarify how the interaction of drought and root types affects rhizosphere extent and enzyme activities. To fill these knowledge gaps, we grew wheat plants under drought (30% WHC, three weeks) and optimum (70% WHC) conditions and then coupled soil zymography with enzyme kinetics for studying β-glucosidase (GLU), acid phosphatase (ACP), and leucine aminopeptidase (LAP). The results showed that drought considerably decreased the hotspot percentage of GLU and ACP activities by 63% and 67% in comparison with optimum moisture. In the regions of the seminal root, the rhizosphere extent of GLU and LAP, normalized rhizosphere extent, and total activity of GLU, ACP, and LAP were higher in optimum than in drought treatment. For the lateral root regions, no difference in the rhizosphere extent and total enzyme activities was found between drought and optimum moisture. Besides, we found higher normalized total enzyme activity in lateral roots regions than in seminal roots regions. Drought elevated the maximum enzymatic reaction velocity (Vmax) and Michaelis–Menten constant (Km) of GLU whereas dropped the Vmax and Km (non-significant) of ACP and LAP in comparison with optimum moisture. The catalytic efficiency of GLU and ACP were 37% and 27% higher in optimum than in drought treatment. Our results revealed that effects of drought on rhizosphere extent and total enzyme activities in lateral roots regions were weaker than in seminal roots regions. Drought decreased microbial hotspots area, rhizosphere expansion of enzyme activities in seminal roots regions, and catalytic efficiency of enzyme systems. In conclusion, drought decreases root biomass and root exudations in the rhizosphere, leading to narrower microbial hotspots area and rhizosphere extent and thus to less enzyme catalytic efficiency.

Inclusion of Mannan-Oligosaccharides in Diets for Tropical Gar Atractosteus tropicus Larvae: Effects on Growth, Digestive Enzymes, and Expression of Intestinal Barrier Genes

Mannan-oligosaccharides (MOS) are non-digestible carbohydrates, and their use in aquaculture as prebiotics is well documented. The objective of this work was to test whether MOS supplemented in the diet of A. tropicus larvae (2, 4, and 6 g kg−1) influence growth parameters, the activity of digestive enzymes, and the expression of genes related to the intestinal barrier. The highest total length was observed in larvae fed 6 g kg−1 MOS compared to control larvae. Trypsin activity increased with the addition of MOS to the diets, but leucine aminopeptidase activity only increased with 6 g kg−1 MOS. Lipase and α-amylase activities increased in larvae fed with 2 and 4 g kg−1 MOS. The expression of zo-2 was higher with the 6 g kg−1 MOS treatment. The cl-3 transcripts were lower with 2 g kg−1 MOS but higher with 6 g kg−1 MOS. All tested concentrations of MOS increased the expression of muc-2. In this study, incorporating mannan-oligosaccharides into the diet of A. tropicus larvae had a positive effect, and the concentration of 6 g kg−1 produced the best results. Therefore, including this prebiotic in the diets for the culture of A. tropicus larvae is suitable.

Dietary Use of the Microalga Chlorella fusca Improves Growth, Metabolism, and Digestive Functionality in Thick-Lipped Grey Mullet (Chelon labrosus, Risso 1827) Juveniles

In recent years, a clear emphasis has been placed on replacing fishmeal and fish oil in aquafeeds with other alternative ingredients, including algae, particularly in low trophic omnivorous fish species. This work aimed at evaluating the effects of moderate dietary supplementation with the green microalga Chlorella fusca on growth, metabolism, and digestive functionality in juvenile thick-lipped grey mullet (Chelon labrosus). Fish were fed a control diet (CT) or a diet containing 15% C. fusca (C-15) biomass during 90 days. C. labrosus fed with the C-15 diet showed higher growth performance (in terms of final weight and length, weight gain, and specific growth rate) than the control group. Somatic indices and muscle proximate composition were similar at the end of the feeding trial. Regarding fatty acids profile, C. fusca-fed fish showed a selective retention of docosahexaenoic acid (DHA) in the liver, and arachidonic acid (ARA), eicosapentaenoic acid (EPA), and DHA in the muscle. Dietary inclusion of this microalga significantly increased intestinal total alkaline protease, leucine aminopeptidase, and alkaline phosphatase activities in specimens fed with C-15 diet. Furthermore, intestine histological analysis revealed the absence of damage signs on gut morphology in fish fed the microalgae supplemented diet. Thick-lipped grey mullets fed the C-15 diet increased plasma glucose and decreased plasma lactate. Overall, the effects observed on liver (lipid metabolism, glycolysis and glycogenolysis) enzyme activities, together with adequate fatty acid profile, metabolic response, and gut morphology, and a significant increase in the intestinal mucosa’s digestive and absorptive capacity, could explain the positive effects on growth performance obtained in fish fed the microalgae-supplemented diet. In conclusion, the results obtained showed that C. fusca is suitable as dietary ingredient for feeding thick-lipped grey mullet juveniles.

Abalone under moderate heat stress have elevated metabolic rates and changes to digestive enzyme activities

Abalone around the world are subject to increasing frequency of marine heatwaves, yet we have a limited understanding of how acute high temperature events impact the physiology of these commercially and ecologically important species. This study examines the impact of a 5 °C temperature increase over ambient conditions for six weeks on the metabolic rates, digestive enzyme activities in the digestive gland, and digestive efficiency of Red Abalone (Haliotis rufescens) and Pāua (H. iris) on their natural diets. We test the hypothesis that abalone digestive function can keep pace with this increased metabolic demand in two separate experiments, one for each species. H. iris had higher food intake in the heat treatment. Both species had higher metabolic rates in the heat treatment with Q10 = 1.73 and Q10 = 2.46 for H. rufescens and H. iris, respectively. Apparent organic matter digestibility, protein digestibility, and carbohydrate digestibility did not differ between the heat treatment and the ambient (control) treatment in either experiment. H. rufescens exhibited higher maltase, alanine-aminopeptidase, and leucine-aminopeptidase activities in the heat treatment. Amylase, β-glucosidase, trypsin, and alkaline phosphatase activities in the digestive gland tissue did not differ between temperature treatments. H. iris exhibited lower amylase and β-glucosidase activities in the heat treatment, while maltase, trypsin, leucine-aminopeptidase, and alkaline phosphatase activities did not differ between treatments. We conclude that over six weeks of moderate heat stress both abalone species were able to maintain digestive function, but achieved this maintenance in species-specific ways.

Effects of the removal of invasive Moso bamboo on soil microbial biomass and enzyme activities in subtropical forests

Removal of invasive plant species is the first step to restoring the invaded ecosystems. The soil microbial biomass and extracellular enzyme activities were measured in Moso bamboo (Phyllostachys edulis) pure forest (completely invasion), invasive P. edulis removal forest (secondary succession 5 years after clear cutting), and the evergreen broadleaved forest (no invasion) in Tianmu Mountain. The results showed that compared with P. edulis pure forest, invasive P. edulis removal significantly increased the contents of soil organic carbon (SOC), nitrate nitrogen, available phosphorus and potassium, as well as microbial biomass carbon (MBC) and microbial biomass phosphorus (MBP), while significantly decreased microbial biomass nitrogen (MBN). The activities of α-glucosidase (AG), β-glucosidase (BG), leucine aminopeptidase (LAP) and phenol oxidase (POX) in the forest with removal of invasive P. edulis were significantly higher than those in P. edulis pure forest, while invasive P. edulis removal did not change the activities of cellodisaccharide hydrolase (CBH), β-N-acetyl-glucosaminopeptidase (NAG), acid phosphatase (ACP) and peroxidase (PER). Furthermore, the activities of AG, BG and LAP were positively correlated with SOC and MBC, while the increase in POX activity was positively correlated with soil nitrate content. In addition, MBC, MBN and MBP, and activities of AG, BG, NAG, LAP and ACP in P. edulis removal forest forest were significantly higher than those in evergreen broadleaved forests. Taken together, the removal of invasive P. edulis could increase soil nutrient contents, microbial biomass and extracellular enzyme activities, thus could be considered as an effective way to restore the invaded forests. Our results provide important theoretical basis for controlling P. edulis invasion in subtropical forests.

Aboveground litter input alters the effects of understory vegetation removal on soil microbial communities and enzyme activities along a 60-cm profile in a subtropical plantation forest

Changes in litter input and understory vegetation due to climate change and human activities affect belowground processes in plantation forests. However, it is unclear whether and how aboveground litter interacts with understory vegetation for their effects on soil microbial communities and functions, especially in deep soils. A field manipulation experiment was conducted, including four treatments of understory vegetation removal plus aboveground litter removal (UVR + ALR), understory vegetation preservation plus aboveground litter addition (UVP + ALA), UVR + ALA, and UVP + ALR in a subtropical Chinese fir (Cunninghamia lanceolata) plantation. After seven years of continuous treatment, the soil samples along a 60-cm profile were used to evaluate microbial community compositions, major microbial enzyme activities, and several physicochemical properties in the four treatments. UVR resulted in significant negative effects on microbial biomass and enzyme activities at different soil layers with or without litter input, but it did not change the soil microbial communities such as F/B, G+/G−. Compared with ALR, ALA amplified the negative understory vegetation removal effects (UVREs) on β-D-cellobioside (CB), β-N-acetylglucosaminidase (NAG), and leucine aminopeptidase (LAP) activities, but did not affect UVREs on total soil PLFAs. Lesser negative UVREs on β-1,4-glucosidase (BG), LAP, and acid phosphatase (AP) activities were observed in top soils compared to deep soils in the ALR treatment. UVREs on soil microbial biomass were not significantly different among soil depths. These results indicate that aboveground litter and soil depth altered UVREs on soil physicochemical properties, primarily through soil microbial biomass and enzyme activities. UVREs were generally more sensitive to major enzyme activities than microbial biomass and were interactively influenced by aboveground litter and soil depth due to synergistic effects within soil ecosystems. This study highlights the integrated feedbacks of microbial structures and functions along soil profiles in response to litter and vegetation interactions in subtropical forests.

Biochemical composition and function of subalpine shrubland and meadow soil microbiomes in the Qilian Mountains, Qinghai-Tibetan plateau, China.

Microorganisms participate in the soil biogeochemical cycle. Therefore, investigating variations in microbial biomass, composition, and functions can provide a reference for improving soil ecological quality due to the sensitivity of microorganisms to vegetation coverage changes. However, the differences in soil microorganisms between shrubland and meadow have not been investigated in ecologically vulnerable subalpine areas. This study aimed to investigate the biochemical composition and functions of the soil microbial community under two shrublands and a meadow at high altitudes (3,400–3,550 m). Three sites under two shrublands, Rhododendron thymifolium (RHO) and Potentilla fruticosa (POT), and one meadow dominated by Kobresia myosuroides (MEA), were selected on the southern slope of the Qilian Mountains on the northeastern edge of the Qinghai–Tibetan Plateau, China. Soil physicochemical properties, the microbial community composition expressed by the phospholipid fatty acid (PLFA) biomarker, and enzyme activities were analyzed as well as their relationships. The results showed that water holding capacity and the soil carbon, nitrogen, and potassium content in RHO and POT were higher than those in the MEA. Moreover, the soil active carbon, dissolved organic carbon, total nitrogen, and dissolved total nitrogen content in RHO were higher than those in POT. The abundance of total PLFAs, bacteria, and fungi beneath the shrublands was considerably higher than that in the MEA. The PLFA abundance in RHO was significantly higher than that in POT. The fungal-to-bacterial ratio of RHO and POT was significantly higher than that in the MEA. The activities of β-glucosidase, cellobiohydrolase, and leucine aminopeptidase were the highest in RHO among the three vegetation types, followed by POT and MEA. The redundancy analysis indicated that the biochemical composition of the soil microorganisms and enzyme activities were driven by total nitrogen, dissolved organic carbon, water holding capacity, and soil organic carbon. Therefore, shrublands, which have higher biomass, can improve soil moisture status, increase soil carbon and nitrogen content (especially active carbon and active nitrogen), and further increase the abundance of total PLFAs, bacteria, and fungi. The increase of microbial biomass indirectly enhances the activity of relevant soil enzymes. The variations in PLFA abundance and enzyme activities can be attributed to shrub species, especially evergreen shrubs, which create more favorable conditions for soil microorganisms. This study provides a theoretical basis for investigating the soil biogeochemical cycle and a scientific basis for soil management and vegetation restoration in the subalpine regions.

Effects of Irrigation Strategy and Plastic Film Mulching on Soil N2O Emissions and Fruit Yields of Greenhouse Tomato

Agriculture is a major source of global greenhouse gas emissions. Approximately 1/3 of vegetables in China are produced in greenhouses. However, the effects of different irrigation strategies and plastic film (PF) mulching combinations on N2O emissions and tomato fruit yields in greenhouses are unclear. The aims of this study were to explore the effects of micro-sprinkler irrigation under plastic film (MSPF), drip irrigation under plastic film (DIPF) and micro-sprinkler irrigation (MSI) on the soil nutrients, enzyme activity, nirS-type denitrifying bacterial community, N2O emissions and fruit yields of tomato. The results showed that MSPF could improve the uniformity of soil water distribution and surface (0–40 cm) soil water content. Film mulching could increase soil temperature at depths of 5–25 cm. Both MSPF and DIPF increased microbial nitrogen, promoted the activity of rhizosphere soil urease and leucine aminopeptidase, changed the community of denitrifying bacteria, accelerated the turnover of soil nutrients and improved yield and water use efficiency. PF mulching had a greater impact on the nirS-type denitrifying bacterial community when compared to irrigation strategy. We conclude that MSPF can be used to configure commercially available installation and operation. The comprehensive benefit of MSPF treatment is that it is more profitable than that of DIPF.

Soil Enzyme Stoichiometric Characteristics of Pinus massoniana Plantations at Different Stand Ages in Mid-subtropical Areas

Soil enzyme activity is an important index to characterize the nutrient requirements and nutrient limitations of soil microorganisms. In this study, Pinus massoniana plantations of different stand ages (9, 17, 26, 34, and 43 a) in mid-subtropical China were taken as the research object; the activities of β-glucosidase (BG), N-acetyl-β-glucosaminidase (NAG), leucine amino-peptidase (LAP), acid phosphatase (AP), polyphenol oxidase (POX), and peroxidase (POD) were determined; and soil enzyme stoichiometric ratios were also calculated to investigate the soil microbial nutrient limitations of P. massoniana plantation development. The results showed that the activities of BG, NAG, AP, POX, and POD were enhanced with the increase in stand age, and the activity of LAP was the lowest at 17 a, which showed a significant difference and fluctuated among the neighboring stand ages. The soil enzyme C:N:P stoichiometric ratio was 1:1:0.56, which deviated from the global ecosystem enzyme C:N:P stoichiometric ratio (1:1:1). The enzyme C:N increased, whereas the enzyme N:P decreased, with increasing stand age, and both ratios tended to be stable after 17 a. There was no significant difference in enzyme N:P among different stand ages. The vector length of enzyme stoichiometry was not significantly different among the five stand ages. The vector angles increased with the increase in stand ages and tended to be stable after 17 a of stand age, but the angles were less than 45°. Redundancy analysis (RDA) revealed that soil carbon quality index and pH were the main factors influencing soil enzyme activity and the associated stoichiometric ratio. Our findings indicated that P. massoniana plantation soil microorganisms at different growth stages were all subjected to N limitation, and the N limitation was alleviated with the increase in stand age; however, the P requirement was gradually enhanced. Therefore, the management of P. massoniana plantations should take care to increase nitrogen fertilizer at the early growth stage of P. massoniana, and more phosphorus fertilizers need to be applied with nitrogen at the late growth stage in order to maintain the productivity and sustainable development of P. massoniana plantations.

Perennial cereal grain cultivation: Implication on soil organic matter and related soil microbial parameters

Novel perennial grains in agriculture have the potential of providing soil ecosystem services and boosting plant-soil microbial relationships via increasing soil organic matter (SOM). Hence, we evaluated the implication of perennial intermediate wheatgrass agroecosystems (+/− alfalfa intercrop) in contrast to organic and conventional rotation systems on SOM and related soil microbial parameters. Soil samples were obtained from perennial intermediate wheatgrass (IWG), sole (IWG-SC) or intercropped with alfalfa (IWG-IC), as well as organic (ORG-A) and conventional (CON-A) rotation systems of annual wheat. Soil samples were taken in two depths (0–30 cm and 30–60 cm) and analyzed for total and fractionated soil organic C (SOC), microbial biomass C and N (MBC and MBN), basal microbial respiration (Rb), potential enzyme activities related to C and N cycling (Cellobiohydrolase (CBH), Leucine-aminopeptidase (LAP)), and abundance of functional genes related to N cycling (amoA, nifH) and 16S rRNA genes. Despite the lack of fertilization in the IWG systems, SOC concentration and the amount of C in the particulate organic matter were higher in the 30–60 cm depth of IWG systems than ORG-A and CON-A. Higher MBN and LAP activities were observed in the 0–30 cm depth of ORG-A than both IWG and CON-A. Mass specific CBH activities and metabolic quotient qCO2 were lower in the 30–60 cm depth of IWG systems, implying reduced C losses and high C use efficiency in these systems. Similarly, we observed higher amounts of 16S rRNA in the 30–60 cm depth of IWGs than ORG-A and CON-A. Hence, IWG systems increased SOC and microbial biomass and activities in the 30–60 cm depth than ORG-A and CON-A. However, the more diverse IWG (+ alfalfa intercrop) did not increase microbial biomass and activities than IWG monoculture. Our study provides a reflection of how perennial grain agroecosystems, in conjunction with organic rotation systems, induce a positive influence on SOC and microbial activities than CON-A. It further suggested that perennial grain cultivation goes beyond the reach of organic farming dominated by annual crops to induce SOC and support relevant soil microbial functions in subsoils.

Permafrost Active Layer Microbes From Ny Ålesund, Svalbard (79°N) Show Autotrophic and Heterotrophic Metabolisms With Diverse Carbon-Degrading Enzymes.

The active layer of permafrost in Ny Ålesund, Svalbard (79°N) around the Bayelva River in the Leirhaugen glacier moraine is measured as a small net carbon sink at the brink of becoming a carbon source. In many permafrost-dominating ecosystems, microbes in the active layers have been shown to drive organic matter degradation and greenhouse gas production, creating positive feedback on climate change. However, the microbial metabolisms linking the environmental geochemical processes and the populations that perform them have not been fully characterized. In this paper, we present geochemical, enzymatic, and isotopic data paired with 10 Pseudomonas sp. cultures and metagenomic libraries of two active layer soil cores (BPF1 and BPF2) from Ny Ålesund, Svalbard, (79°N). Relative to BPF1, BPF2 had statistically higher C/N ratios (15 ± 1 for BPF1 vs. 29 ± 10 for BPF2; n = 30, p < 10–5), statistically lower organic carbon (2% ± 0.6% for BPF1 vs. 1.6% ± 0.4% for BPF2, p < 0.02), statistically lower nitrogen (0.1% ± 0.03% for BPF1 vs. 0.07% ± 0.02% for BPF2, p < 10–6). The d13C values for inorganic carbon did not correlate with those of organic carbon in BPF2, suggesting lower heterotrophic respiration. An increase in the δ13C of inorganic carbon with depth either reflects an autotrophic signal or mixing between a heterotrophic source at the surface and a lithotrophic source at depth. Potential enzyme activity of xylosidase and N-acetyl-β-D-glucosaminidase increases twofold at 15°C, relative to 25°C, indicating cold adaptation in the cultures and bulk soil. Potential enzyme activity of leucine aminopeptidase across soils and cultures was two orders of magnitude higher than other tested enzymes, implying that organisms use leucine as a nitrogen and carbon source in this nutrient-limited environment. Besides demonstrating large variability in carbon compositions of permafrost active layer soils only ∼84 m apart, results suggest that the Svalbard active layer microbes are often limited by organic carbon or nitrogen availability and have adaptations to the current environment, and metabolic flexibility to adapt to the warming climate.

Evaluation of a novel methanotroph bacteria meal grown on natural gas as fish meal substitute in rainbow trout, Oncorhynchus mykiss

In the present study, we evaluated the dietary fishmeal (FM) replacement potential of String Pro®, a novel methanotrophic bacterial protein meal (BM), in rainbow trout, based on growth, feed utilization, digestibility, nutrient balance and histological observations. Five isonitrogenous (49.4–51.1%) and isoenergetic (21–22.1 MJ/kg) practical diets containing 0, 12.5, 25, 37.5 and 48% BM, corresponding to 0, 25, 50, 75 and 100% FM replacement, respectively, were fed to triplicate groups of rainbow trout juveniles for 9 weeks. At the end of the feeding trial, growth rate and feed utilization were high in all the dietary groups (i.e. thermal growth coefficient of 2.55–2.96 and feed efficiency of 1.2–1.3). Feed efficiency and protein efficiency ratio did not differ between dietary groups; however, the decreasing growth trend due to reduction in feed intake was reflected in body condition and carcass estimates of protein and lipid retention, gain and whole-body content, which decreased with increasing BM inclusion levels. There was no significant difference in apparent digestibility of protein, although, dry matter digestibility was reduced at the 48% dietary BM inclusion. Intestinal leucine aminopeptidase activity decreased; whereas, intestinal alkaline phosphatase activity and phosphorus retention increased with BM inclusion, with favourable reduction in phosphorus loss. Histological observations of the gastrointestinal tract, liver and kidney did not show any differences between dietary treatments. Based on these findings, we conclude that 12.5% of this novel BM ingredient can be safely included in the diets of rainbow trout, as a sustainable substitute for fish meal.

Extracellular Enzyme Stoichiometry and Microbial Metabolism Limitation During Vegetation Restoration Process in the Middle of the Qinling Mountains, China

Clarifying the characteristics of soil microbial nutrient limitation and its driving mechanisms during vegetation restoration after farmland abandonment has important implications for revealing soil nutrient cycling and maintaining ecosystem stability. To determine the limitation of soil microbial nutrients and its relationship with soil properties along a chronosequence of abandoned farmland in the middle of the Qinling Mountains, the soil physicochemical properties and five enzyme activities (β-1,4-glucosidase (BG), cellobiohydrolase (CBH), β-1,4-N-acetylglucosaminidase (NAG), leucine aminopeptidase (LAP), and acid phosphatase (AP)) were measured, and models of extracellular enzymatic activity were applied. The results showed that the activities of BG, CBH, NAG, LAP, and AP were significantly increased following farmland abandonment. With the increasing years of abandonment, the ratios of (BG+CBH)/(NAG+LAP) and (BG+CBH)/AP significantly decreased, whereas the ratio of (NAG+LAP)/AP increased. Correlation analysis showed that most soil physicochemical properties were significantly correlated with extracellular enzyme activities and extracellular enzymatic stoichiometry. The vector length of extracellular enzymatic stoichiometry decreased with the increase in abandonment years, indicating that the limitation of soil microorganisms on carbon (C) was reduced. Moreover, the vector angles (>45°) showed a decreasing trend, indicating that microbial metabolisms were limited by phosphorus (P) and gradually decreased. Regression analysis showed that the C and P limitations were significantly related to total nutrients, available nutrients, nutrient ratio, and soil physical properties. Partial least squares path modeling (PLS-PM) revealed that the C and P limitations were directly regulated by nutrient ratio. PLS-PM further showed that soil total nutrients indirectly affected soil microbial C and P limitations by affecting nutrient ratio, and nutrient ratio affected the soil metabolism limitation via available nutrients and pH. Our study suggests that the characteristics of microbial metabolism during the vegetation restoration process reflect the mechanism of microorganism-mediated soil nutrient cycling, which provides a theoretical basis for revealing the community dynamics and stability during the vegetation restoration process and maintaining the regional ecological environment security in the Qinling Mountains.

Co-exposures of acid rain and ZnO nanoparticles accelerate decomposition of aquatic leaf litter

The pattern of acid rain is dependent on the ratio of SO42− and NO3−, which change may affect the dissolution activity of dull heavy metals in the aquatic environment and further complicate the ongoing challenge of ecosystem stability and increase risks. In this study, we assessed the combined effects of acid rain (SO42-: NO3- was 2:1, 1:1, and 1:2) and ZnO nanoparticles (30 ng L-1) on plant litter decomposition through a microcosm experiment. The highest dissolution of ZnO nanoparticles was achieved when the SO42-: NO3- ratio was 1:2, and there were no significant differences among other treatments. The fungal biomass showed significant decreases under acute exposures but tended to be adaptive during chronic exposures. The co-exposure significantly stimulated the activities of leucine-aminopeptidase, glycine-aminopeptidase, polyphenol oxidase, and cellobiohydrolase. Besides, the fungal diversity and the relative abundance of some functional genera (e.g. Anguillospora) were enhanced when the SO42-: NO3- ratio was 1:2 and 2:1. In conclusion, the decomposition rate of plant litter was increased by 123–204% by co-exposures. Collectively, the findings underline the importance of considering environmental context to assess nanoparticle toxicity.