Leucine Aminopeptidase Activity Research Articles

How do tree species with different successional stages affect soil organic nitrogen transformations?

Organic nitrogen (N) is the most important N component of soil organic matter. However, knowledge on how tree species with different successional stages affect organic N transformations in soils remains limited. To address this issue, we sampled mineral soils (0–10 cm) under monocultures composed of tree species from different successional stages, including early (black alder and silver birch), early to mid (sycamore and European ash), and late (sweet chestnut, pedunculate oak and European beech), and measured the potential protease activity, the microbial uptake and respiration of 14C-labeled organic N (l-alanine and l-trialanine), and the mineralization of l-alanine N. The activities of alanine aminopeptidase and leucine aminopeptidase (153.8–341.9 and 91.6–147.9 nmol/g/h, respectively), the half-life of the uptake of alanine and trialanine (26.7–39.6 and 60.8–78.6 min, respectively), the half-life of the mineralization of alanine and trialanine (1.98–2.45 and 2.98–4.13 h, respectively) by soil microbes were altered by tree species from different successional stages, systematically changing the transformation chain of soil organic N. The turnover rates of soil organic N under trees of early to late successional stage appeared to decrease and the half-life appeared to increase significantly. The C:N ratio of soil microbial biomass was positively related to the half-life of 14C-labeled alanine and trialanine mineralization, and was negatively related to the carbon (C) use efficiency of alanine, suggesting that microbial demand for C could partially drive the assimilation of soil organic N. These results suggest that the successional stage of tree species play an important role in regulating the turnover rates of soil organic N. An improved understanding of how tree species from different successional stages influence microbial function and soil organic N cycling is beneficial to future afforestation and forest management, alleviating the impacts of global change on the ecosystem.

Potential implications of biochar and compost on the stoichiometry-based assessments of soil enzyme activity in heavy metal-polluted soils

Biochar and compost application in soil has been proved as an environmental management and soil remediation strategy for upgrading soil quality and growth-promoting soil microorganisms. A detailed examination of the fluctuations and stoichiometric interactions between β-glucosidase (BG), N-acetylglucosaminidase (NAG), leucine aminopeptidase (LAP), and acid phosphatase (AP) after applying biochar and compost to the heavy metal soils was conducted in the paper. However, biochar stimulated the activity of AP and inhibited BG, NAG, and LAP. Compost and biochar-compost might strengthen BG, NAG, LAP, and AP activities. Redundancy analysis discovered that the crucial parameters that affected soil enzyme activity were TN, NO3−-N, and TP. However, the stoichiometric ratio of C/N, C/P, and N/P with the application of the biochar, compost, and biochar-compost could be changed. While alleviating the P-limitation in soils under Cu stress, biochar may also alleviate the C-limitation in soils under As, Cd, and Zn stress. Compost and biochar-compost application alleviated C-limitation in heavy-metal (Cu, As, Cd, and Zn) polluted soil. This work will help us further understand the stoichiometry and energy restrictions of biochar and compost on ecological functional enzymes.

Mixed-litter effects of fresh leaf semi-decomposed litter and fine root on soil enzyme activity and microbial community in an evergreen broadleaf karst forest in southwest China.

Litter decomposition is the main process that affects nutrient cycling and carbon budgets in mixed forests. However, knowledge of the response of the soil microbial processes to the mixed-litter decomposition of fresh leaf, semi-decomposed leaf and fine root is limited. Thus, a laboratory microcosm experiment was performed to explore the mixed-litter effects of fresh leaf, semi-decomposed leaf and fine root on the soil enzyme activity and microbial community in an evergreen broadleaf karst forest in Southwest China. Fresh leaf litter, semi-decomposed litter and fine root in the Parakmeria nitida and Dayaoshania cotinifolia forests, which are unique protective species and dominant species in the evergreen broadleaf forest, were decomposed alone and in all possible combinations, respectively. Our results showed that the mass loss of fresh leaf litter in three mixed-litter treatment was significantly higher than that in two mixed-litter treatment in the P. nitida and D. cotinifolia forests. Mass loss of fine root in the single litter treatment was significantly lower in the P. nitida forest and higher in the D. cotinifolia forest compared to that in the other litter treatments. There were insignificant differences in the activities of β-glucosidase (BG) and leucine aminopeptidase (LAP) between control and mixed-litter treatment in the P. nitida forest and between control and single litter treatment in the D. cotinifolia forest. The N-acetyl-β-D-glucosaminidase (NAG) activity was significantly increased by the single litter decomposition of fresh leaf and fine root and three mixed-litter decomposition in the P. nitida and D. cotinifolia forests. The activity of acid phospomonoesterase (AP) in the decomposition of fresh leaf litter was lower in the P. nitida forest and higher in the D. cotinifolia forest compared to that in control. The most dominant soil bacteria were Proteobacteria in the P. nitida forest and were Actinobacteria and Proteobacteria in the D. cotinifolia forest. Shannon, Chao1, ACE and PD indexes in the mixed-litter decomposition of fresh leaf and semi-decomposition litter were higher than that in control in P. nitida forest. There were insignificant differences in observed species and indexes of Chao1, ACE and PD between litter treatments in the D. cotinifolia forest. Richness of mixed-litter significantly affected mass loss, soil enzyme activity and microbial diversity in the P. nitida forest. Litter N concentration and the presence of fresh leaf litter were significantly correlated with the mass loss and soil enzyme activity in the P. nitida and D. cotinifolia forests. These results indicated that the presence of fresh leaf litter showed a non-negligible influence on mixed-litter decomposition and soil enzyme activity, which might be partly explained by litter initial quality in the P. nitida and D. cotinifolia forests.

Living grass mulching improves soil enzyme activities through enhanced available nutrients in citrus orchards in subtropical China.

Living grass mulching (LGM) is an important orchard floor management that has been applied worldwide. Although LGM can effectively enhance soil nutrient availability and fertility, its effects on microbial-mediated soil nutrient cycling and main drivers are unclear. Meanwhile, the variation of enzyme activities and soil nutrient availability with LGM duration have been rarely studied. This study aims to explore the effects of mulching age and soil layer on enzyme activities and soil nutrients in citrus orchards. In this study, three LGM (Vicia villosa) treatments were applied, i.e., mulching for eight years, mulching for four years, and no mulching (clean tillage). Their effects on the enzyme activities and soil nutrients were analyzed in different soil layers of citrus orchards in subtropical China, i.e., 0-10, 10-20, and 20-40cm. Compared to clean tillage, mulching for four years had fewer effects on enzyme activities and soil nutrients. In contrast, mulching for eight years significantly increased available nitrogen (N), phosphorus (P) nutrients, β-glucosidase, and cellobiohydrolase activities in the soil layer of 0-20cm. In the soil layer of 0-40cm, microbial biomass carbon (C), N, P, N-acetylglucosaminidase, leucine aminopeptidase, and acid phosphatase activities also increased (P < 0.05). Mulching for eight years significantly promoted C, N, and P-cycling enzyme activities and total enzyme activities by 2.45-6.07, 9.29-54.42, 4.42-7.11, and 5.32-14.91 times, respectively. Redundancy analysis shows that mulching treatments for eight and four years had soil layer-dependent positive effects on soil enzyme activities. Microbial C and P showed the most significant positive correlation with enzyme activities, followed by moisture content, organic C, and available N (P < 0.05). Available nutrients contributed almost 70% to affect enzyme activities significantly and were the main drivers of the enzyme activity variation. In summary, LGM could improve soil enzyme activities by increasing available nutrients. The promotion effect was more significant under mulching for eight years. Therefore, extending mulching age and improving nutrient availability are effective development strategies for sustainable soil management in orchard systems. Our study can provide valuable guidelines for the design and implementation of more sustainable management practices in citrus orchards.

Distinct roles of bacteria and fungi in mediating soil extracellular enzymes under long-term nitrogen deposition in temperate plantations

Soil microbes decompose soil organic matter by producing various extracellular enzymes, and also regulate soil carbon (C) and nutrient cycles. However, how shifts in soil microbial community under long-term nitrogen (N) deposition govern soil extracellular enzyme activities (EEAs) is not known. Here, we conducted a decade-long N fertilization experiment consisting of control, low-N (2 g N m−2 year−1), medium-N (5 g N m−2 year−1), and high-N (10 g N m−2 year−1) treatments in a N-limited temperate plantation in northern China. The diversity and community composition of fungi and bacteria were determined with bacterial 16S rRNA genes and fungal internal transcribed spacer genes sequencing. Soil EEAs involved in C, N, and phosphorus (P) cycles were also measured. The results showed that high-N addition increased fungal diversity and altered microbial community composition. In particular, rare fungal diversity and rare bacterial community composition were more sensitive to N addition. N addition improved β-1,4-glucosidase (BG) and acid phosphatase (AP) activities by altering bacterial community composition, and inhibited leucine aminopeptidase (LAP) and polyphenolic oxidase (PPO) activities by affecting fungal functional groups. Specifically, shifts in bacterial community composition caused by N addition were reflected in the significant increase in the dominant class Thermoleophilia, which could be an indicator of secreted hydrolase involved in C and P cycles. Symbiotrophic fungi were more closely associated with LAP for acquiring N and PPO. Our data provide a novel view of the link between major extracellular enzymes and specific microbial taxa under the background of N deposition.

Contrasting distribution of enzyme activities in the rhizosphere of European beech and Norway spruce.

Recent policies and silvicultural management call for forest regeneration that involve the selection of tree species able to cope with low soil nutrient availability in forest ecosystems. Understanding the impact of different tree species on the rhizosphere processes (e.g., enzyme activities) involved in nutrient mobilisation is critical in selecting suitable species to adapt forests to environmental change. Here, we visualised and investigated the rhizosphere distribution of enzyme activities (cellobiohydrolase, leucine-aminopeptidase, and acid phosphomonoesterase) using zymography. We related the distribution of enzyme activities to the seedling root morphological traits of European beech (Fagus sylvatica) and Norway spruce (Picea abies), the two most cultivated temperate tree species that employ contrasting strategies in soil nutrient acquisition. We found that spruce showed a higher morphological heterogeneity along the roots than beech, resulting in a more robust relationship between rhizoplane-associated enzyme activities and the longitudinal distance from the root apex. The rhizoplane enzyme activities decreased in spruce and increased in beech with the distance from the root apex over a power-law equation. Spruce revealed broader rhizosphere extents of all three enzymes, but only acid phosphomonoesterase activity was higher compared with beech. This latter result was determined by a larger root system found in beech compared with spruce that enhanced cellobiohydrolase and leucine-aminopeptidase activities. The root hair zone and hair lengths were significant variables determining the distribution of enzyme activities in the rhizosphere. Our findings indicate that spruce has a more substantial influence on rhizosphere enzyme production and diffusion than beech, enabling spruce to better mobilise nutrients from organic sources in heterogeneous forest soils.

Linkages between bacterial community and extracellular enzyme activities crossing a coastal front

Exploring the responses of microbial communities and their functions to marine frontal systems are the foci of marine ecologists. However, most of the existing studies have focused on only microbial communities or their functions. The relationships between microbial communities and their functions across coastal fronts therefore remain unclear. Here we studied a coastal front in the Taiwan Strait during late spring and examined the bacterial community structure, extracellular enzymatic activity (EEA), and their linkages. The results showed that the coastal front strongly delineated zones of bacterial community composition and diversity and functioned as a transitional zone rather than a hotspot between the coastal water and offshore water. Co-occurrence network analysis indicated that the interaction of bacterial communities was much weaker in the frontal zone than in the water masses on both sides. The suggestion was that the highly fluctuating environment in the frontal zone reduced the complexity and stability of the bacterial co-occurrence network. The activities of β-glucosidase (BGA) and leucine aminopeptidase (LAPA) were as high in the frontal zone as in the coastal water and much higher than those in offshore waters. We therefore suggest that, despite obvious shifts in the bacterial community structure in the frontal zone, the bacterial community was able to maintain its ability to hydrolyze organic substrates via functional redundancy. The composition of key microbial assemblies differed among the three water masses, and the relative abundance of module 2 in the frontal zone was positively correlated with BGA and LAPA. The indication was that changing the bacterial assemblage was the mechanism that made it possible for the bacterial community to maintain EEA in the frontal zone. Our results help to improve understanding on how bacterial community-function linkages vary in marine frontal systems.

Biochar and nitrogen fertilizer promote rice yield by altering soil enzyme activity and microbial community structure

AbstractBiochar can significantly change soil properties and improve soil quality. However, the effects of long‐term combined application of biochar (B) and nitrogen (N) fertilizer on relationships between soil enzyme activity, microbial community structure, and crop yield are still obscure. We characterized these relationships in a long‐term (8 years) field experiment with rice, two biochar rates of 0 and 13.5 t ha−1 year−1 (B0 and B) and two N fertilizer rates of 0 and 300 kg N ha−1 year−1 (N0 and N). The repeated, long‐term combined applications of biochar and N fertilizer significantly increased microbial biomass carbon (MBC) and microbial biomass nitrogen (MBN), but biochar decreased the abundance of total bacteria, fungi, actinomycetes, and gram‐positive and gram‐negative bacteria as well as the amount of total phospholipid fatty acids. The activity of leucine aminopeptidase (LAP) decreased significantly in the biochar‐amended and N fertilized treatment, but the LAP activity either remained unchanged or increased with biochar amendment at N0. The relative abundance of bacterial phylum Chloroflexi was increased in the combined biochar and N fertilizer treatment. The changes in soil organic matter and the activity of α‐1,4‐xylosidase were the major properties influencing soil bacterial community composition, whereas the structure of fungal community was governed by MBC, MBN, and LAP activities. In addition, long‐term biochar and N fertilizer applied together significantly increased rice yield (more than biochar and nitrogen fertilizer applied alone). Yield was significantly positively correlated with LAP activity, but significantly negatively correlated with the relative abundance of Chloroflexi. In conclusion, long‐term biochar and nitrogen fertilizer applications increased rice yield, which was associated with altered soil microbial community and enhanced activity of some enzymes.

Influence of earthworms on the microbial properties and extracellular enzyme activities during vermicomposting of raw and distilled grape marc

The treatment of winery wastes by using appropriate management technologies is of utmost need in order to reduce to a minimum their disposal and avoid negative environmental impacts. This is of particular interest for grape marc, the main solid by-product of the winery industry. However, comparative studies on a pilot-scale dealing with the impact of earthworms on marc derived from both red and white grape varieties during vermicomposting are still scarce. The present study sought to evaluate the changes in the biochemical and microbiological properties of red and white raw marc in the presence and the absence of the earthworm species Eisenia andrei. The distilled marc obtained through distillation of the red grape marc was also considered under this scenario. Samples were taken after 14, 28, 42, and 63 days of vermicomposting. On day 14 earthworms led to a pronounced increase in most of the enzymatic activities, but only in those vermireactors fed with raw marc from the red grape variety. Alfa- and beta-glucosidase as well as chitinase and leucine-aminopeptidase activities were between 3 to 5-times higher relative to the control, while alkaline phosphomonoesterase was even up to 14-fold higher with earthworm presence. From day 28 onwards the magnitude of earthworms' effect on the studied enzymes was also dependent on the type of grape marc. Reduced values of basal respiration, ranging between 200 and 350mg CO2 kg OM h-1 and indicative of stabilized materials were found in the resulting vermicomposts. Moreover, the content of macro- and micronutrients in the end products matched with those considered to have the quality criteria of a good vermicompost. Altogether, these findings reinforce the effectiveness of vermicomposting for the biological stabilization of grape marc with the dual purpose of fertilizer production and environmental protection.

Intercropping-driven nitrogen trade-off enhances maize productivity in a long-term experiment

Intercropping improves land productivity by exploiting species complementarities to achieve sustainable agriculture. Intercropping systems, therefore, require a nitrogen (N) management approach that matches temporal and spatial N supply with crop requirements. Present field-based study (started in 2013) explored the effects of intercropping of maize ( Zea mays L. ) with legumes [peanut ( Arachis hypogaea ) and soybean ( Glycine max )] and non-legumes [gingelly ( Sesamum indicum L. ) and sweet potato ( Dioscorea esculenta )] on maize productivity and N trade-off. Compared with maize monoculture, intercropping with gingelly, peanut, soybean, and sweet potato all significantly improved maize average yield (2013 and 2021), average N use efficiency (NUE), average N partial factor productivity (PFPN), and plant N uptake by 8–29%, 28–49%, 20–80%, and 16–35%, respectively. The N losses via ammonia volatilization and runoff in intercropping systems decreased by 12–19% and 11–43% respectively, compared with monoculture. Soil total N, inorganic N (NH 4 + -N and NO 3 - -N), and microbial biomass N (MBN) contents as well as the urease, β-1,4-N-acetyl-glucosaminidase (NAG), and leucine aminopeptidase (LAP) activities varied among different intercropping systems and maize growth periods. Maize productivity (yield, PFPN, and NUE) was positively correlated with plant N uptake (R 2 = 0.66–0.92, P < 0.001) and soil MBN content (R 2 = 0.41–0.47, P < 0.01), while it was found negatively associated with N losses (R 2 = 0.35–0.56, P < 0.05), soil N availability (R 2 = 0.27–0.67, P < 0.05), and NAG activity (R 2 = 0.36–0.55, P < 0.05). These findings collectively indicated that regulating the balance between soil N supply and plant N uptake in an intercropping system with low N fertilization could achieve high productivity. This study highlighted the importance of understanding the complex interaction between crop development and intercropping practices in improving maize productivity and N trade-off. • Intercropping increased average (2013 and 2021) maize yield by 8–29%. • Intercropping improved average NUE by 28–49% and maize N uptake by 16–35%. • N loss via ammonia volatilization and runoff decreased by 11–43% duo to intercropping. • Maize productivity was primarily related to plant N uptake, soil N dynamics and N loss. • Intercropping-driven plant-soil N trade-offs contributed to increased maize productivity.

Contrasting effects of maize litter and litter-derived biochar on the temperature sensitivity of paddy soil organic matter decomposition.

Organic matter input regulates the rate and temperature sensitivity (expressed as Q10) of soil organic matter (SOM) decomposition by changing microbial composition and activities. It remains unclear how the incorporation of litter-made biochar instead of litter affects the Q10 of SOM decomposition. Using a unique combination of two-and three-source partitioning methods (isotopic discrimination between C3/C4 pathways and 14C labeling), we investigated: (1) how maize litter versus litter-made biochar (of C4 origin) addition influenced the Q10 of SOM (C3 origin) under 10°C warming, and (2) how the litter or biochar amendments affected the Q10 of 14C-labeled fresh organic matter (FOM) after long-term incubation. Compared with biochar addition, litter increased the rates and Q10 of mass-specific respiration, SOM and FOM decomposition, as well as the contents of SOM-derived dissolved organic C (DOC) and total phospholipid fatty acids (PLFA). Litter-amended soils have much higher activities (Vmax) of β-glucosidase, N-acetyl-β-glucosaminidase, and leucine aminopeptidase, suggesting larger enzyme pools than in soils with biochar. The Q10 of enzyme Vmax (1.6–2.0) and Km (1.2–1.4) were similar between litter-and biochar-amended soils, and remained stable with warming. However, warming reduced microbial biomass (PLFA) and enzyme activity (Vmax), suggesting decreased enzyme production associated with smaller microbial biomass or faster enzyme turnover at higher temperatures. Reductions in PLFA content and enzyme Vmax due to warming were larger in litter-amended soils (by 31%) than in the control and biochar-amended soils (by 4–11%), implying the active litter-feeding microorganisms have a smaller degree of heat tolerance than the inactive microorganisms under biochar amendments. The reduction in enzyme activity (Vmax) by warming was lower in soils with biochar than in the control soil. Our modeling suggested that the higher Q10 in litter-amended soils was mainly caused by faster C loss under warming, linked to reductions in microbial biomass and growth efficiency, rather than the slightly increased SOM-originated substrate availability (DOC). Overall, using straw-made biochar instead of straw per se as a soil amendment lowers the Q10 of SOM and FOM by making microbial communities and enzyme pools more temperature-tolerant, and consequently reduces SOM losses under warming.

Effects of fecal bacteria on growth, digestive capacity, antioxidant capacity, intestinal health of large yellow croaker (Larimichthys crocea) larvae

Marine fish at early life is vulnerable to the threat of external pathogenic bacteria with unestablished intestinal flora. Early life intervention could contribute to healthy development of larvae gut tract. Consequently, the present study examined how large yellow croaker larvae (Larimichthys crocea) (mean body weight 3.64 ± 0.20 mg) responde to fecal microbiota came from 12-month-old large yellow croaker by oral administration. The intestinal contents of the donor were prepared into bacterial solution and sprayed on the Micro-diet. After 30 days feeding, growth performance, intestinal development, digestive ability and gut microbiota of larvae were detected. Results showed that there was no significant difference in survival rate. After fecal microbiota transplantation (FMT) treatment fish larvae had significantly higher final body weight, weight gain rate and specific growth rate. FMT treatment remarkably enhanced the activities of α-amylase and trypsin in intestinal segment and the activity of lipase and trypsin in pancreatic segment. Furthermore, FMT treatment significantly increased the activity of leucine-aminopeptidase on brush border membrane and the mRNA expression of pcna and occludin compared with the control group. Similarly, gut in larvae of FMT group had significantly higher villus height and muscular thickness. Moreover, FMT treatment significantly improved the activity of lysozyme and decreased malondialdehyde concentration in visceral mass. FMT treatment increased intestinal microbial diversities and altered intestinal microbiota profile of larvae. Spearman analysis revealed that FMT profoundly influenced the function of gut microbiota. Bacteria introduced by FMT may play a role in digestion and intestinal tract development. In conclusion, our results revealed that FMT treatment improved microbial diversity and modulated intestinal microecology of large yellow coraker larvae, which could significantly affect the digestive capacity, antioxidant capacity, intestinal development and intestinal health, thus improved its growth performance.

Effects of Chitosan-Coated Microdiet on Dietary Physical Properties, Growth Performance, Digestive Enzyme Activities, Antioxidant Capacity, and Inflammation Response of Large Yellow Croaker (Larimichthys crocea) Larvae.

A 30-day feeding trial was designed to investigate the physical properties of chitosan-coated microdiet (CCD) and the effect of CCD on survival, growth performance, activities of digestive enzymes, intestinal development, antioxidant capacity, and inflammatory response of large yellow croaker larvae (initial weight: 3.81 ± 0.20 mg). Four isonitrogenous (50% crude protein) and isolipidic (20% crude lipid) microdiets were prepared with different concentrations of chitosan wall material by spray drying method (0.00%, 0.30%, 0.60%, and 0.90%, weight (chitosan) : volume (acetic acid)). Results showed that the lipid encapsulation efficiency (control: 60.52%, Diet1: 84.63%, Diet2: 88.06%, Diet3: 88.65%) and nitrogen retention efficiency (control: 63.76%, Diet1: 76.14%, Diet2: 79.52%, Diet3: 84.68%) correlated positively with the concentration of wall material (P < 0.05). Furthermore, the loss rate of CCD was significantly lower than the uncoated diet. Larvae fed the diet with 0.60% CCD had significantly higher specific growth rate (13.52 and 9.95%/day) and survival rate (14.73 and 12.58%) compared to the control group (P < 0.05). Larvae fed the diet with 0.30% CCD had significantly higher trypsin activity in pancreatic segments than the control group (4.47 and 3.05 U/mg protein) (P < 0.05). Larvae fed the diet with 0.60% CCD had significantly higher activity of leucine aminopeptidase (7.29 and 4.77 mU/mg protein) and alkaline phosphatase (83.37 and 46.09 U/mg protein) in the brush border membrane than those of the control group (P < 0.05). The intestinal epithelial proliferation- and differentiation-related factors (zo-1, zo-2, and pcna) in larvae fed the diet with 0.30% CCD had higher expression than those of the control group (P < 0.05). When the concentration of wall material reached 0.90%, the larvae had significantly higher superoxide dismutase activity than that of the control group (27.27 and 13.72 U/mg protein) (P < 0.05). Meanwhile, malondialdehyde contents were significantly lower in larvae fed the diet with 0.90% CCD than that of the control group (8.79 and 6.79 nmol/mg protein) (P < 0.05). 0.30%~0.60% CCD significantly increased the activity of total nitric oxide synthase (2.31, 2.60, and 2.05 mU/mg protein) and inducible nitric oxide synthase (1.91, 2.01, and 1.63 mU/mg protein) and had significantly higher transcriptional levels of inflammatory factor genes (il-1β, tnf-α, and il-6) than those of the control group (P < 0.05). The results indicated chitosan-coated microdiet had great potential in feeding large yellow croaker larvae in addition to reducing nutrition loss.

Effects of Nitrogen Addition on Soil Microbial Functional Diversity and Extracellular Enzyme Activities in Greenhouse Cucumber Cultivation

Greenhouses, commonly used for vegetable production, are experiencing large nitrogen (N) inputs in North China, which leads to soil acidification, increases soil N availability, and affects microbial community structure and composition. However, it remains unclear how N enrichment influences soil microbial functional activities in this region. In this study, we conducted a two-year pot experiment in a greenhouse to evaluate the effects of four different rates of N addition (0, 334, 668, and 1002 kg N ha−1 year−1) on cucumber soil properties, extracellular enzyme activities, and community level physiological profiles (CLPP). We found that high-N addition (1002 kg N ha−1) caused a massive accumulation of inorganic nitrogen and soil acidification, which was not beneficial to soil microbial activities. The color development (AWCD) values for the metabolism of microbial carbon sources and the activities of soil extracellular enzymes also showed a significant decrease in high N(N3) treatment. Additionally, the activity of leucine aminopeptidase (LAP) and polyphenol oxidase (PPO) of N3 decreased by 36% and 50% compared to the N0 and could be a good predictor for microbial functional diversity and microbial biomass carbon (MBC). Structural equation modeling (SEM) confirmed that the reduction of microbial functional diversity is mainly coregulated by the decline of soil pH and the change of cucumber BGB (belowground biomass) resulting from soil C and N imbalance. Overall, excessive N-fertilizer amendment can be more dangerous to microbial community functional diversity, especially for carbohydrate utilization which adversely affects cucumber yield in current intensive management.

Pollock liver oil extraction using a new enzyme obtained from the Aspergillus oryzae strain

A distinctive feature of fish fats from fats of plants and terrestrial animals is the presence in their composition of highly unsaturated and polyunsaturated omega-3 fatty acids (eicosapentaenoic and docosahexaenoic acids), which causes their high biological value. A typical type of raw material for obtaining edible fat is cod liver, which makes up about 10% of the weight of fish and contains an average of about 50% lipids. The Far Eastern pollock liver with a high content of polyunsaturated fatty acids has similar characteristics. From a technological point of view, the complexity of extracting these biologically valuable components is due to the high sensitivity of fish oil lipids to oxidation. The article discusses the use of new enzyme Protorizin-LAP obtained at Russian scientific research institute of food biotechnology using Aspergillus oryzae strain - a producer of a complex of exo- and endopeptidases with an increased activity of leucine aminopeptidase. Oil yield (%) and acid value (mg KOH/g oil) are considered as efficiency criteria for enzymatic hydrolysis. Optimization results of enzymatic hydrolysis of the pollock liver using a full-factorial experiment for two factors are presented. The optimal values of the factors are established: the dosage of the enzyme preparation Protorizin-LAP (0.4% by weight of the raw material) and the duration of the enzymatic hydrolysis process (1 hour). The study of the comparative enzymatic analysis of pollock liver showed that the use of the enzyme preparation "Protorizin LAP" in the technological process of processing fat-containing fish. The laboratory sample is not inferior to commercial analogues in terms of the ability to extract fat from pollock liver and may be promising in the processing of other raw materials of animal origin.

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.