Extracellular Enzyme Activities Research Articles

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.

Extracellular enzyme activity and stoichiometry reveal P limitation in the wild panda habitat of the Qinling Mountains

AbstractSoil microbial extracellular enzymes play crucial roles in soil carbon and nutrient cycling by catalyzing soil biochemical processes. However, the activity and stoichiometry of enzymes involved in the carbon, nitrogen, and phosphorus cycles in the environment with the highest density of wild giant pandas on the southern slopes of the Qinling Mountains is unknown. We have established eight research areas at an elevation of 1090–2621 m. The β‐1,4‐glucosidase (BG), β‐1,4‐N‐acetylglucosaminidase (NAG), and acid phosphatase (APase) in soil samples were measured. We used redundancy analysis and structural equation model to evaluate the driving factors of metabolic restriction of soil microorganisms along the elevational gradient, and four models were used to cross‐evaluate the nutrient restriction status of soil microorganisms. The results showed that most soil physiochemical properties, soil microbial biomass, and microbial extracellular enzymes exhibited a hump‐shaped trend with increasing elevation. Elevation indirectly affected soil enzyme activity and stoichiometry by C, N, and P status. Microorganisms are limited by C at lower and higher elevations but limited by N at medium elevations. These results could help strengthen the conservation and management of the wild panda's natural habitat.

Vegetation drives soil microbial metabolic limitation through modifications of soil properties and microbial biomass during desert grassland-shrubland state anthropogenic transition

Shrub encroachment became widespread in grass-dominated ecosystem regions. However, there is still limited attention on the changes in plant-soil system C:N:P stoichiometry and soil microbial metabolic limitation status caused by anthropogenic encroachment of shrubs in the desert grassland of northwest China, as well as their driving mechanisms. In this study, we quantified the C:N:P stoichiometry of plant-litter-soil, soil physicochemical properties, microbial biomass, extracellular enzyme activities, and microbial metabolic limitation status during the anthropogenic transition from grassland to shrubland. Here, our results showed that anthropogenic shrub encroachment reduced plant C, soil water, and soil nutrient contents while increasing N and P contents in both plants and litter. The correlation also suggested that litter nutrient content relied on plant nutrients, while plants depleted soil nutrients. Secondly, with the gradient of shrub encroachment, the stoichiometry (except for soil C:N ratio) of plant-litter-soil showed a decreasing trend and a positive correlation. Furthermore, our study indicated that shrub encroachment reduced soil microbial biomass and extracellular enzyme activities, altering microbial stoichiometry and patterns of enzyme allocation. Redundancy analysis revealed that microbial biomass, extracellular enzyme activities, and stoichiometry were mainly driven by soil water content, nutrient content, and nutrient stoichiometry. The vector-threshold element ratio model revealed that along the transition from desert grassland to shrubland, soil microbial nutrient limitation shifted from P to N, while energy (C) limitation intensified. The soil microbial metabolic limitation was driven jointly by plants and litter through modifications of soil properties and microbial biomass. Additionally, soil properties played a crucial role among these factors. In summary, over the past 40 years, shrub anthropogenic encroachment has formed a desert grassland-shrub mosaic landscape, depleted soil water and nutrient contents, and altered ecological stoichiometry and microbial metabolic limitation patterns in northwest China. This study provides new insights into the C, N, and P cycling in plant-soil systems following shrub encroachment caused by global climate change and anthropogenic disturbances.

Understanding the differential impact of PFOS and F-53B pollution on reed-mediated soil organic matter decomposition: Insights from rhizosphere priming effect

Plants affect soil microorganisms through the release of root exudates under pollution stress. This process may affect rhizosphere priming effect (RPE) and alter the rate of soil organic matter decomposition. However, the influence of plants on the decomposition of organic matter in soil subjected to pollution stress remains unclear. We studied the effects of exposure to perfluorooctanesulfonic (PFOS) and its alternative, chlorinated polyfluoroalkyl ether sulfonic (F-53B), at concentrations of 0.1 mg/kg and 50 mg/kg on the RPE of reed. We conducted our experiments in an artificial climate chamber and used the natural 13C tracer method to determine RPE. In the PFOS-exposed groups, the RPE was negative, with values of −11.45 mg C kg−1 soil d−1 in the low PFOS group and −8.04 mg C kg−1 soil d−1 in the high PFOS group. In contrast, in the F-53B-exposed groups, the RPE was positive, with values of 8.26 mg C kg−1 soil d−1 in the low F-53B group and 12.18 mg C kg−1 soil d−1 in the high F-53B group. Exposure of reeds to PFOS/F-53B stress resulted in differential effects on extracellular enzyme activities. The observed positive and negative RPE phenomena could be attributed to variations in extracellular enzyme activities. In conclusion, RPE responded differently under PFOS/F-53B exposure.

Isolation and Identification of Swan (Cygnus olor) Digestive Tract Cellulothic Bacteria to Support Fiber Degradation

Cellulolytic bacteria are bacteria that produce cellulase enzymes capable of degrading cellulose substrates. This study aimed to isolate and identify as well as measure the cellulase enzyme activity of cellulolytic bacteria isolated from the digestive tract of swans (Cygnus olor) in Mataram. The bacteria were isolated using de Man Rogosa Sharpe (MRS) selective media with the spread plate method. Then, the extracellular enzyme activity test was conducted by growing selected pure isolates on 1% Carboxy Methyl Cellulose (CMC 1%) media, followed by pouring 0.1% congo red solution and rinsing with 1M NaCl solution to determine cellulolytic activity (cellulolytic potential indicated by the appearance of a clear zone around the colony) and measuring the cellulolytic index. The bacterial isolation results obtained 4 isolates with the potential to support fiber degradation in animal feed. The highest cellulolytic index was produced by the isolate coded S1, reaching 34.5 mm, while the lowest cellulolytic index was produced by 2 isolates coded J21 and I2, reaching 18.8 mm.

Postfire extracellular enzyme activity in a temperate montane forest

AbstractWildfire is a disturbance expected to increase in frequency and severity, changes that may impact carbon (C) dynamics in the soil ecosystem. Fire changes the types of C sources available to soil microbes, increasing pyrogenic C and coarse downed wood, and if there is substantial tree mortality, decreasing C from root exudates and leaf litter. To investigate the impact of this shift in the composition of C resources on microbial processes driving C cycling, we examined microbial activity in soil sampled from an Oregon burn 1 year after fire from sites spanning a range in soil burn severity from unburned to highly burned. We found evidence that postfire rhizosphere priming loss may reduce soil C loss after fire. We measured the potential activity of C‐acquiring and nitrogen (N)‐acquiring extracellular enzymes and contextualized the microbial resource demand using measurements of mineralizable C and N. Subsurface mineralizable C and N were unaltered by fire and negatively correlated with hydrolytic extracellular enzyme activity (EEA) in unburned, but not burned sites. EEA was lower in burned sites by up to 46%, but only at depths below 5 cm, and with greater decreases in sites with high soil burn severity. These results are consistent with a subsurface mechanism driven by tree mortality. We infer that in sites with high tree mortality, subsurface EEAs decreased due to loss of rhizosphere priming and that inputs of dead roots contributed to mineralizable C stabilization.

Characteristics of Rhizosphere Microbiome, Soil Chemical Properties, and Plant Biomass and Nutrients in Citrus reticulata cv. Shatangju Exposed to Increasing Soil Cu Levels.

The prolonged utilization of copper (Cu)-containing fungicides results in Cu accumulation and affects soil ecological health. Thus, a pot experiment was conducted using Citrus reticulata cv. Shatangju with five Cu levels (38, 108, 178, 318, and 388 mg kg-1) to evaluate the impacts of the soil microbial processes, chemistry properties, and citrus growth. These results revealed that, with the soil Cu levels increased, the soil total Cu (TCu), available Cu (ACu), organic matter (SOM), available potassium (AK), and pH increased while the soil available phosphorus (AP) and alkali-hydrolyzable nitrogen (AN) decreased. Moreover, the soil extracellular enzyme activities related to C and P metabolism decreased while the enzymes related to N metabolism increased, and the expression of soil genes involved in C, N, and P cycling was regulated. Moreover, it was observed that tolerant microorganisms (e.g., p_Proteobacteria, p_Actinobacteria, g_Lysobacter, g_Sphingobium, f_Aspergillaceae, and g_Penicillium) were enriched but sensitive taxa (p_Myxococcota) were suppressed in the citrus rhizosphere. The citrus biomass was mainly positively correlated with soil AN and AP; plant N and P were mainly positively correlated with soil AP, AN, and acid phosphatase (ACP); and plant K was mainly negatively related with soil β-glucosidase (βG) and positively related with the soil fungal Shannon index. The dominant bacterial taxa p_Actinobacteriota presented positively correlated with the plant biomass and plant N, P, and K and was negatively correlated with plant Cu. The dominant fungal taxa p_Ascomycota was positively related to plant Cu but negatively with the plant biomass and plant N, P, and K. Notably, arbuscular mycorrhizal fungi (p_Glomeromycota) were positively related with plant P below soil Cu 108 mg kg-1, and pathogenic fungi (p_Mortierellomycota) was negatively correlated with plant K above soil Cu 178 mg kg-1. These findings provided a new perspective on soil microbes and chemistry properties and the healthy development of the citrus industry at increasing soil Cu levels.

Effects of long-term nighttime warming on extractable soil element composition in a Mediterranean shrubland

Understanding the soil biogeochemical responses to increasing global warming in the near future is essential for improving our capacity to mitigate the impacts of climate change on highly vulnerable Mediterranean ecosystems. Previous studies have primarily focused on the effects of warming on various biogeochemical processes. However, there is limited knowledge about how the changes in water availability associated to high temperatures can alter the bioavailability and dynamics of soil elements, thereby impacting ecosystem productivity, species composition, and pollution through soil biogeochemical and hydrological processes. In this study, we investigated the effects of long-term nighttime warming on the extractable concentrations of organic carbon (EOC), total nitrogen (ETN), total phosphorus (ETP), and 17 mineral elements (arsenic (As), calcium (Ca), cadmium (Cd), chromium (Cr), copper (Cu), iron (Fe), mercury (Hg), potassium (K), magnesium (Mg), manganese (Mn), molybdenum (Mo), nickel (Ni), lead (Pb), sulfur (S), strontium (Sr), vanadium (V), and zinc (Zn)) through environmental experiments in a semi-arid Mediterranean shrubland. We explored the potential biotic and abiotic mechanisms underlying the seasonal and long-term changes in extractable-mobilizable elemental composition and concentrations. Our findings revealed that prolonged warming led to higher mean annual soil temperature (with an average increase of 0.67 °C from 1999 to 2014), accumulation of soil organic matter (EOC) and extractable concentrations of soil elements (particularly increased ETP and extractable Ca, Mg, Cu, Sr, Mn, and As). These changes were attributed to uniformly higher activities of extracellular soil enzymes and/or lower plant photosynthetic and nutrient uptake capacity linked to more water deficit under warmer conditions. Seasonality unevenly altered element extractable concentrations, with soil microclimate (temperature and water content) and biological (soil microbial and plant) activity being the main drivers of this variability, thus influencing soil element composition. These results suggest significant fluctuations in the extractable concentrations of specific mineral elements in these soils, implying potential future variations in soil element composition as well as the loss of total element concentrations/contents in semi-arid Mediterranean ecosystems due to increasing warming. Therefore, these findings enhance our ability to predict ecosystem management strategies and mitigate the observed negative impacts on plant-soil systems and water quality in the context of climate change.

The influence of soil depth and tree age on soil enzyme activities and stoichiometry in apple orchards

The indispensable role of soil microorganisms in apple orchards, impacting soil quality, nutrient cycling, and crop productivity, cannot be overstated. Nonetheless, as apple trees mature, the potential for soil nutrient imbalances arises, affecting apple growth and yield. Research on how deep soil properties evolve with tree age is limited. Soil samples were gathered from apple orchards of various ages, utilizing the microplate fluorescence method to examine soil enzyme activity, microbial stoichiometry, and their correlation with soil nutrients. Results reveal that soil properties, microbial biomass, and enzymes are influenced by both tree age and depth. Microbial biomass decreases with depth, peaking in young orchards with the highest MBN:MBP ratio. Initially declining, microbial entropy nitrogen rises with tree age. Redundancy analysis identifies OP as pivotal in explaining extracellular enzyme activity. The enzyme C:N ratio exhibits a negative correlation with the MBN:MBP ratio. Additionally, ratios of ln(NAG) to ln(BG), ln(AKP) to ln(BG), and ln(AKP) to ln(NAG) demonstrate positive correlations. Enzyme activity ratios for C, N, and P transformation deviate from the global ecosystem ratio, indicating nuanced nutrient dynamics. Soil vector angles are influenced by tree age, suggesting limitations on microbial metabolism by carbon and phosphorus. Structural equation modeling unveils that soil depth negatively impacts pH, AKP, and Enzyme N:P, while tree age adversely affects Enzyme N:P. Furthermore, MBN:MBP is directly influenced by AKP and soil depth, indirectly by tree age and pH. This study underscores the significance of microbial stoichiometry in evaluating soil resource ecology relations, offering insights into soil microbe-orchard ecosystem dynamics.

Enzymatic Stoichiometry Reveals the Metabolic Limitations of Soil Microbes under Nitrogen and Phosphorus Addition in Chinese Fir Plantations.

Increasing nitrogen (N) deposition alters the availability of soil nutrients and is likely to intensify phosphorus (P) limitations, especially in P-limited tropical and subtropical forests. Soil microorganisms play vital roles in carbon (C) and nutrient cycling, but it is unclear whether and how much N and P imbalances affect the soil's microbial metabolism and mechanisms of nutrient limitations. In this study, a 3-year field experiment of N and P addition (control (CK), 100 kg N ha-1 yr-1 (N), 50 kg P ha-1 yr-1 (P), and NP) was set up to analyze the extracellular enzyme activities and stoichiometry characteristics of the top mineral soils in Chinese fir plantations with different stand ages (7, 20, and 33 years old). The results showed that the enzyme activities associated with the acquisition of C (β-1,4-glucosidase (BG) and β-d-cellobiohydrolase (CBH)) and P (acid phosphatases (APs)) in the N treatment were significantly higher than those in the CK treatment. Moreover, vector analysis revealed that both the vector's length and angle increased in stands of all ages, which indicated that N addition aggravated microbial C and P limitations. The P and NP treatments both significantly decreased the activity of AP and the enzymes' N:P ratio, thereby alleviating microbial P limitations, as revealed by the reduction in the vector's angle. Stand age was found to promote all enzymatic activities but had no obvious effects on the limitation of microbial metabolism with or without added nutrients in the soils under Chinese fir. Available N, Olsen-P, and pH were the main drivers of microbial metabolic limitations related to C nutrients. These results provide useful data for understanding the change in soil microbial activity in response to environmental changes, and suggest that P fertilization should be considered for management to improve productivity and C sequestration in Chinese fir plantation in the context of increased deposition of N.

Ozone strengthens the ex vivo but weakens the in vivo pathway of the microbial carbon pump in poplar plantations

Elevated ozone (eO3) and atmospheric nitrogen (N) deposition are important climate change components that can affect plant growth and plant-soil-microbe interactions. However, the understanding of how eO3 and its interaction with N deposition affect soil microbially mediated carbon (C) cycling and the fate of soil C stocks is limited. This study aimed to test how eO3 and N deposition affected soil microbial metrics (i.e., respiration, enzyme activities, biomass, necromass, and community composition) and resulting soil organic C (SOC) fractions in the rhizosphere of poplar plantations with different sensitivity to O3. Exposure to O3 and/or N deposition for four years was conducted within a free-air O3 concentration-enrichment facility. Elevated O3 reduced soil microbial respiration and biomass C but enhanced the enzymatic acquisition of C (i.e., potential soil hydrolase and oxidase activity) and shifted to a fungi-dominated community composition. These responses suggest that microbial C availability decreased and microbes allocated more energy to obtain C and nutrients from biochemically resistant substrates under eO3. Elevated O3 decreased bacterial necromass C and total necromass C, which could explain the observed decreases in mineral-associated organic C and SOC. The effects of eO3 on soil microbial C availability and community composition were strengthened by N addition, whereas there were no differences in the below-ground effects of eO3 between the two poplar clones. Taken together, the increased soil extracellular enzyme activities and slightly increased particulate organic C content suggest that the microbial C pump pathway via microbial ex vivo modification was strengthened by eO3, whereas the pathway via microbial in vivo turnover was weakened, as suggested by the decreases in soil microbial respiration, biomass, necromass, and mineral-associated organic C. Our study provides evidence that aboveground eO3 effects on trees may affect belowground microbial processing of organic matter and ultimately the persistence of SOC.

Extracellular Enzymatic Activities of Endophytic Bacteria Isolates Obtained from Dioclea reflexa Hook Seeds

Extracellular Enzymatic Activities of Endophytic Bacteria Isolates Obtained from Dioclea reflexa Hook Seeds

Effects of fertilization on soil ecological stoichiometry and fruit quality in Karst pitaya orchard

Pitaya (Hylocereus undulatus) is a significant cash crop in the karst region of Southwest China. Ecological stoichiometry is an essential method to research biogeochemical cycles and limiting elements. The purpose of this study was to explore the stoichiometric characteristics of C, N, and P in Karst pitaya orchards and fruit quality and to elucidate the mechanism and process of nutrient cycling. The results showed that: (1) Fruit quality was highest under the combination of chemical and organic fertilizers. Compared to the control, the contents of per-fruit weight, vitamin C, and soluble sugar increased significantly by 55.5%, 60.7%, and 23.0%, respectively, while the content of titratable acidity decreased significantly by 22.0%. (2) The content of soil nutrients under fertilization stress showed a downward trend in general, as did microbial biomass and extracellular enzyme activities. (3) Different fertilization treatments significantly affected the soil-microbial stoichiometry C:N ratio, C:P ratio, with research areas being significantly limited by C and P. (4) Spearman and PLS-SEM (partial least squares-structural equation model) analysis results showed that under the influence of fertilization, there was a significant positive effect between microorganisms and soil nutrients, but a significant negative effect between soil nutrients and quality. The results of this study offer an innovative perspective on pitaya quality research in Karst areas.

Litter application increases soil multinutrient cycling in alpine meadow ecosystems on the Tibetan Plateau

In soil, multiple nutrients are cycled simultaneously (multinutrient cycling), rather than a single measurable process. While the impact of litter decomposition on individual soil nutrient cycling in alpine meadow ecosystems on the Tibetan Plateau is well-documented, its effects on soil multinutrient cycling remain unclear. We deployed a three-year litter application experiment in an alpine meadow on the Tibetan Plateau to examine the responses of soil microclimate, extracellular enzyme activities, and bacterial and fungal communities to litter applications, as well as the correlations between these vital factors with soil multinutrient cycling induced by litter application. We showed that litter application raised the soil multinutrient cycling index, temperature, and moisture content by an average of 190 %, 1.2 °C, and 12 %, respectively. Litter application increased the activities of soil extracellular enzyme β-1,4-glucosidase, β-1,4-xylosidase, β-D-cellobiosidase, L-leucine aminopeptidase, acid phosphatase, and phenol oxidase. Moreover, litter application increased the richness and diversity of both soil bacterial and fungal communities, and altered their community structure, but with larger effects on bacterial communities (R2 = 0.43 for bacterial community and R2 = 0.19 for fungal community). This indicates that bacterial communities are more responsive to litter application than fungal communities in alpine meadow soils on the Tibetan plateau. Partial least-square path modeling indicated that soil bacterial and fungal communities and extracellular enzyme activities were significantly positively correlated with soil multinutrient cycling after litter application. The relative abundances of bacterial phyla of Proteobacteria, Actinobacteria, and Verrucomicrobiota, and fungal phyla of Basidiomycota were positively related to most of the critical soil nutrients, indicating that those microbial taxa are the main drivers of soil multinutrient cycling. Overall, this study provides explicit evidence that litter application accelerates Tibetan meadow soil multinutrient cycling, which contributes to an enhanced understanding of the role of litter in sustaining the functions and services of alpine meadow ecosystems on the Tibetan Plateau.

Pore connectivity and anisotropy affect carbon mineralization via extracellular enzymes in > 2 mm aggregates under conservation tillage of Mollisols

Soil aggregates, which are the basic units of soil structure, play an important role in the carbon cycle of ecosystems. The pore characteristics of aggregates influence soil organic carbon sequestration. However, studies on SOC mechanisms in aggregates have been limited to Mollisols. This study was conducted as a long-term experiment established in 2004 with a corn-soybean rotation in Mollisols. There are three treatments, including rotary tillage without straw return (conventional tillage, CT), subsoiling without straw return (reduced tillage, RT), and no tillage with straw return (NT). The soil pore size distribution, shape parameters, extracellular enzymes activity, and carbon mineralization were measured. The results showed that 15-year no tillage and reduced tillage increased the total porosity and proportion of larger pores, but significantly decreased the proportion of smaller pores in situ soil columns. Conventional tillage exhibited the most complex pores because of the highest pore fractal dimension (2.75–2.90), anisotropy (0.366–0.516), and the lowest sphericity (5.1–28.7). As for the soil columns filled with > 2 mm aggregates, reduced tillage significantly increased the pore connectivity by 3.02–3.62 %, whereas no tillage had no effect. The structural equation modelling indicated that in soil columns filled with > 2 mm aggregates, pore shape parameters, particularly connectivity and anisotropy, positively influenced the activities of β-glucosidase and β-xylosidase directly, and positively affected soil carbon mineralization by influencing extracellular enzymes activity indirectly. The findings emphasize the importance of pore shape parameters effect on soil carbon sequestration, and will be helpful in comprehending the microscopic mechanisms of soil carbon sequestration in > 2 mm aggregates.

A kinetic modelling approach to explore mechanism of Cr(VI) detoxification by a novel strain Pseudochrobactrum saccharolyticum NBRI-CRB 13 using response surface methodology.

A novel Pseudochrobactrum saccharolyticum strain NBRI-CRB 13, isolated from tannery sludge, was studied to grow up to 500 mgL-1 of Cr(VI) and showed Cr(VI) detoxification by reducing > 90% of Cr(VI) at different concentrations 25, 50 and 100 mgL-1. Kinetic studies showed that first-order models were fitted (R2 = 0.998) to the time-dependent Cr(VI) reduction with degradation rate constant (k) (1.03-0.429h-1). Cr(VI) detoxification was primarily related to the extracellular fraction of microbial cells, which showed a maximum extracellular reductase enzyme activity led to 94.6% reduction of Cr(VI). Moreover, the strain showed maximum extracellular polymeric substances (EPS) production at 100 mgL-1 Cr(VI), which is presumably the reason for Cr(VI) removal as EPS serves as the metal binding site for Cr(VI) ions. Further, an optimization study using Box-Behnken design was conducted considering parameters viz., pH, temperature, and initial concentration of Cr(VI). The maximum percent reduction of Cr(VI) was obtained at pH 6.5, temperature 30°C with 62.5 mgL-1Cr(VI) concentration. Further, the Cr(VI) reduction and adsorption ability of strain P. saccharolyticum NBRI-CRB13 were confirmed by SEM-EDS, FTIR, and XRD analyses. FTIR analysis confirmed the presence of functional groups (-OH, -COOH, -PO4) on bacterial cell walls, which were more likely to interact with positively charged chromium ions. The study elucidated the reduction of Cr(VI) by the novel bacterium within 24h using the response surface methodology approach and advocated its application in real-time situations.

Activin A Is a Master Regulator of Phenotypic Switch in Adipose Stromal Cells Initiated by Activated Immune Cell-Secreted Interleukin-1β.

Prolonged tissue ischemia and inflammation lead to organ deterioration and are often accompanied by microvasculature rarefaction, fibrosis, and elevated systemic Activin A (ActA), the level of which frequently correlates with disease severity. Mesenchymal stromal cells are prevalent in the perivascular niche and are likely involved in tissue homeostasis and pathology. This study investigated the effects of inflammatory cells on modulation of phenotype of adipose mesenchymal stromal cells (ASC) and the role of ActA in this process. Peripheral blood mononuclear cells were activated with lipopolysaccharide (activated peripheral blood mononuclear cells [aPBMC]) and presented to ASC. Expression of smooth muscle/myofibroblast markers, ActA, transforming growth factors beta 1-3 (TGFβ1-3), and connective tissue growth factor (CTGF) was assessed in ASC. Silencing approaches were used to dissect the signaling cascade of aPBMC-induced acquisition of myofibroblast phenotype by ASC. ASC cocultured with aPBMC or exposed to the secretome of aPBMC upregulated smooth muscle cell markers alpha smooth muscle actin (αSMA), SM22α, and Calponin I; increased contractility; and initiated expression of ActA. Interleukin (IL)-1β was sufficient to replicate this response, whereas blocking IL-1β eliminated aPBMC effects. ASC-derived ActA stimulated CTGF and αSMA expression in ASC; the latter independent of CTGF. Induction of αSMA in ASC by IL-1β or ActA-enriched media relied on extracellular enzymatic activity. ActA upregulated mRNA levels of several extracellular matrix proteins in ASC, albeit to a lesser degree than TGFβ1, and marginally increased cell contractility. In conclusion, the study suggests that aPBMC induce myofibroblast phenotype with weak fibrotic activity in perivascular progenitors, such as ASC, through the IL-1β-ActA signaling axis, which also promotes CTGF secretion, and these effects require ActA extracellular enzymatic processing.

Characteristics of biodepolymerized coal and insights into depolymerization pathway catalyzed by fungal extracellular enzymes

ABSTRACT The bio-depolymerization of coal is a promising technology for converting coal into low-molecular-weight industrial chemicals. In this study, we investigated the characteristics of biodepolymerized coal (BDC) and the associated depolymerization pathway. We analyzed the extracellular enzyme activities of Hypocrea lixii AH in the biodepolymerization system, performed Fourier transform infrared spectroscopy (FTIR) and 13C nuclear magnetic resonance spectroscopy (NMR) of BDC, and performed gas chromatography and mass spectrometry (GC/MS) of the BDC methanol extract. Results showed that the activities of peroxidase (POD), polyphenol oxidase (PPO), laccase (Lac), and lignin peroxidase (LiP) were 1377, 162, 10, and 1.919 U, respectively. Following biodepolymerization by Hypocrea lixii AH, 20 organic compounds were identified. Benzaldehyde 4-hydroxy-3,5-dimethoxy was the most dominant at 33.16%, followed by (Z)-9-octadecenamide, accounting for 14.73%. The aromaticity (f a) decreased from 54.3% to 23.7%, suggesting ring-opening reactions catalyzed by LiP and Lac. The ratio of carbonyl carbon (f a C) increased from 8.3% to 16.2%, suggesting oxidation reactions catalyzed by POD. By comparing the structure of the Fushun oxidized coal model and BDC, a biodepolymerization pathway was elucidated. Biodepolymerization of Fushun oxidized coal is an oxidation–depolymerization process catalyzed by a series of enzymes, which breaks the aromatic rings and oxidizes side chains. Altogether, our findings provide a theoretical support for the chemical application of BDC.

Functional analysis of FlbA-regulated transcription factor genes in Aspergillus niger using a multiplexed CRISPRoff system

FlbA of Aspergillus niger (indirectly) regulates 36 transcription factor (TF) genes. As a result, it promotes sporulation and represses vegetative growth, protein secretion and lysis. In this study, the functions of part of the FlbA-regulated TF genes were studied by using CRISPRoff. This system was recently introduced as an epigenetic tool for modulating gene expression in A. niger. A plasmid encompassing an optimized CRISPRoff system as well as a library of sgRNA genes that target the promoters of the 36 FlbA-regulated TF genes was introduced in A. niger. Out of 24 transformants that exhibited a sporulation phenotype, 12 and 18 strains also showed a biomass and secretion phenotype, respectively. The transforming sgRNAs, and thus the genes responsible for the phenotypes, were identified from five of the transformants. The results show that the genes dofA, dofB, dofC, dofD, and socA are involved in sporulation and extracellular enzyme activity, while dofA and socA also play roles in biomass formation. Overall, this study shows that the multiplexed CRISPRoff system can be effectively used for functional analysis of genes in a fungus.

A Membrane-Confined Signal Amplification Strategy for Sensitive Monitoring of Extracellular Enzymatic Activity Upon Drug Stimulus.

Extracellular enzymes are not only strongly correlated with disease development but also play critical roles in modulating immune responses. Therefore, real-time monitoring of extracellular enzymatic activity can afford straightforward insights into their spatiotemporal dynamics upon drug stimulus, and provide promising tools to unravel their key roles in modulating the cell signaling. Although DNA-based sensing probes have been frequently developed for the detection of a variety of biomolecules, there still lacks a modular design strategy for amplified imaging of extracellular enzymatic activity associated with live cells. Herein, we developed an enzymatically triggerable signal amplification strategy for real-time dynamic imaging of extracellular enzyme activity through a cell membrane-confined hybrid chain reaction (HCR). We demonstrated that, by modifying the initiator DNA with enzyme-specific incision sites and cholesterol tail, extracellular enzyme-trigged HCR could be fulfilled on the surface of the cellular membrane, facilitating amplified detection of extracellular enzymatic activity. Dynamic monitoring of enzyme secretion of cancer cells upon stimulus or macrophage cells upon inflammation challenge has also been achieved. We envision that the design strategy could provide valuable information for dissecting the role of extracellular enzymes in modulating cell responses to drug treatment.