Cellobiohydrolase Research Articles

Co-expression of endoglucanase and cellobiohydrolase from yak rumen in lactic acid bacteria and its preliminary application in whole-plant corn silage fermentation.

Endoglucanase (EG) and cellobiohydrolase (CBH) which produced by microorganisms, have been widely used in industrial applications. In order to construct recombinant bacteria that produce high activity EG and CBH, in this study, eg (endoglucanase) and cbh (cellobiohydrolase) were cloned from the rumen microbial genome of yak and subsequently expressed independently and co-expressed within Lactococcus lactis NZ9000 (L. lactis NZ9000). The recombinant strains L. lactis NZ9000/pMG36e-usp45-cbh (L. lactis-cbh), L. lactis NZ9000/pMG36e-usp45-eg (L. lactis-eg), and L. lactis NZ9000/pMG36e-usp45-eg-usp45-cbh (L. lactis-eg-cbh) were successfully constructed and demonstrated the ability to secrete EG, CBH, and EG-CBH. The sodium carboxymethyl cellulose activity of the recombinant enzyme EG was the highest, and the regenerated amorphous cellulose (RAC) was the specific substrate of the recombinant enzyme CBH, and EG-CBH. The optimum reaction temperature of the recombinant enzyme CBH was 60°C, while the recombinant enzymes EG and EG-CBH were tolerant to higher temperatures (80°C). The optimum reaction pH of EG, CBH, and EG-CBH was 6.0. Mn2+, Fe2+, Cu2+, and Co2+ could promote the activity of CBH. Similarly, Fe2+, Ba2+, and higher concentrations of Ca2+, Cu2+, and Co2+ could promote the activity of EG-CBH. The addition of engineered strains to whole-plant corn silage improved the nutritional quality of the feed, with the lowest pH, acid detergent fiber (ADF), and neutral detergent fiber (NDF) contents observed in silage from the L. lactis-eg group (p < 0.05), and the lowest ammonia nitrogen (NH3-N), and highest lactic acid (LA) and crude protein (CP) contents in silage from the L. lactis-eg + L. lactis-cbh group (p < 0.05), while the silage quality in the L. lactis-cbh group was not satisfactory. Consequently, the recombinant strains L. lactis-cbh, L. lactis-eg, and L. lactis-eg-cbh were successfully constructed, which could successfully expressed EG, CBH, and EG-CBH. L. lactis-eg promoted silage fermentation by degrading cellulose to produce sugar, enabling the secretory expression of EG, CBH, and EG-CBH for potential industrial applications in cellulose degradation.

Preparation of water-soluble dietary fiber from bamboo shoots by fungi fermentation and its supplementation in biscuits

Expanding the utilization of bamboo shoots is extremely meaningful in China because of the high output every year. In this study, the bamboo shoot powder was used as a substrate to produce water-soluble dietary fiber (WSDF) by fermentation with a screened fungi strain Trichoderma dorotheae MLG23, and the prepared WSDF was utilized in the production of biscuits. The bamboo shoots and their residues, WSDF, and biscuits were analyzed on Fourier transform infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS), scanning electron microscope (SEM), inductively coupled plasma mass spectrometry (ICP-MS), and texture analyzer. The results showed that the fungi exhibited a strong ability to express extracellular cellulase. The enzymatic activities of filter paper activity (FPA), cellobiohydrolase (CBH), and ꞵ-glucosidase (BG) secreted by the strain showed the highest at 50 °C, reaching 0.13, 0.19, and 1.20 U/mL, respectively, and the highest enzymatic activity of endoglucanase (EG) with 0.37 U/mL was obtained at 60 °C. The highest WSDF yield of 45.97 wt% was obtained under the fermentation conditions of bamboo shoot loading 30 g/L, strain inoculum 1010 CFU/L, and fermentation time 4 d at 30 °C. The cellulose and hemicellulose in bamboo shoots were partially utilized by the fungi to produce WSDF. The main metal elements in WSDF were Mg, K, Ca, and Zn. The hardness, cohesion, chewiness, resilience, and adhesion of the biscuits showed a gradual decrease with the increase of WSDF addition in the range from 0 to 10 g per 100 g of low gluten flour. The incorporation of WSDF disrupted the network structure of the gluten, leading to the reduction of some textural properties and the change of microstructure. These results showed a potential utilization of bamboo shoot in production of WSDF and functional biscuits.

Effects of Incubation Temperature and Sludge Addition on Soil Organic Carbon and Nitrogen Mineralization Characteristics in Degraded Grassland Soil

Elucidating the characteristics and underlying mechanisms of soil organic carbon (SOC) and nitrogen mineralization in the context of sludge addition is vital for enhancing soil quality and augmenting the carbon sink capacity of soil. This study examined the chemical properties, enzyme dynamics, and organic carbon and nitrogen mineralization processes of soil from degraded grasslands on the Loess Plateau at various incubation temperatures (5, 15, 25, and 35 °C) and sludge addition rates (0%, 5.0%, 10.0%, and 20.0%) through a laboratory incubation experiment. The results showed that incubation temperature, sludge addition, and their interactive effects significantly altered the soil enzyme C:N, C:P, and N:P stoichiometries. The cumulative mineralization rates of SOC and nitrogen increased significantly with increasing incubation temperature and sludge addition rate. Principal component analysis revealed a significant linear correlation between cumulative SOC and nitrogen mineralization. Random forest analysis indicated that β-1,4-Glucosidase (BG), β-1,4-N-acetyglucosaminidase (NAG), cellobiohydrolase (CBH), ammonium nitrogen (NO3−), enzyme C:P ratio, alkaline phosphatase (ALP), and incubation temperature were crucial determinants of cumulative SOC mineralization. Structural equation modeling demonstrated that sludge addition, NO3−, NAG, ALP, and enzyme C:P positively impacted SOC mineralization, whereas dissolved organic carbon and BG had negative impacts. Conversely, incubation temperature negatively affected soil nitrogen mineralization, whereas NO3−, available phosphorus, and ALP contributed positively. Sludge addition and temperature indirectly modulated soil net nitrogen mineralization by altering soil chemical properties and enzyme activities. These findings underscore the role of SOC and nitrogen mineralization as indicators for evaluating soil nutrient retention capabilities.

Maize straw increases while its biochar decreases native organic carbon mineralization in a subtropical forest soil

Organic soil amendments have been widely adopted to enhance soil organic carbon (SOC) stocks in agroforestry ecosystems. However, the contrasting impacts of pyrogenic and fresh organic matter on native SOC mineralization and the underlying mechanisms mediating those processes remain poorly understood. Here, an 80-day experiment was conducted to compare the effects of maize straw and its derived biochar on native SOC mineralization within a Moso bamboo (Phyllostachys edulis) forest soil. The quantity and quality of SOC, the expression of microbial functional genes concerning soil C cycling, and the activity of associated enzymes were determined. Maize straw enhanced while its biochar decreased the emissions of native SOC-derived CO2. The addition of maize straw (cf. control) enhanced the O-alkyl C proportion, activities of β-glucosidase (BG), cellobiohydrolase (CBH) and dehydrogenase (DH), and abundances of GH48 and cbhI genes, while lowered aromatic C proportion, RubisCO enzyme activity, and cbbL abundance; the application of biochar induced the opposite effects. In all treatments, the cumulative native SOC-derived CO2 efflux increased with enhanced O-alkyl C proportion, activities of BG, CBH, and DH, and abundances of GH48 and cbhI genes, and with decreases in aromatic C, RubisCO enzyme activity and cbbL gene abundance. The enhanced emissions of native SOC-derived CO2 by the maize straw were associated with a higher O-alkyl C proportion, activities of BG and CBH, and abundance of GH48 and cbhI genes, as well as a lower aromatic C proportion and cbbL gene abundance, while biochar induced the opposite effects. We concluded that maize straw induced positive priming, while its biochar induced negative priming within a subtropical forest soil, due to the contrasting microbial responses resulted from changes in SOC speciation and compositions. Our findings highlight that biochar application is an effective approach for enhancing soil C stocks in subtropical forests.

Thermotolerance Mechanism of Fungal GH6 Cellobiohydrolase. Part I. Characterization of Thermotolerant Mutant from the Basidiomycete Phanerochaete chrysosporium.

Cellobiohydrolase (CBH), belonging to glycoside hydrolase family 6 (GH6), plays an essential role in cellulose saccharification, but its low thermotolerance presents a challenge in improving the reaction efficiency. Based on a report that chimeric CBH II (GH6) engineered to remove non-disulfide-bonded free Cys shows increased thermotolerance, we previously mutated the two free Cys residues to Ser in GH6 CBH from the basidiomycete Phanerochaete chrysosporium (PcCel6A) and obtained a thermotolerant double mutant, C240S/C393S (Yamaguchi et al., J. Appl. Glycosci. 2020; 67: 79-86). Here, characterization of the double mutant revealed that its activity towards both amorphous and crystalline cellulose was higher than that of the wild-type enzyme at elevated temperature, suggesting that the catalytic domain is the major contributor to the increased thermotolerance. To investigate the role of each free Cys residue, we prepared both single mutants, C240S and C393S, of the catalytic domain of PcCel6A and examined their residual activity at high temperature and the temperature-dependent changes of folding by means of circular dichroism measurements and thermal shift assay. The results indicate that the C393S mutation is the main contributor to both the increased thermotolerance of C240S/C393S and the increased activity of the catalytic domain at high temperature.

Alterations of bacterial community related C cycle by affecting soil carbon fractions under aged biochar application

AbstractBiochar is a beneficial substance for sequestering organic carbon in soil. Application of fresh biochar (FB) to the soil leads to an increase in soil organic carbon (SOC). However, the effects of aged biochar on SOC stabilization in acidic soils are unclear. A 90‐day incubation study was assessed to evaluate the impact of adding rice straw biochar on acid soil. The study aimed to assess the alterations in carbon fractions, enzyme activity and bacterial communities in soil. We used different ageing processes (water‐washed biochar [WB] and H2O2 oxidation biochar [OXB]) at a rate of 2%, with control at 0% of biochar. The results indicated that aged biochar reduced CO2 efflux and increased microbial biomass carbon (MBC), reducing the microbial metabolic quotient (qCO2). The SOC and its fractions increased with ageing relative to the FB treatment. Compared with the FB treatment, the carbon pool management index (CPMI) also increased with the OXB application. Additionally, the application of OXB stimulated the soil enzymes involved in carbon cycling, specifically β‐1,4‐glucosidase (βG), β‐1,4‐xylosidase (βX) and cellobiohydrolase (CBH) relative to the FB and WB treatments. Moreover, biochar applications improved the structure and functions of the bacterial population in soil with high acidity. Proteobacteria, Acidobacteria, Actinobacteria and Chloroflexi were the most prevalent species in all treatments, as shown by their relative abundances. The redundancy analysis revealed that the DOC (dissolved organic carbon) and MBC significantly impacted on the microbial community. Furthermore, adding OXB altered the Bacterial community involved in the C cycle by affecting SOC. These findings help us learn more about how applying fresh or aged rice straw biochar changes different carbon fractions and microbial communities in soil. It may serve as an instance for studying the provision of soil organic carbon after applying biochar in acidic soil.

Nitrogen deposition-induced stimulation of soil heterotrophic respiration is counteracted by biochar in a subtropical forest

Both atmospheric nitrogen (N) deposition and biochar application can markedly impact soil heterotrophic respiration (RH), an important component of the global carbon cycle. However, the interactive effects of N deposition and biochar application on soil RH in subtropical forest ecosystems remain unclear. Here, we conducted a three-year (2019–2022) field trial within a bamboo forest in subtropical China to examine the responses of soil physicochemical and microbial properties to N deposition and biochar application, and to elucidate how biochar regulates N deposition-induced change in soil RH. Nitrogen deposition stimulated soil RH by 8.1–9.8 % annually over three years compared to the control, and this stimulation was mitigated (by 8.1–8.9 % annually) with biochar addition. In the context of N deposition, the decrease of soil RH by biochar application was not through changing soil temperature, moisture or labile organic carbon content. Biochar treatment reduced the abundances of bacterial glycoside hydrolase family 48 gene (GH48) and fungal glycoside hydrolase family 7 cellobiohydrolase I gene (cbhI) and the activities of β-glucosidase and cellobiohydrolase (CBH), but increased the abundance of cbbL gene and activity of RubisCO enzyme. Furthermore, the RH was correlated positively (P < 0.01) with β-glucosidase and CBH activities and negatively (P < 0.01) with RubisCO enzyme activity. Structural equation modeling revealed that the biochar-induced reduction of soil RH under N deposition was associated with decreases in the abundance of cbhI gene and the activity of CBH in soils. We highlight that management practices can mitigate soil carbon loss in forests through modulating soil microbial functions under atmospheric N deposition, and that biochar application in Moso bamboo forests has the potential to reduce RH by approximately 7.6 × 106 t CO2 yr−1 under atmospheric N deposition.

Engineering of glycoside hydrolase family 7 cellobiohydrolases directed by natural diversity screening

Protein engineering and screening of processive fungal cellobiohydrolases (CBHs) remain challenging due to limited expression hosts, synergy-dependency, and recalcitrant substrates. In particular, GH7 CBHs are a family critically important for the bioeconomy as well as a methodology for improving these and other difficult to engineer enzymes. Here, we target the discovery of highly active natural GH7 CBHs and engineering of variants with improved activity. Using experimentally assayed activities of genome mined CBHs, we applied sequence and structural alignments to top performers to identify key point mutations linked to improved activity. From ∼1500 known GH7 sequences, an evolutionarily diverse subset of 57 GH7 CBH genes was expressed in Trichoderma reesei and screened using a multiplexed activity screening assay. Ten catalytically enhanced natural variants were identified, produced, purified, and tested for efficacy using industrially-relevant conditions and substrates. Three key amino acids in CBHs with performance comparable or superior to Penicillium funiculosum Cel7A were identified and combinatorically engineered into P. funiculosum cel7a, expressed in T. reesei, and assayed on lignocellulosic biomass. The top performer generated using this combined approach of natural diversity genome mining, experimental assays, and computational modeling produced a 41% increase in conversion extent over native P. funiculosum Cel7A, a 55% increase over the current industrial standard T. reesei Cel7A, and 10% improvement over Aspergillus oryzae Cel7C, the best natural GH7 CBH previously identified in our laboratory.

Plant Diversity Changes and Its Driving Factors of Abandoned Land at Different Restoration Stages in the Middle of the Qinling Mountains

The process of vegetation restoration is often accompanied by significant changes in aboveground plant diversity. To explore the driving mechanism of litter nutrient-soil nutrient-enzyme activity stoichiometry on aboveground vegetation change is of great importance for maintaining regional biodiversity conservation and ecological stability. Taking typical abandoned farmland of different restoration years （1， 8， 16， 31， and 50 a） in the Qinling Mountains as the research object， the variation characteristics of plant community diversity during vegetation restoration were analyzed through field investigation. Litter nutrients， soil nutrients， and the activities of five extracellular enzymes， including β-1，4-glucosidase （BG）， cellobiohydrolase （CBH）， β-1，4-N-acetylglucosaminidase （NAG）， leucine aminopeptidase （LAP）， and acid phosphatase （AP）， were determined. The characteristics of litter nutrients， soil nutrients， and enzyme stoichiometric ratios during vegetation restoration and the driving mechanism of plant diversity changes were discussed. The results showed that the plant community diversity index firstly decreased and then increased with the increase in vegetation restoration years， and the minimum was reached at 16 years after restoration. The results of principal component analysis showed that there were significant differences between total plant community diversity index and litter-soil-enzyme stoichiometric characteristics in different years of vegetation restoration. The plant community diversity index had a strong positive correlation with litter C∶P ratio and litter N∶P ratio but had a negative correlation with soil enzyme C∶P ratio （EEA C∶P）. The results of redundancy analysis showed that soil EEA C∶P had the highest explanation rate of plant diversity changes during vegetation restoration （25.93%）， followed by soil TP （5.94%）， which was the key factor regulating plant diversity changes. In conclusion， plant species and quantity increased significantly in abandoned farmland in the middle part of the Qinling Mountains at the late stage of vegetation restoration. Changes in the soil environment affected microbial metabolic activities and thus changed enzyme activities. Litter-soil-soil extracellular enzymes affected the community environment and plant diversity through feedback and regulation. EEA C∶P and TP were the main driving factors of aboveground plant diversity change during vegetation restoration.

Culture of Trichoderma sp. with biochar to produce high-activity cellulase in a laboratory

Biochar (BC) was used in Trichoderma sp. culture to produce high-activity cellulase on a laboratory scale. The biochar was added into the flask before being applied to the fermenter to identify the enhancement effect and to determine the best amount of addition and the most suitable incubation period. Cellulase production was performed with a working volume of 4 L, and enzymatic hydrolysis was conducted to evaluate the saccharification ability of the enzyme. During incubation, the activities of three enzymes (Endoglucanase (EG), β-glucosidase (BGL), and cellobiohydrolase (CBH)) were measured for three days, and the cellulase activity was determined using a filter paper unit (FPU). In flask scale, EG, BGL, and CBH activities were increased by 1.4, 2.1, and 1.8 folds, respectively, and the incubation period was shortened by adding BC. In the fermenter scale, EG, BGL, and CBH activities were noticeably enhanced by 12.1, 5.8, and 7.2 folds, respectively, and FPU was 42.1 (9.8 folds). Additionally, the conversion rates of cellulose and steam exploded softwood and hardwood were 109.4%, 75.4%, and 87.3%, which were similar to a commercial enzyme (Cellic CTecⅡ). This study demonstrated that biochar could be used to produce high-activity cellulase in a shorter period and suggests a novel method for effective cellulase production.

Effects of stand ages on soil enzyme activities in Chinese fir plantations and natural secondary forests.

Forest type and stand age are important biological factors affecting soil enzyme activities. However, the changes in soil enzyme activities across stand ages and underlying mechanisms under the two forest restoration strate-gies of plantations and natural secondary forests remain elusive. In this study, we investigated the variations of four soil enzyme activities including cello-biohydrolase (CBH), β-1,4-glucosidase (βG), acid phosphatase (AP) and β-1,4-N-acetylglucosaminidase (NAG), which were closely associated with soil carbon, nitrogen, and phosphorus cycling, across Cunninghamia lanceolata plantations and natural secondary forests (5, 8, 21, 27 and 40 years old). The results showed that soil enzyme activities showed different patterns across different forest types. The acti-vities of AP, βG and CBH in the C. lanceolata plantations were significantly higher than those in the natural secon-dary forests, and there was no significant difference in the NAG activity. In the plantations, AP activity showed a decreasing tendency with the increasing stand ages, with the AP activity in the 5-year-old plantations significantly higher than other stand ages by more than 62.3%. The activities of NAG and CBH decreased first and then increased, and βG enzyme activity fluctuated with the increasing stand age. In the natural secondary forests, NAG enzyme activity fluctuated with the increasing stand age, with that in the 8-year-old and 27-year-old stand ages being significantly higher than the other stand ages by more than 14.9%. βG and CBH enzyme activities increased first and then decreased, and no significant difference was observed in the AP activity. Results of the stepwise regression analyses showed that soil predictors explained more than 34% of the variation in the best-fitting models predicting soil enzyme activities in the C. lanceolata plantations and natural secondary forests. In conclusion, there would be a risk of soil fertility degradation C. lanceolata plantations with the increasing stand age, while natural secondary forests were more conducive to maintaining soil fertility.

Promoters and Synthetic Promoters in Trichoderma reesei.

Trichoderma reesei holds immense promise for large-scale protein production, rendering it an excellent subject for deeper exploration using genetic engineering methods to achieve a comprehensive grasp of its cellular physiology. Understanding the genetic factors governing its intrinsic regulatory network is crucial, as lacking this knowledge could impede the expression of target genes. Prior and ongoing studies have concentrated on advancing new expression systems grounded in synthetic biology principles. These methodologies involve utilizing established potent promoters or engineered variations. Genomic and transcriptomic analyses have played a pivotal role in identifying robust promoters and expression systems, including light-responsive, copper-inducible, L-methionine-inducible, and Tet-On systems, among others. This chapter seeks to highlight various research endeavors focusing on tunable and constitutive promoters, the impact of different promoters on both native and foreign protein expression, the discovery of fresh promoters, and strategies conducive to future research aimed at refining and enhancing protein expression in T. reesei. Characterizing new promoters and adopting innovative expression systems hold the potential to significantly expand the molecular toolkit accessible for genetically engineering T. reesei strains. For instance, modifying potent inducible promoters such as Pcbh1 by replacing transcriptional repressors (cre1, ace1) with activators (xyr1, ace2, ace3, hap2/3/5) and integrating synthetic expression systems can result in increased production of crucial enzymes such as endoglucanases (EGLs), β-glucosidases (BGLs), and cellobiohydrolases (CBHs). Similarly, robust constitutive promoters such as Pcdna1 can be converted into synthetic hybrid promoters by incorporating activation elements from potent inducible promoters, facilitating cellulase induction and expression even under repressive conditions. Nevertheless, further efforts are necessary to uncover innovative promoters and devise novel expression strategies to enhance the production of desired proteins on an industrial scale.

Effects of Mycorrhizal and Extraradical Hyphae of Subtropical Native Tree Species on Soil Enzyme Activities and Their Stoichiometric Ratios

We aimed to study the effects of mycorrhizal and extraradical hyphae on soil physical and chemical properties and enzyme activity characteristics in a subtropical plantation and to explore its indicative effect on the effectiveness of soil nutrients. In this study, three native afforestation tree species, Cunninghamia lanceolata, Schima superba, and Liquidambar formosana, with different biological characteristics, root functional traits, and nutrient acquisition strategies in subtropical regions were selected as the research objects. Based on the method of in-growth soil cores, the nylon mesh with different pore sizes was used to limited the root system and hypha into the soil column. The soil physical and chemical properties of five kinds of hydrolase related to the carbon (C), nitrogen (N), and phosphorus (P) cycles were determined in this study. The correlation of different tree species, roots, and mycelia with soil physicochemical properties, enzyme activity, and stoichiometric ratios was analyzed. The results revealed that mycorrhizal treatment significantly affected the soil total carbon (TC) and pH but had no significant effect on hydrolase activity and its stoichiometric ratio. Tree species significantly affected soil physical and chemical properties, soil β-1,4-N-acetylglucosaminidase (NAG), β-1,4-glucosidase (βG), and cellobiohydrolase (CB) activities and soil enzyme stoichiometric ratios. The soil enzyme activity and stoichiometric ratio of the Chinese fir forest had higher values than in monoculture broad-leaved stands of both Schima superba and Liquidambar formosana. There was no significant interaction effect of mycorrhizal treatments and tree species on all soil properties, enzyme activities, and stoichiometric ratios. In addition, the soil enzyme activity and stoichiometric characteristics were mainly affected by the pH. In this study, the soil enzyme activity ratios In(BG + CB):In(AP) and In(NAG + LAP):In(AP) were lower values than the global scale, while the ratios of In(βG + CB):In(NAG + LAP) were higher than the average, indicating that the soil microorganisms in this area were limited by C and P. Moreover, the soil enzyme activity and chemical metrology characteristics were mainly affected by the pH change. In conclusion, differences in litter quality and root functional traits of tree species affected the soil enzyme activity and its stoichiometric characteristics through the shaping of the forest environment by organic matter input, and the influence of pH was the main regulating factor.

The mechanism of polysaccharide synthesis of Sanghuangporus sanghuang based on multi-omic analyses and feedback inhibition

S. sanghuang polysaccharide has various biological roles in promoting human health, however, the underlying mechanism of polysaccharide synthesis in S. sanghuang remain elusive. In the present study, the molecular structure of novel polysaccharide in the mutant S. sanghuang strain A130 with high yield of polysaccharide was characterized. The critical genes/proteins and pathways involved in polysaccharide synthesis were investigated via comparative transcriptomic, proteomic, and integrative analysis between wildtype strain SH-1 and A130. An integrated analysis of transcriptomic and proteomic results was also performed to locate potential regulators in the production of polysaccharides. The genes of cellobiohydrolase1 (CBH1) and MutS Homolog 6 (MSH6) related to glycolysis/gluconeogenesis were differentially expressed between A130 and SH-1, suggesting the potential involvement of these genes in regulating the production of polysaccharide. Proteomic analysis revealed that the abundance of Tyrosinase (TYR) and Trehalase (TREH) were substantially different between A130 and SH-1. The potential involvement of TYR in polysaccharide production was confirmed by transcriptomic-proteomic integrated analysis. The biological role of TYR and TREH in polysaccharide production was further verified by feedback inhibition of kojic acid and validamycin A, respectively. Overall, our study provides critical insights for the polysaccharide synthesis and high yield of polysaccharide through genes/pathways regulating in S. sanghuang.

Variation in microbial biomass and enzymatic activities in metal contaminated soils during storage at low temperature (4°C)

ABSTRACT Microbial response to soil storage at low temperature in limed and unlimed samples has not been investigated. For this study, soil samples were kept at 4°C for six weeks. Soil microbial biomass was determined using phospholipid fatty acid analysis (PLFA). Nine enzymes were targeted including β-glucosidase (BG), cellobiohydrolase (CBH), β-N-acetylglucosaminidase (NAGase), aryl sulfatase (AS), acid phosphatase (AP), alkaline phosphatase (AlP), glycine aminopeptidase (GAP), leucine aminopeptidase (LAP), and peroxidase (PER). PLFA results revealed a significant increase in total microbial biomass in limed area compared to unlimed soil samples. Microbial biomass decreased during the first two weeks of storage and remained unchanged thereafter for both limed and unlimed samples. Analysis of microbial activities revealed that most enzymes inconsistently decreased over time during storage at 4°C. Activities of BG, CBH, NAGase, AlP, AS, GAP, LAP were significantly higher in limed compared to the unlimed samples. Overall, the levels of activities of most enzymes in limed soils decreased significantly after the second week of storage and remained unchanged thereafter. The reduction of enzyme activity in the unlimed soil samples varied over time, with some enzymes such as LAP increasing on the sixth week. PER activity decreased after two weeks and increased thereafter.

Effect of Biochar on Labile Organic Carbon Fractions and Soil Carbon Pool Management Index

Biochar is useful for soil organic carbon (SOC) sequestration. However, the effects of biochar aging and addition rates on SOC stabilization are unclear. A field experiment with four biochar application rates (0% (control), 1% (LB), 2% (MB), and 4% (HB) of dry fluvo-aquic soil) was conducted. Soil samples were sampled after 8, 12, and 24 months of its application to clarify the question. In general, SOC gradually increased with the biochar application rate. SOC with HB was higher than that in other treatments, while the ratio of microbial biomass carbon (MBC)/SOC and readily oxidizable carbon (ROC)/SOC with HB was lower than that in other treatments (p &lt; 0.05), indicating a positive effect of HB for C stabilization over time. The effects of biochar on the soil carbon pool management index (CPMI) changed from negative to positive after 8 and 24 months of biochar application. The activities of β-D-glucosidase (βG), cellobiohydrolase (CBH), and β-N-acetylglucosaminidase (NAG) under HB were higher than with other treatments after 12 and 24 months of biochar application (p &lt; 0.05) and negatively correlated with the ratio of MBC/SOC and ROC/SOC over time. The CPMI was positively related with βG and CBH activities after 8 and 24 months of biochar application, respectively (p &lt; 0.05). HB increased the relative abundance of oligotrophs, including Acidobacteria, Actinobacteria, and Chloroflexi, but decreased the relative abundance of copiotrophs, including γ-Proteobacteria and Bacteroidetes over time (p &lt; 0.05). The ratio of dissolved organic carbon (DOC)/SOC was positively correlated with the bacterial oligotroph/copiotroph ratio and significantly affected the oligotrophic and copiotrophic bacterial communities, especially after 8 and 12 months of biochar application (p &lt; 0.05). These findings reinforce that increasing the biochar application rate and time enhances SOC stabilization by decreasing the proportions of labile organic carbon and making oligotrophic/copiotrophic communities and enzyme activities more conducive to C sequestration.

Nitrogen addition increases topsoil carbon stock in an alpine meadow of the Qinghai-Tibet Plateau

Soil carbon (C) sequestration plays a critical role in mitigating climate change. Nitrogen (N) deposition greatly affects soil C dynamics by altering C input and output. However, how soil C stocks respond to various forms of N input is not well clear. This study aimed to explore the impact of N addition on soil C stock and to elucidate the underlying mechanisms in an alpine meadow on the eastern Qinghai-Tibet Plateau. The field experiment involved three N application rates and three N forms, using a non-N treatment as a control. After six years of N addition, the total C (TC) stocks in the topsoil (0–15 cm) were markedly increased by an average of 12.1 %, with a mean annual rate of 20.1 ‰, and no difference was found between the N forms. Irrespective of rate or form, N addition significantly increased the topsoil microbial biomass C (MBC) content, which was positively correlated with mineral-associated and particulate organic C content and was identified as the most important factor that affecting the topsoil TC. Meanwhile, N addition significantly increased the aboveground biomass in the years with moderate precipitation and relatively high temperature, which leads to higher C input into soils. Owing to decreased pH and/or activities of β-1,4-glucosidase (βG) and cellobiohydrolase (CBH) in the topsoil, organic matter decomposition was most likely inhibited by N addition, and this inhibiting effect varied under different N forms. Additionally, TC content in the topsoil and subsoil (15–30 cm) exhibited parabolic and positive linear relationship with the topsoil dissolved organic C (DOC) content, respectively, indicating that DOC leaching might be an important influencing factor for soil C accumulation. These findings improve our understanding of how N enrichment affects C cycles in alpine grassland ecosystems and suggest that soil C sequestration in alpine meadows probably increases with N deposition.

Effects of arbuscular mycorrhizae and extraradical mycelium of subtropical tree species on soil nitrogen mineralization and enzyme activities.

Through symbiosis with plants, arbuscular mycorrhizal (AM) fungi effectively improve the availability of soil nitrogen (N). However, the mechanism through which AM and associated extraradical mycelium affect soil N mineralization remains unknow. We carried out an in situ soil culture experiment by using in-growth cores in plantations of three subtropical tree species, Cunninghamia lanceolata, Schima superba, and Liquidambar formosana. We measured soil physical and chemical properties, net N mineralization rate, and the activities of four kinds of hydrolase (leucine aminopeptidase (LAP), β-1,4-N-acetylglucosaminidase (NAG), β-1,4-glucosidase (βG), cellobiohydrolase (CB)) and two kinds of oxidases (polyphenol oxidase (POX) and peroxidase (PER)) involved in soil organic matter (SOM) mineralization in treatments of mycorrhiza (with absorbing roots and hyphae), hyphae (hyphae only), and control (mycorrhiza-free). The results showed that mycorrhizal treatments significantly affected soil total carbon and pH but did not affect N mineralization rates and all enzymatic activities. Tree species significantly affected net ammonification rate, net N mineralization rate and activities of NAG, βG, CB, POX and PER. The net N mineralization rate and enzyme activities in the C. lanceolata stand were significantly higher than that in monoculture broad-leaved stands of either S. superba or L. formosana. There was no interactive effect of mycorrhizal treatment and tree species on any of soil properties, nor on enzymatic activities or net N mineralization rates. Soil pH was negatively and significantly correlated with five kinds of enzymatic activities except for LAP, while net N mineralization rate significantly correlated with ammonium nitrogen content, available phosphorus content, and the activity level of βG, CB, POX, and PER. In conclusion, there was no difference in enzymatic activities and N mineralization rates between rhizosphere and hyphosphere soils of three subtropical tree species in the whole growing season. The activity of particular carbon cycle-related enzymes was closely related to soil N mineralization rate. It is suggested that differences in litter quality and root functional traits among different tree species affect soil enzyme activities and N mineralization rates through organic matter inputs and shaping soil condition.

Comparative assessment on lignocellulose degrading enzymes and bioethanol production from spent mushroom substrate of Calocybe indica and Volvariella volvacea.

In the current study, we compared the production of extracellular lignocellulose degrading enzymes and bioethanol from the spent mushroom substrate (SMS) of Calocybe indica and Volvariella volvacea. From SMS at different stages of the mushroom development cycle, ligninolytic and hydrolytic enzymes were analysed. The activities of lignin-degrading enzymes, including lignin peroxidase (LiP), laccase, and manganese peroxidase (MnP) were maximal in the spawn run and primordial stages, while hydrolytic enzymes including xylanase, cellobiohydrolase (CBH), and carboxymethyl cellulase (CMCase) showed higher activity during fruiting bodies development and at the end of the mushroom growth cycle. SMS of V. volvacea showed relatively lower ligninase activity than the SMS of C. indica, but had the maximum activity of hydrolytic enzymes. The enzyme was precipitated with acetone and further purified with the DEAE cellulose column. The maximum yield of reducing sugars was obtained after hydrolysis of NaOH (0.5M) pretreated SMS with a cocktail of partially purified enzymes (50% v/v). After enzymatic hydrolysis, the total reducing sugars were 18.68 ± 0.34g/l (SMS of C. indica) and 20.02 ± 0.87g/l (SMS of V. volvacea). We observed the highest fermentation efficiency and ethanol productivity (54.25%, 0.12g/l h) obtained from SMS hydrolysate of V. volvacea after 48h at 30 ± 2°C, using co-culture of Saccharomyces cerevisiae MTCC 11,815 and Pachysolen tannophilus MTCC 1077.

Early cellular events and potential regulators of cellulase induction in Penicillium janthinellum NCIM 1366

Cellulase production by fungi is tightly regulated in response to environmental cues, and understanding this mechanism is a key pre-requisite in the efforts to improve cellulase secretion. Based on UniProt descriptions of secreted Carbohydrate Active enZymes (CAZymes), 13 proteins of the cellulase hyper-producer Penicillium janthinellum NCIM 1366 (PJ-1366) were annotated as cellulases- 4 cellobiohydrolases (CBH), 7 endoglucanases (EG) and 2 beta glucosidases (BGL). Cellulase, xylanase, BGL and peroxidase activities were higher for cultures grown on a combination of cellulose and wheat bran, while EG was stimulated by disaccharides. Docking studies indicated that the most abundant BGL- Bgl2- has different binding sites for the substrate cellobiose and the product glucose, which helps to alleviate feedback inhibition, probably accounting for the low level of glucose tolerance exhibited. Out of the 758 transcription factors (TFs) differentially expressed on cellulose induction, 13 TFs were identified whose binding site frequencies on the promoter regions of the cellulases positively correlated with their abundance in the secretome. Further, correlation analysis of the transcriptional response of these regulators and TF-binding sites on their promoters indicated that cellulase expression is possibly preceded by up-regulation of 12 TFs and down-regulation of 16 TFs, which cumulatively regulate transcription, translation, nutrient metabolism and stress response.