Cellulolytic Enzyme Activities Research Articles

Utilization of Agricultural Waste for the Cultivation of Pleurotus Mushrooms in Thailand

Thailand is one of the major agricultural production countries in the world. Therefore, large amounts of agricultural waste are generated as by-products of the agroindustry. The wastes are usually discarded or burnt, resulting in environmental pollution. The main goal of this research was to evaluate the use of agricultural waste for mushroom cultivation. A total of 15 mushroom isolates were recovered from mother spawns and basidiocarps. They were screened for their cellulolytic enzyme activity on Czapek agar using carboxymethyl cellulose (CMC) as the sole carbon source. Two isolates of the oyster mushrooms, Pleurotus pulmonarius PP6 and Pleurotus ostreatus PO3, produced the best enzyme activities. To observe the mycelial growth on agricultural waste, the two oyster mushroom candidates were cultured in jars containing five different types of agricultural waste: corn husk, rice straw, coconut meal, coconut husk and sugarcane bagasse, and the jars were incubated at 25°C for six weeks. The results show that both isolates grew best on coconut meal, producing very densely packed mycelia. Meanwhile, corn husk and rice straw were also good sources for oyster mushroom cultivation. This study shows that these three substrates have the potential to be utilized in mushroom cultivation on a commercial scale.

Preliminary study for evaluation of some fungicides against Aspergillus flavus isolated from historical illuminated paper manuscript dated back to the Mamluk period

Various valuable illuminated paper manuscripts in museums and libraries suffer from fungal deterioration. The treatment and protection of illuminated paper manuscripts from fungal damage is an important process. Therefore, the current study aimed to evaluate different fungicides against fungi isolated from historical illuminated paper manuscript dated back to the Mamluk period (1250–1516 AD). Fungi isolated from infected historical illuminated paper manuscripts were identified and their cellulolytic enzyme activity was performed. The identified fungi were A. fumigatus, A. niger, A. flavus, Alternaria alternate, and Trichoderma sp. Isolate (4-1), identified as Aspergillus flavus, possessed the highest cellulase activity. This isolate was tested against the following fungicides: azoxystrobin, boscalid, difenoconazole, dimethomorph, propiconazole, pyraclostrobin, and thiophanate-methyl at concentrations from 1 to 200 ppm, nanoparticles of TiO2 and SiO2 at concentrations of 0.5%, and 1% and mixtures of fungicides [difenoconazole + propiconazole (1:1)] at 100 and 200 ppm and [boscalid + pyraclostrobin (2:1)] at 150 and 300 ppm. The disc diffusion method (DDM) was used to evaluate the efficiency of the prepared individual and mixed fungicides against Aspergillus flavus (4-1) isolate. The results of this study revealed that treatments with individual difenoconazole at 200 ppm exhibited the best antifungal activity, followed by the propiconazole fungicide. Further research is needed to evaluate the in vitro practical application of these fungicides with infected illuminated paper samples with Aspergillus flavus agents in the field.

Secondary Metabolites from Symbiotic Bacteria of Seagrass and Molluscs as A Reference for Natural Food Preservatives

Symbiotic bacteria from seagrass and molluscs have the potential to be a source of secondary metabolites. Compounds from these secondary metabolites, such as natural food preservatives, can be used in the bioindustry sector. This research aims to identify the potential secondary metabolites from symbiotic bacteria in molluscs and seagrass that can support the Sustainable Development Goals (SDG) public health program. The samples used in this study were symbiotic bacteria of mollusc from seagrass environments, symbiotic bacteria from seagrass, and seagrass samples. The seagrass samples were analysed phytochemically, while the symbiotic bacteria samples from molluscs and seagrass were tested for their antibacterial activity against food spoilage pathogens such as Staphylococcus aureus and Escherichia coli using the disc diffusion method. Bacteria that showed potential antibacterial activity were identified using molecular methods, and the content of active compounds was analysed using gas chromatography-mass spectrometry. The phytochemical analysis revealed that seagrass contains flavones, tannins, steroids, and compounds with potential antibacterial properties. GC-MS analysis indicated that seagrass is predominantly composed of unsaturated fatty acids, which have antimicrobial effects by damaging bacterial cell membranes and disrupting the function of enzymes essential for growth and reproduction. Ten symbiotic bacteria isolates demonstrated proteolytic, cellulolytic, and lipolytic enzyme activity, suggesting their potential as antibacterial agents due to their ability to break down cell walls. Molecular identification of selected bacteria revealed the presence of mollusc symbionts, including Vibrio owensii, Bacillus paramycoides, and Pseudoalteromonas flavipulchra. The symbiotic bacteria found in seagrass in this study included Bacillus velezensis, Bacillus paramycoides, Nocardiopsis alba, Nocardiopsis alba, Bacillus tropicus, Lysinibacillus fusiformis, and Bacillus tropicus. GC-MS analysis of selected symbiotic bacteria from seagrass showed that the dominant compound components were phthalic acid, bis(7-methyl octyl) ester (25.18, 43.84, 35.55 %), and trimethoxy-3-piperidyl-2,2'-binaphthalene-1,1',4,4'-tetrone (36.36, 47.71, 64.48 %). In conclusion, seagrass and its symbiotic bacteria, as well as symbiotic bacteria from molluscs living in seagrass, have the potential to serve as antibacterial agents and can be used as references for food preservatives. HIGHLIGHTS Seagrass and mollusc symbiotic bacteria produce secondary metabolites that have antimicrobial properties, making them suitable for food preservation The component that acts as an antibacterial agent is found in seagrass: Essential fatty acids, which can have antibacterial properties Symbiotic bacteria are microorganisms that live in mutualistic symbiosis with other organisms. The enzymatic activities of symbiotic bacteria can involve the production of enzymes that break down complex molecules into simpler molecules In food preservation, symbiotic bacteria, with their enzymatic activity, can be used as natural preservatives Using natural food preservatives derived from seagrass and mollusc symbiotic bacteria can contribute to a more sustainable and environmentally friendly approach to food preservation GRAPHICAL ABSTRACT

Effects of α-amylase and coated α-amylase supplementation on growth performance, nutrient digestion, and rumen fermentation in Holstein bulls.

This study evaluated the impacts of α-amylase (AM) and coated α-amylase (CAM) on bull performance, nutrient digestibility, and ruminal fermentation. This study randomized 60 Holstein bulls of 365 ± 11.5 days of age and 457.5 ± 9.35 kg body weight into three groups: without AM addition, adding AM 0.6 g/kg dry matter (DM), and adding CAM 0.6 g AM/kg DM, separately. This whole experimental period was 80 days, including a 20-day adaptation period and a 60-day data and sample acquisition period. In comparison with the unsupplemented control, dry matter intake (DMI) was unaltered; however, average daily gain (ADG) and feed efficiency (FE) were greater for AM or CAM addition. Bulls receiving AM or CAM supply had greater total-tract nutrient digestibility, ruminal total volatile fatty acids (VFA) content, propionate molar proportion, cellulolytic enzyme and AM activities, and the number of microorganisms. In addition, the activities of AM and trypsin in the jejunum and ileum and glucose, albumin, and total protein concentrations in serum were greater for AM or CAM addition compared to the control. When comparing the supplementation mode of AM, bulls receiving CAM addition had greater ADG and FE. The crude protein and starch digestibility and intestinal AM and trypsin activity were higher, while acid detergent fiber (ADF) digestibility was lower for CAM addition than for AM addition. The lower propionate molar proportion and cellobiase and carboxymethyl cellulase activities, together with Ruminococcus albus, Ruminococcus flavefaciens, and Fibrobacter succinogenes populations were observed for CAM addition compared with AM addition. However, there were greater glucose, albumin, and total protein concentrations in serum after adding CAM. According to the data, the supply of AM improved ADG, nutrient digestion, and rumen fermentation. Notably, the optimum supplementation mode was in the form of CAM in bulls.

Enzymatic Valorization of Lignocellulosic Biomass—The Influence of Deep Eutectic Solvents and Ionic Liquids on the Activity of Cellulolytic Enzymes

In recent years, there has been a steady increase in the interest in changing lignocellulose processing technologies from harmful and nonecological to more ecological and sustainable. Deep eutectic solvents (DESs) and ionic liquids (ILs) are green, efficient solvents/catalysts for biomass pretreatment as well as its further transformation. In many cases, they are used together with biocatalysts. Additionally, in processes where DESs and ILs are used for the pretreatment of biomass, before enzymatic transformation, traces of these solvents can influence the activity of biocatalysts. For this reason, it is important to evaluate the effect of novel, green solvents on the activity of enzymes widely applied in the biomass valorization processes. In this work, we present the impact of chosen DESs and ILs, most often applied in biomass pretreatment and transformation, on the enzymatic activity of popular cellulolytic enzymes and enzymatic preparations (namely, cellulases from Aspergillus niger, Trichoderma reesei, and Viscozyme L). The enzymatic activity was investigated in the process of transformation of cellulose or biomass to glucose. The results showed that many of the solvents tested had a negative effect on enzymatic activity, suggesting a separation of pretreatment and enzymatic processes.

Dairy manure acidogenic fermentation at hyperthermophilic temperature enabled superior activity of thermostable hydrolytic enzymes linked to the genus Caldicoprobacter

In this study, fermentation experiments were conducted under mesophilic, thermophilic, and hyperthermophilic conditions to investigate adaptation of microbial communities and its effect on extracellular enzyme activities toward degradation of cellulose, hemicellulose and proteins in dairy manure. Hyperthermophilic conditions transformed the microbiome structure and stimulated activity of extracellular proteolytic, cellulolytic, and hemicellulolytic enzymes. Specifically, the activities of protease, cellulose 1,4-β-cellobiosidase, and β-glucosidase secreted by hyperthermophilic microbes were higher by 22%, 47% and 49% compared to those produced by mesophilic and thermophilic communities. Enhanced hydrolytic activity of hyperthermophilic microbes enabled improved feedstock solubilization and production of 39% and 22% more soluble COD than mesophilic and thermophilic microbes, respectively. Connections between hydrolytic function and microbial community structure at various temperatures were assessed using the PICRUSt2 computational tool. Genus Caldicoprobacter was identified as the primary candidate responsible for increased production of thermostable endo-1,4-β-glucanase, β-glucosidase and endo-1,4-β-xylanase, and enhanced hydrolytic performance of hyperthermophilic microbial community.

Designing of recombinant hydrolytic enzymes cocktail for effective saccharification of delignified rice straw

Enzymatic saccharification is the most crucial and challenging step in lignocellulosic biorefineries for bioethanol production. The enzymatic saccharification requires a cocktail of hydrolytic enzymes, cellulases and hemicellulases for effective degradation of biomass. However, the complete bioconversion of biomass is governed by the proportion and synergistic action of hydrolytic enzymes present in the cocktail. Thus, this study aims to formulate an enzyme cocktail consisting of recombinant cellulases (bifunctional cellulolytic chimeric enzyme, CtGH1-L1-CtGH5-F194A and cellobiohydrolase, CtCBH5A) and xylanases (endo-1,4-β-xylanase, CtGH11A, and β-xylosidase, BoGH43), all in crude form (unpurified enzyme present in the total cellular proteins) for producing high yield of total reducing sugars using choline chloride:acetic acid pretreated rice-straw (CApRS). D-optimal mixture design approach was used for statistically designing the optimal proportion of crude recombinant enzyme cocktail for hydrolysis of delignified rice straw. The developed enzyme cocktail comprising chimera, CtCBH5A, CtGH11 and BoGH43 in ratio 35.1:41.8:10.0:13.1 resulted in cellulolytic enzyme activity, 5.6 FPU/mL and xylanase activity, 102 U/mL. Enzymatic saccharification of 3% (w/v) delignified CApRS using developed enzyme cocktail at 187 FPU/gCApRS, pH 5.8 and 35 °C, yielded total reducing sugar of 498 mg/gbiomass, glucose yield (410 mg/g) and xylose yield (71.3 mg/g) resulting in 72% saccharification efficiency. Whereas, significantly low total reducing sugar yield of 80 mg/g and 218 mg/g were released from raw RS and partially pretreated RS, respectively, by the developed cocktail under the same hydrolytic conditions, thereby inferring the effectiveness of the enzyme cocktail against CApRS biomass. The developed enzyme cocktail was compared with a commercial enzyme cocktail and prospects its applicability in lignocellulosic biorefineries.

Effect of Blue LED Light on Bioemulsifier Production in Bioreactor by Aureobasidium pullulans LB83 in Solid State Fermentation

This study analyzed the impact of LED light on bioemulsifier production by Aureobasidium pullulans LB83 in solid-state fermentation (SSF) using pre-treated sugarcane bagasse (PSB). The biomass was subjected to alkaline pre-treatment and conducted fermentations in Erlenmeyer flasks containing 2 g of PSB that were immersed in a humectant solution with a cell concentration of 108 cells/mL. The screening involved varying LED light wavelengths (green, red, orange, and blue) over a 7-day period at 28 °C. Notably, under the influence of blue light, the process achieved maximum production, yielding an EI24% of 63.9% and 45.1% for soybean oil and kerosene, respectively. Prolonged exposure to blue light for 11 days at 28 °C resulted in maximum bioemulsifier production (75%) and cellulolytic enzyme activity (3.67 IU g−1 for endoglucanase and 0.41 IU g−1 for exoglucanase) with soybean oil and kerosene. Experiments in a bioreactor, with varying light conditions (dark, white light, and blue LED light), demonstrated that the blue LED bioreactor outperformed others, achieving EI24% values of 55.0% and 45.7% for soybean oil and kerosene, respectively. The scanning electron microscopy (SEM) confirmed yeast growth under these conditions after 9 days. Our findings highlight the significant potential of LED light to enhance bioemulsifier production by A. pullulans LB83 from PSB.

Biomass conversion through optimization of cellulase from Chryseobacterium junjuense Bp17 and their utility in bioethanol production

The search for economically viable and less laborous strategy for bioethanol production has increased globally in this quest bacterial cellulase treated biomass has been promising. Potential bacterium isolated from the forest soil near Bandipur of Karnataka, India exploiting its cellulolytic activity on the CMC agar medium. Molecular identification and characterization of isolate Bp17 revealed as Chryseobacterium junjuense Bp17. An optimization study, enhance the yield of the enzyme through varying parameters such as time, temperature, and pH. Enzymatic assays like β 1, 4 endoglucanase (148.55 U/ml), β 1, 4 exoglucanase (37.21 U/ml), and β glucosidase (42.21 U/ml) exhibit the enzyme activity. Endo, exo and β-glucosidase activities of hydrolyzed biomass broth like coconut husk; 212.88 ± 1.24 U/ml, 151.65 ± 1.24 U/ml and 176.63 ± 1.24 U/ml, rice bran; 223.72 ± 1.42 U/ml, 112.88 ± 1.42 U/ml and 133.43 ± 1.42 U/ml, Pongamia leaves; 389.69 ± 2.54 U/ml, 205.92 ± 2.54 and 187.99 ± 2.54 U/ml, and wood waste; 196.86 ± 0.2 U/ml, 138.90 ± 0.2 U/ml and 168.84 ± 0.2 U/ml, respectively. Hydrolyzed and control biomass differences analyzed through Scanning electron microscope (SEM), Energy dispersive X-ray (EDX), and Fourier transform infrared (FTIR) spectroscopy. Zymography reveals the activity and molecular weight of cellulolytic enzymes (40-130 KB). Hydrolyzed broth filtrate produced from biomass used as crude enzyme and further used for bioethanol production through the yeast fermentation process.

The Impact of Peatland Fires on Cellulolytic Microbial Populations in Several Landuses

Studies on the impact of peatland fires on agricultural land, especially oil palm, and rubber, are still limited. This study aims to calculate the total population of cellulolytic bacteria and determine the activity of cellulolytic enzymes qualitatively in each microbial isolate found and to determine the level of peatland degradation after the fire. The research was conducted by survey in the field. The research location is in the long jungle village of Tambang Subdistrict, Kampar Regency. At the same time, the laboratory analysis was carried out in the Pathology, Entomology, and Microbiology Laboratory, Faculty of Agriculture and Animal Husbandry, Sultan Syarif Kasim Riau University. Determination of sample points using the purposive sampling method, which is to choose a location per the study's objectives at 4 locations. The first location is oil palm land that is not burnt, the second is burning oil palm land, the third is unburned rubber land, and the fourth is burned rubber land. Each location was repeated three times to obtain 12 sample points for observation. Furthermore, at each observation sample point, two soil depths were observed, 0-15 cm and 15-30 cm. The total population of cellulolytic bacteria in oil palm and rubber plantations that were burned and not burned with different soil depths ranged from 5.3 x 109 cfu / g soil to 14.0 x 109 cfu / g soil. The clear zone ratio (Z / K) of 8 cellulolytic bacterial isolates observed was high criteria, namely> 1.76 (100%). The highest Z / K ratio was produced by bacterial isolates originating from burnt rubber plantations at a soil depth of 0-15 cm, namely 5.25.

Sucrose-nonfermenting 1 kinase activates histone acetylase GCN5 to promote cellulase production in Trichoderma.

Trichoderma serves as the primary producer of cellulases and hemicellulases in industrial settings as it readily secretes a variety of cellulolytic enzymes. The protein kinase SNF1 (sucrose-nonfermenting 1) can enable cells to adapt to changes in carbon metabolism by phosphorylating key rate-limiting enzymes involved in the maintenance of energy homeostasis and carbon metabolism within cells. Histone acetylation is an important epigenetic regulatory mechanism that influences physiological and biochemical processes. GCN5 is a representative histone acetylase involved in promoter chromatin remodeling and associated transcriptional activation. Here, the TvSNF1 and TvGCN5 genes were identified in Trichoderma viride Tv-1511, which exhibits promising activity with respect to its ability to produce cellulolytic enzymes for biological transformation. The SNF1-mediated activation of the histone acetyltransferase GCN5 was herein found to promote cellulase production in T. viride Tv-1511 via facilitating changes in histone acetylation. These results demonstrated that cellulolytic enzyme activity and the expression of genes encoding cellulases and transcriptional activators were clearly enhanced in T. viride Tv-1511 mutants in which TvSNF1 and TvGCN5 were overexpressed, with concomitant changes in histone H3 acetylation levels associated with these genes. GCN5 was also found to be directly recruited to promoter regions to alter histone acetylation, while SNF1 functioned upstream as a transcriptional activator that promotes GCN5 upregulation at the mRNA and protein levels in the context of cellulase induction in T. viride Tv-1511. These findings underscore the important role that this SNF1-GCN5 cascade plays in regulating cellulase production in T. viride Tv-1511 by promoting altered histone acetylation, offering a theoretical basis for the optimization of T. viride in the context of industrial cellulolytic enzyme production. KEY POINTS: • SNF1 kinase and GCN5 acetylase promoted cellulase production in Trichoderma by increasing the expression of genes encoding cellulases and transcriptional activators • SNF1 and GCN5 promoted cellulase production by driving H3ac modifications, and GCN5 directly band to the promoter regions to catalyze distinct H3ac modifications • SNF1 acts upstream of GCN5 as a transcriptional activator in the cellulase production of Trichoderma.

Cultural and enzymatic activity studies of a pathogenic wood-decaying fungus Fomitiporia hippophaeicola (Hymenochaetales, Basidiomycota), recollected in the Eastern Caucasus.

Stenotrophic basidiomycete fungus Fomitiporia hippophaeicola, being a wood-decaying pathogen of sea buckthorn (Hippophaë rhamnoides), has been recollected after 48years in the Eastern Caucasus during the mycological and phytopathological investigations in the inner-mountainous part of the Republic of Dagestan, Russia. The identity of the species was confirmed by both morphological and ITS1-5.8S-ITS2 nrDNA data. We introduced and characterized the dikaryotic strain of F. hippophaeicola deposited for permanent storage to the Basidiomycete Culture Collection of the Komarov Botanical Institute RAS (LE-BIN). The morphological features and growth parameters of this xylotrophic fungus with phytopathogenic activity under cultivation on different agarized media (BWA, MEA, PDA) are described for the first time. The LE-BIN 4785 strain of F. hippophaeicola showed differences in growth rate and macromorphology, while the microscopic characteristics remained more robust during growth on the media tested. Qualitative analyses of oxidative and cellulolytic enzyme activities and assessment of the degradation potential of the strain examined in vitro were carried out. As a result, the newly obtained strain of F. hippophaeicola was found to exhibit medium enzyme activities and a moderate capacity to degrade the polyphenol dye azur B.

Residue‐ and nitrogen‐induced carbon mineralization varies with soil fertility status

AbstractBy increasing the input of corn (Zea mays L.) residues, synthetic nitrogen (N) fertilization is often assumed to enhance soil storage of organic carbon (C), which could be especially beneficial for improving the fertility of depleted soils. To ascertain whether such a strategy can be effective, C mineralization was compared for two soils with different indigenous N contents by conducting a 60‐day laboratory incubation experiment that involved continuous monitoring of CO2 emissions with periodic sampling for atmospheric δ13C analysis and for determination of soil microbial biomass and cellulolytic enzyme activities. The addition of exogenous N had a stimulatory effect on cumulative CO2 production that was greater for the low than high N supplying soil and more prominent in the first than in the second month of incubation. During residue decomposition, microbial activities were maximized by incubating the low N soil with exogenous N, whereas cellulolytic enzyme activities were greater for the high N soil. Although intensive N fertilization can substantially increase the productivity of low‐fertility soils, the additional residue inputs thereby generated are more effective for promoting C mineralization than sequestration.

Cellulase Activity Bacillus sp. Which Was Grown on A Medium with Aluminum Salt and Magnetic Field Exposure

Aluminum is the best metal cofactor enzyme. Exposure to the 0,2mT magnetic field on Aluminium salt is said to be able to increase cellulolytic activity. The purpose of the research is to know if the best aluminum salt can increase cellulolytic activity. This can be tested by Cellulolytic Index Test and Enzyme Activity Test. This experiment is descriptive research with three treatments; control, the addition of aluminum salt without an exposed magnetic field, and added aluminum salt exposed to a magnetic field. Salt aluminum used are AlCl3, Al2(PO4)3, and Al2(SO4)3. The concentration was 0.01% and exposed to a magnetic field of 0.2mT. The final results tell that aluminum is an effective cofactor. Exposure to a magnetic field on aluminum salts showed an increase in enzyme activity. The best result is AlCl3 exposed to a 0.2mT magnetic field. This was able to increase the cellulolytic activity by 0.69U/mL. Then Al2(PO4)3 increased enzyme activity by 0.41U/mL and Al2(SO4)3 increased activity by 0.39U/mL.

Intracellular Nitric Oxide and cAMP Are Involved in Cellulolytic Enzyme Production in Neurospora crassa.

Although molecular regulation of cellulolytic enzyme production in filamentous fungi has been actively explored, the underlying signaling processes in fungal cells are still not clearly understood. In this study, the molecular signaling mechanism regulating cellulase production in Neurospora crassa was investigated. We found that the transcription and extracellular cellulolytic activity of four cellulolytic enzymes (cbh1, gh6-2, gh5-1, and gh3-4) increased in Avicel (microcrystalline cellulose) medium. Intracellular nitric oxide (NO) and reactive oxygen species (ROS) detected by fluorescent dyes were observed in larger areas of fungal hyphae grown in Avicel medium compared to those grown in glucose medium. The transcription of the four cellulolytic enzyme genes in fungal hyphae grown in Avicel medium was significantly decreased and increased after NO was intracellularly removed and extracellularly added, respectively. Furthermore, we found that the cyclic AMP (cAMP) level in fungal cells was significantly decreased after intracellular NO removal, and the addition of cAMP could enhance cellulolytic enzyme activity. Taken together, our data suggest that the increase in intracellular NO in response to cellulose in media may have promoted the transcription of cellulolytic enzymes and participated in the elevation of intracellular cAMP, eventually leading to improved extracellular cellulolytic enzyme activity.

Statistics-Based Optimization of Cellulase and Xylanase Production by the Endophytic Fungus Talaromyces Funiculosus using Agricultural Waste Materials

Lignocellulosic biomass can be utilized as a low-cost, renewable, and sustainable feedstock for obtaining non-fossil energy sources with low CO2 emission. One of the most promising technologies for producing 2G biofuels is the saccharification of agricultural waste materials with the help of cellulolytic enzymes, followed by yeast fermentation of sugars into cellulosic ethanol. Cellulases are multi-component enzymes involved in the degradation of cellulose, which can synergistically degrade cellulose and includes three major categories: endoglucanase (EC 3.2.1.4), exoglucanase or cellobiohydrolase (EC 3.2.1.91), and β-glucosidase (EC 3.2.1.21). The core enzyme used for the degradation of the xylan skeleton of hemicellulose is endo-β-1,4-xylanase (EC 3.2.1.8). The high cost of enzymes synthesized by fungi is a bottleneck for the production of cellulosic ethanol. Optimization of the nutrient medium composition is an important factor in increasing the production of enzymes and the efficiency of lignocellulosic biomass hydrolysis. The aim of the current study was to optimize the production of cellulolytic and xylanolytic enzymes through cultivation of filamentous fungus Talaromyces funiculosus on low-cost nutrient media with non-pretreated agricultural waste materials. Methods. Filamentous fungus Talaromyces funiculosus was grown on potato-dextrose agar for 10—14 days at 26±2 °С. To obtain the culture filtrate, the fungus was cultivated under submerged conditions in an Erlenmeyer flask for 4 days. The nutrient medium composition was varied according to the factor experiment design. A two-step optimization of the nutrient medium composition was used. A screening experiment with the Plackett-Burman fractional factorial design and response surface methodology with the Box-Behnken design were used to optimize cellulase production. The enzymatic activity was determined by measuring the reduced sugar production after the enzymes hydrolysis with specific substrates: exoglucanase with filter paper, endoglucanase with carboxymethylcellulose, and xylanase with beech wood xylan, using the colorimetric DNS method with glucose or xylose as a standard. The activity of β-glucosidase was determined by the hydrolysis reaction of p-nitrophenyl-β-D-glucopyranoside, which results in the formation of p-nitrophenol, quantified at 410 nm. Results. As a result of experiments with using agricultural waste, including wheat straw, corn stalk, and corn cob as carbon sources of the culture medium, it was shown that T. funiculosus is able to grow and produce cellulase and xylanase on all non-pretreated substrates studied. The two-step sequential optimization of the nutrient medium composition for T. funiculosus cultivation according to the Plackett-Berman and Box-Behnken designs made it possible to increase the activity of cellulolytic and xylanolytic enzymes by 2.4—2.6 times. The optimized cultivation medium does not contain such expensive components as Avicel, peptone, and yeast extract and has the following composition, g/L: corn stalks — 50.0; urea — 0.86; NaNO3 — 1.0; KH2PO4 — 6.0; KCl — 0.25; MgSO4 — 0.25; FeSO4 — 0.01. Conclusions. The studied strain of T. funiculosus produces a lignocellulosic enzyme complex with a high level of β-glucosidase activity when cultivated on an optimized nutrient medium with untreated agricultural waste and is promising for the conversion of lignocellulosic biomass into fermentable sugars.

Characterization of Lignin Peroxidase from Cryptococcus neoformans for Bioremediation of Pulp and Paper Mill Effluent

Eight fungal strains (F1–F8) isolate from the sediment core and degraded wood of the Pulp and Paper Mill effluent and Phanerochaete chrysosporium are tested for different cellulolytic (CMcase, FPase, Xylanases) and lignolytic (lignin peroxidase) enzyme activity in pulp. F-3 (Cryptococcus neoformans) strain shows higher enzyme activity compare to other fungi isolated from effluent. Weight loss, cellulose loss, and organic carbon contents are found maximum in F-3 (C. neoformans) strain. Reducing sugar, protein content, and color removing potential are also higher in C. neoformans. The culture extract of F-3 strain grows in presence of pulp fractionated into five protein fractions has indicated two fractions of molecular weight, 38,000 and 40,000 Da, having lignin peroxidase activity. Lignin peroxidase has optimum activity at pH 2–3 and temperature 20–30 °C, maximum activity at 6–48 mM veratryl alcohol concentration and 256 mM H2O2, however, sodium azide inhibits the enzyme activity. Different metals (CoCl2, HgSO4, CaCl2, SnCl2, FeSO4, CuSO4, and ZnSO4) also affect the lignin peroxidase activity. Characterization study is performed for significant analysis.

The Mixture of Saccharomyces cerevisiae and Clostridium butyricum Could Promote Rumen Fermentation and Improve the Growth Performance of Goats in Hot Summer.

This study aimed to investigate the effects of multiple mixing ratio pairs of Saccharomyces cerevisiae (SC) and Clostridium butyricum (CB) on rumen fermentation and growth performance of goats in hot summer. Thirty goats were divided into five groups: 0.00% probiotics (control), 0.30% SC and 0.05% CB (P1), 0.30% SC and 0.10% CB (P2), 0.60% SC and 0.05% CB (P3), and 0.60% SC and 0.10% CB (P4) of the dry matter (DM) weight of the basal diet and were assigned to a 5 × 5 Latin square experimental design. The results showed the pH values, the activities of ruminal cellulolytic enzymes, and the concentrations of ammonia nitrogen, acetic acid, propionic acid, total volatile fatty acids, vitamins B1 and B2, and niacin were significantly increased (p < 0.05) by probiotics. Moreover, the DM intake, average daily gain, the digestibilities of DM, neutral detergent fiber, and acid detergent fiber were significantly increased (p < 0.05) in probiotic-supplemented groups. Additionally, among all probiotic supplementation groups, the P3 group had the most beneficial effect on rumen fermentation parameters and the growth performance of goats. These results suggested that the mixture of 0.60% Saccharomyces cerevisiae and 0.05% Clostridium butyricum of the DM concentration was beneficial to improve rumen fermentation and promote the growth of goats in hot summer.

Inhibition of cellulase activity by liquid hydrolysates from hydrothermally pretreated soybean straw

The one-pot biomass conversion process is a promising strategy to minimize potential product loss and reduce processing costs. However, this strategy has technical limitations due to the inhibitory effects of biomass components like lignin as well as the generated inhibitors (e.g., furans, phenols) during biomass processing. In this study, the inhibitory effects of liquid hydrolysates formed by hydrothermal pretreatment of soybean straw with either sodium hydroxide (NaOH) or hydrogen peroxide (H2O2) on cellulolytic enzyme activity were investigated. Hydrothermal pretreatment of soybean straw (10% w/v) was carried out with either sodium hydroxide (1% v/v) or hydrogen peroxide (1% v/v) at 121°C for 60 min to evaluate the effect of water-soluble inhibitors released from soybean pretreatment on cellulolytic enzyme activity. The fraction of cellulose in pretreated solids (1% w/v glucan) was enzymatically hydrolyzed for 72 h with 45 IU/g glucan (corresponding to 25 mg enzyme protein/g glucan) in the presence of either buffer or liquid hydrolysate generated from the pretreatments. Hydrolysis of NaOH and H2O2 pretreated solids resulted in 57% and 39% of glucose yields in buffer, respectively. In the presence of the liquid hydrolysates, NaOH and H2O2 pretreated biomass showed 20% and 30% glucose yield, respectively, indicating the enzyme suppression by inhibitors in the liquid hydrolysates. Of the enzyme activities in hydrolysates tested, NaOH hydrolysate showed a higher inhibitory effect on enzyme activities (mainly β-glucosidase) compared to H2O2 liquid, where enzyme deactivation has a first-order correlation and the manner in which the vacuum-filtered inhibitors were generated from pretreated soybean straw.

Prophylactic Feeding of Clostridium butyricum and Saccharomyces cerevisiae Were Advantageous in Resisting the Adverse Effects of Heat Stress on Rumen Fermentation and Growth Performance in Goats.

Simple SummaryHeat stress occurs when goats are exposed to high environmental temperatures and humidity for a long period. Heat stress could adversely affect the rumen fermentation and growth performance of goats. Dietary supplementation with Clostridium butyricum, Saccharomyces cerevisiae, and their mixture were effective ways to alleviate the effects on the rumen fermentation and growth performance of heat-stressed goats. In this study, these two probiotics and their mixture were supplemented in goats for a period before heat stress. The results showed that these probiotics effectively alleviate the adverse effects of heat stress by promoting rumen fermentation and growth performance. Therefore, this study provides a reference for applying these two probiotics at the optimum timing to alleviate the adverse effects of heat stress on goats.This study aimed to investigate the effect of the prophylactic feeding of Clostridium butyricum (CB), Saccharomyces cerevisiae (SC), and their mixture before the onset of heat stress on the rumen fermentation and growth performance of goats, and subsequently, on heat stress status. Forty-eight male Macheng Black × Boer crossed goats (22.25 ± 4.26 kg) were divided into four groups—the control group (fed the basal diet), and the CB (0.05% CB added to the basal diet), SC (0.60% SC added to the basal diet), and Mix (0.05% CB and 0.60% SC added to the basal diet) groups—and fed for fourteen days. Then, these goats were kept in a heat stress environment (with a temperature–humidity index of 87.04) for fourteen days. Then, the parameters of rumen fermentation and growth performance were measured. The results showed that the pH values, the activities of cellulolytic enzymes (avicelase, CMCaes, cellobiase, and xylanase), and the concentrations of ammonia-N, total volatile fatty acid, acetic acid, propionic acid, and butyric acid were significantly increased (p < 0.05) in the rumens of the CB, SC, and Mix groups compared to those of the control group. Moreover, the average daily gain and the digestibility of dry matter, neutral detergent fiber, and acid detergent fiber were significantly increased (p < 0.05) in the CB, SC, and Mix groups compared to those of the control group. These results suggest that these two probiotics and their mixture effectively alleviate the adverse effects of heat stress on rumen fermentation and growth performance via prophylactic feeding.