Carboxymethylcellulase Activity Research Articles

Effects of yak rumen anaerobic fungus Orpinomyces sp. YF3 fermented on in vitro wheat straw fermentation and microbial communities in dairy goat rumen fluid, with and without fungal flora.

Rumen fungi play an essential role in the breakdown of dietary fibrous components, facilitating the provision of nutrients and energy to the host animals. This study investigated the fermentation characteristics and effects on rumen microbiota of yak rumen anaerobic fungus Orpinomyces sp. YF3 in goat rumen fluid, both with and without fungal flora, utilizing anaerobic fermentation bottles. Crushed and air-dried wheat straw served as the fermentation substrate, and cycloheximide was used to eradicate microorganisms from the rumen fluid of dairy goats. The experiment compromised four treatment groups (2×2 factorial design): control (C); yak fungus group (CF, Orpinomyces sp. YF3); goat fungi eliminated group (CA, antibiotic: 0.25 mg/mL cycloheximide); goat fungi eliminated+yak fungus group (CAF). Each treatment had six replicates. Fermentation characteristics and microbial composition of the fermentation media were analyzed using one-way analysis of variance and high-throughput sequencing technology. The findings revealed that in the Orpinomyces sp. YF3 addition group (CF and CAF groups), there were significant increases in ammonia nitrogen concentration by 70%, total volatile fatty acids (VFA) by 53%, as well as acetate, isobutyrate, and valerate concentrations, and the ratio of acetate to propionate (p < 0.05), while the propionate proportion declined by 13%, alongside a reduction of butyrate concentration (p < 0.05). Similarly, in the CF and CAF groups, there were a notable increase in the relative abundance of Bacteroidota, Synergistota, Desulfobacterota, Actinobacteria, and Fusobacteriota, alongside a decrease in the relative abundance of Fibrobacterota and Proteobacteria (p < 0.05). Bacteria exhibiting increased relative abundance were positively correlated with the activity of carboxymethyl cellulase and avicelase, total VFA concentration, and acetate proportion, while showing a negatively correlation with propionate proportion. In conclusion, supplementing rumen fermentation media with yak rumen anaerobic fungus Orpinomyces sp. YF3 led to an increase in bacteria associated with fibre degradation and acetic acid production, a decrease in propionate-producing bacteria, enhanced the activity of plant cell wall degrading enzymes, and promoted cellulose degradation, ultimately elevating total VAF concentration and acetate proportion. This presents a novel approach to enhance roughage utilization in ruminants.

Isobutyramide and Slow-Release Urea as Substitutes for Soybean Meal in the Finishing Diet of Beef Cattle.

Two experiments were conducted to investigate the effects of isobutyramide (IBA) and slow-release urea (SRU) as substitutes for soybean meal (SBM) in the finishing diet of beef cattle. The completely randomized design in vitro experiment with five treatments, i.e., control, 0.9% SRU group, 0.6% SRU + 0.3% IBA group (SRU-I), 0.3% SRU + 0.6% IBA group (IBA-S), 0.9% IBA group was conducted. The results showed that the IBA-S and IBA increased (p ≤ 0.05) substrate disappearance of dry matter (DM), neutral detergent fiber (NDF), acid detergent fiber (ADF), total gas, and total volatile fatty acids (TVFA). The SRU group had the highest (p < 0.01) crude protein disappearance and ammonia nitrogen concentration, but the IBA contrarily decreased (p < 0.01) them compared with the control. Inclusion of IBA increased isobutyrate concentrations (p = 0.01) with the highest value for the IBA group. Then, an 84-day replicate 4 × 4 Latin square design with 8 Angus steers and four treatments, i.e., control, SRU, SRU-I, IBA-S was performed. The results showed that the treatments did not affect DM intake (p > 0.05) but tended (p = 0.09) to increase average daily gain. The inclusion of IBA increased (p < 0.05) the apparent digestibility of DM, organic matter, NDF, ADF, TVFA, and microbial crude protein with the highest values for the IBA-S group. The IBA-contained groups also increased (p ≤ 0.01) isobutyrate concentration, activities of carboxymethyl cellulase and xylanase, and the relative abundance of Butyrivibrio fibrisolvens with the highest values for the IBA-S group. The SRU had no effect on animal growth and nutrient apparent digestibility. In conclusion, IBA was developed as a new substitute for SBM in the finishing diet of beef cattle, and the optimal strategy was the isonitrogenous substitution of SBM with 0.3% SRU and 0.6% IBA of the diet.

Whole-plant silage maize to improve fiber digestive characteristics and intestinal microbiota of Hezuo pigs.

Utilizing roughage resources is an effective approach to alleviate the shortage of corn-soybean feed and reducing the costs in the swine industry. Hezuo pig is one group of plateau type local Tibetan pig with strong tolerance to crude feeding. Nevertheless, current research on the roughage tolerance in Hezuo pigs and the microbiological mechanisms behind it is still minimally.This study explored the impact of various ratios of whole-plant silage (WPS) maize on the pH, cellulase activity, short-chain fatty acids (SCFAs), and intestinal microbiota in Hezuo pigs. Thirty-two Hezuo pigs were randomly divided into four groups (n = 8). The control group received a basal diet, while experimental groups I, II, and III were given diets with incremental additions of 5%, 10%, and 15% air-dried WPS maize, respectively, for 120 days. The findings revealed that compared with the control group, in Group II, the pH of cecum and colon were notably decreased (p < 0.05), while acid detergent fiberdigestibility, the concentration of propionic and isobutyric acid in the cecum, and the concentration of isobutyric acid in the colon were significantly increased (p < 0.05). Also, carboxymethyl cellulase activity in the cecum in group II of Hezuo pigs was significantly higher than that in the other three groups (p < 0.05). Furthermore, the cecum microbiota showed a higher diversity in the group II of Hezuo pigs than that in the control group, as shown by the Simpson and Shannon indices. Specifically, 15 and 24 bacterial species showed a significant difference in relative abundance at the family and genus levels, respectively. Correlation analyses revealed significant associations between bacterial genera and SCFAs concentrations in the cecum. The abundance of Bacteroides and NK4A214_group was positively correlated with amounts of valeric and isovaleric acid but negatively with propionic acid (p < 0.05). The abundance of UCG-010 was positively linked with acetic acid and negatively correlated with butyric acid (p < 0.05). Actinobacillus abundance was positively associated with butyric acid levels (p < 0.05). In conclusion, a 10% WPS maize diet improved crude fiber digestibility by lowering cecal and colonic chyme pH, enhancing intestinal cellulase activity, improving SCFA production, and increasing intestinal microbiota diversity.

Ficus hookeri tree leaves as herbal feed additives to enhance ruminal fermentation and reduced protozoal population in growing crossbred cattle

An experiment was conducted to evaluate the effect of dietary supplementation of Ficus hookeri leaf meal as herbal feed additive on rumen protozoal population, enzyme profile and fermentation characteristics in growing crossbred calves. Ten growing male crossbred (Jersey × Tharparker) calves with similar initial body weight of 91.8+1.37 kg, were randomly divided into two equal groups (T1 and T2) and fed individually under stall feeding for 140 days on a mixed ration containing rice straw and concentrate mixture in 50:50 ratio. Two types (C1 and C2) of iso-nitrogenous concentrate mixtures were prepared. Wheat bran in concentrate mixture (C2) of test group (T2) was partially replaced (4 parts w/w) with Ficus hookeri leaf meal. Daily dry matter intake between two experimental groups was similar. Rumen pH, NH3-N concentration and total rumen protozoal population decreased while ruminal TVFA and propionic acid production increased due to dietary supplementation of Ficus hookeri leaf meal as herbal feed additive. Activity of carboxymethyl cellulase, xylanase and b-glucosidase enzymes were significantly higher in the rumen liquor of calves fed Ficus hookeri leaf meal. It could be concluded that dietary supplementation of Ficus hookeri leaf meals as herbal feed additive have a potential for reducing rumen protozoal population and ammonia nitrogen concentration with improving ruminal TVFA and propionate production in growing crossbred calves.

Construction of cellulose-degrading microbial consortium and evaluation of their ability to degrade spent mushroom substrate.

Spent mushroom substrate (SMS) is a solid waste in agricultural production that contains abundant lignocellulosic fibers. The indiscriminate disposal of SMS will lead to significant resource waste and pollution of the surrounding environment.The isolation and screening of microorganisms with high cellulase degradation capacity is the key to improving SMS utilization. The cellulose-degrading microbial consortiums were constructed through antagonism and enzyme activity test. The effect of microbial consortiums on lignocellulose degradation was systematically evaluated by SMS liquid fermentation experiments. In this study, four strains of cellulose-degrading bacteria were screened, and F16, F, and F7 were identified as B. amyloliquefaciens, PX1 identified as B. velezensis. At the same time, two groups of cellulose efficient degrading microbial consortiums (PX1 + F7 and F16 + F) were successfully constructed. When SMS was used as the sole carbon source, their carboxymethyl cellulase (CMCase) activities were 225.16 and 156.63 U/mL, respectively, and the filter paper enzyme (FPase) activities were 1.91 and 1.64 U/mL, respectively. PX1 + F7 had the highest degradation rate of hemicellulose and lignin, reaching 52.96% and 52.13%, respectively, and the degradation rate of F16 + F was as high as 56.30%. Field emission scanning electron microscopy (FESEM) analysis showed that the surface microstructure of SMS changed significantly after microbial consortiums treatment, and the change of absorption peak in Fourier transform infrared spectroscopy (FTIR) and the increase of crystallinity in X-ray diffraction (XRD) confirmed that the microbial consortiums had an actual degradation effect on SMS. The results showed that PX1 + F7 and F16 + F could effectively secrete cellulase and degrade cellulose, which had practical significance for the degradation of SMS. In this study, the constructed PX1 + F7 and F16 + F strains can effectively secrete cellulase and degrade cellulose, which holds practical significance in the degradation of SMS. The results can provide technical support for treating high-cellulose solid waste and for the comprehensive utilization of biomass resources.

Fungal cellulolytic enzyme complex immobilized on chitosan-functionalised magnetic nanoparticles for paddy straw saccharification

Utilization of biopolymeric functionalized nanocomposites for enzyme immobilization has been actively explored for their potential role as catalysts in sustainable agro-residue valorization models. In the present study, cellulase immobilization on control and chitosan functionalized magnetic nanoparticles (c-MNP & Ch-MNP, respectively) was investigated. A cellulase binding efficiency of 67.50% on c-MNP and 74.06% on Ch-MNP was achieved. The enzyme immobilization on nano-supports was confirmed using electron microscopic and spectroscopic techniques. The immobilized cellulase (E-Ch-MNP) exhibited carboxymethyl cellulase, cellobiase and avicelase specific activities of 48.68, 25.98 and 33.80 nmol min−1 mg−1 protein. The optimum pH for free and immobilized enzyme were reported to be pH 5 and 6, respectively. An increased thermal stability for E-Ch-MNP was observed over a broader temperature range with a peak relative enzyme activity at 60℃. The binding constant (Km= 5.00 mg ml−1) and maximal rate of reaction (Vmax= 0.12 µmol min−1 ml−1) were also determined for E-Ch-MNP. Molecular docking studies determined the flexibilities of both the receptor cellulase protein and the ligand immobilization support molecules with respect to the binding energy and inhibition constants, reflecting their desired conformation and stability. Maximum reducing sugar content of 242.14 mg g−1 of pre-treated straw was reported at 96 h of incubation for E-Ch-MNP with a saccharification efficiency of 35.78%. The retained relative activity of immobilized cellulase was 82.83, 75.17, 44.61 and 27.95% of initial activity after second, third, fourth and fifth cycle, respectively. Therefore, immobilization of cellulase on chitosan coated magnetic nanoparticles holds promise for further optimization for improved cellulose hydrolysis.

Methane production and lignocellulosic degradation of wastes from rice, corn and sugarcane by natural anaerobic fungi-methanogens co-culture.

Biomass from agriculture, forestry, and urban wastes is a potential renewable organic resource for energy generation. Many investigations have demonstrated that anaerobic fungi and methanogens could be co-cultured to degrade lignocellulose for methane generation. Thus, this study aimed to evaluate the effect of natural anaerobic fungi-methanogens co-culture on the methane production and lignocellulosic degradation of wastes from rice, corn and sugarcane. Hu sheep rumen digesta was used to develop a natural anaerobic fungi-methanogen co-culture. The substrates were rice straw (RS), rich husk (RH), corn stover (CS), corn cobs (CC), and sugarcane baggage (SB). Production of total gas and methane, metabolization rate of reducing sugar, glucose, and xylose, digestibility of hemicellulose and cellulose, activity of carboxymethylcellulase and xylanase, and concentrations of total acid and acetate were highest (P < 0.05) in CC, moderate (P < 0.05) in RS and CS, and lowest (P < 0.05) in SB and RH. The pH, lactate and ethanol were lowest (P < 0.05) in CC, moderate (P < 0.05) in RS and CS, and lowest (P < 0.05) SB and RH. Formate was lowest (P < 0.05) in CC, RS and CS, moderate (P < 0.05) in SB, and lowest (P < 0.05) in RH. Therefore, this study indicated that the potential of methane production and lignocellulosic degradation by natural anaerobic fungi-methanogens co-culture were highest in CC, moderate in RS and CS, and lowest in SB and RH.

Rice straw hydrolysis using in-situ produced enzymes: Feedstock influences fungal enzyme composition and hydrolytic efficiency

Trichoderma reesei RUT-C30 was cultivated on differentially pretreated rice straw and pure cellulose as a carbon source/inducer for cellulase production, and the enzymes were evaluated for hydrolysis of sequential acid and alkali pretreated rice straw. Growth on pretreated rice straw enhanced protein secretion and cellulase activities compared to pure cellulose as a carbon source. The yield of cellulolytic enzymes was higher for alkali pretreated rice straw (ALP-RS), while H2O2-treated (HP-RS) could not induce cellulases to a larger level compared to pure cellulose. Protein concentration was 3.5-fold higher on ALP-RS as compared to pure cellulose, with a maximum filter-paper cellulase (FPase) activity of 1.76 IU/ml and carboxy-methyl cellulase (CMCase) activity of 40.16 IU/ml (2.18 fold higher). Beta-glucosidase (BGL) activity was more or less the same with the different substrates and supplementation of heterologous BGL could result in a quantum jump in hydrolytic efficiencies, which in the case of ALP-RS induced enzymes was 34% (increased from 69.26% to 92.51%). The use of lignocellulosic biomass (LCB) itself as a substrate for the production of cellulase is advantageous not only in terms of raw material costs but also for obtaining a more suitable enzyme profile for biomass hydrolysis.

Regulation of cellulase production via calcium signaling in Trichoderma reesei under PEG8000 stress

In this study, the effect of polyethylene glycol 8000 (PEG8000) stress on cellulase biosynthesis in Trichoderma reesei CICC2626 via calcium signaling was investigated, and a plausible mechanism by which intracellular Ca2+ regulates the transcription of cellulase genes was proposed. The results indicated that the total cellulase (filter paper-hydrolyzing activity [FPase]), endoglucanase (carboxymethyl cellulase activity [CMCase]), and β-glucosidase activities of the strain were 1.3-, 1.2-, and 1.3-fold higher than those of the control (no PEG8000 addition) at a final concentration of 1.5% (w/v) PEG8000. Moreover, the transcriptional levels of cellulase genes, protein concentrations, and biomass increased. With the synergistic use of commercial cellulase and T. reesei CICC2626 cellulase to hydrolyze alkali-pretreated rice straw, the released reducing sugar concentration reached 372.7 mg/g, and the cellulose content (22.7%, 0.32 g) was significantly lower than the initial content (62.5%, 1.88 g). Transcriptome data showed that 12 lignocellulose degradation–related genes were significantly upregulated in the presence of 1.5% PEG8000. Furthermore, the addition of Ca2+ inhibitors and deletion of crz1 (calcineurin-responsive zinc finger 1-encoding gene, which is related to the calcium signaling pathway) demonstrated that calcium signaling plays a dominant role in PEG8000-induced cellulase genes overexpression. These results revealed a link between PEG8000 induction and calcium signaling transduction in T. reesei CICC2626. Moreover, this study also provides a novel inducer for enhanced cellulase production.Key points• Cellulase biosynthesis in Trichoderma reesei could be enhanced by PEG8000• PEG8000 could induce a cytosolic Ca2+ burst in Trichoderma reesei• The activated calcium signaling was involved in cellulase biosynthesisGraphical 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.

Effect of dietary supplementation with zinc-methionine on ruminal enzyme activities, fermentation characteristics, methane production, and nutrient digestibility: An in vitro study.

The principal objective of this research was to examine the influence of zinc-methionine (Zn-Met) addition on feed on the in vitro ruminal enzyme activities, fermentation characteristics, methane production, and digestibilities of feed nutrients. The dosage of Zn-Met as a source of organic Zn was added to feed based on dry matter (DM) as follows: 0-without Zn addition (CON), 30 Zn mg/kg-low (LZM), 60 Zn mg/kg-medium (MZM), and 90 Zn mg/kg-high (HZM). The results indicated significant impacts of Zn-Met addition on various parameters. Compared to the CON group, all Zn-Met treatments resulted in increased total volatile fatty acids (VFA) (linear; p < 0.001), carboxymethyl-cellulase activity (linear; p < 0.001), and gas production at 48-h of incubation (linear; p < 0.001, quadratic; p < 0.001). Additionally, the MZM and HZM groups exhibited higher levels of amylase activity (linear; p < 0.001), protease activity (linear; p = 0.006), microbial protein (linear; p = 0.009), DM digestibility (linear; p < 0.001), organic matter (OM) digestibility (linear; p < 0.001), crude protein (CP) digestibility (linear; p = 0.004), and crude fiber (CF) digestibility (linear; p = 0.003) than CON treatment. However, the treatments did not have any noteworthy effects on pH, the individual VFA (acetate, propionate, and butyrate) proportions, NH3-N concentration, and methane production (p > 0.05). It could be summarized that supplementing 60 and 90 Zn mg/kg DM as Zn-Met could improve the in vitro ruminal enzyme activities, fermentation characteristics, and nutrient digestibility without affecting methane production.

Mixed fermentation of navel orange peel by Trichoderma viride and Aspergillus niger: Effects on the structural and functional properties of soluble dietary fiber

Navel orange peel is a potential source of soluble dietary fiber. In order to make full use of this resource and improve its physiological activities, the effects of single and mixed strains fermentation modifications were compared in this study. The structural, physicochemical, and functional properties of soluble dietary fiber fermented by Trichoderma viride and/or Aspergillus niger were investigated. The results showed that mixed fermentation resulted in significantly higher activity of complex cellulase than single fermentation. On the second day, when T. viride and A. niger were inoculated at a ratio of 3:1, the activities of carboxymethyl cellulase, β-glucosidase, and filter paper lyase were 1.54, 1.61, and 1.30 times those of single fermentation. Mixed fermentation increased the yield of soluble dietary fiber from 6.63% to 15.73%. Scanning electron microscopy and Fourier transform infrared spectroscopic analyses suggested that soluble dietary fiber produced by mixed fermentation had smaller particle size, lower crystallinity, and more disrupted structure. Moreover, the functional properties of soluble dietary fiber were remarkably improved. After mixed fermentation, the glucose and cholesterol adsorption amounts, and α-amylase inhibition activity increased by 1.51, 1.63, and 1.76 times, respectively. In summary, mixed fermentation was an efficient and environmentally friendly modification method. Modified SDF can be utilized in the food processing industry, expanding the range of by-product applications.

Enzyme production mechanism of anaerobic fungus Orpinomyces sp. YF3 in yak rumen induced by different carbon source

In order to investigate the enzyme production mechanism of yak rumen-derived anaerobic fungus Orpinomyces sp. YF3 under the induction of different carbon sources, anaerobic culture tubes were used for in vitro fermentation. 8 g/L of glucose (Glu), filter paper (Flp) and avicel (Avi) were respectively added to 10 mL of basic culture medium as the sole carbon source. The activity of fiber-degrading enzyme and the concentration of volatile fatty acid in the fermentation liquid were detected, and the enzyme producing mechanism of Orpinomyces sp. YF3 was explored by transcriptomics. It was found that, in glucose-induced fermentation solution, the activities of carboxymethyl cellulase, microcrystalline cellulase, filter paper enzyme, xylanase and the proportion of acetate were significantly increased (P < 0.05), the proportion of propionate, butyrate, isobutyrate were significantly decreased (P < 0.05). The results of transcriptome analysis showed that there were 5 949 differentially expressed genes (DEGs) between the Glu group and the Flp group, 10 970 DEGs between the Glu group and the Avi group, and 6 057 DEGs between the Flp group and the Avi group. It was found that the DEGs associated with fiber degrading enzymes were significantly up-regulated in the Glu group. Gene ontology (GO) function enrichment analysis identified that DEGs were mainly associated with the xylan catabolic process, hemicellulose metabolic process, β-glucan metabolic process, cellulase activity, endo-1,4-β-xylanase activity, cell wall polysaccharide metabolic process, carbohydrate catabolic process, glucan catabolic process and carbohydrate metabolic process. Moreover, the differentially expressed pathways associated with fiber degrading enzymes enriched by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were mainly starch and sucrose metabolic pathways and other glycan degradation pathways. In conclusion, Orpinomyces sp. YF3 with glucose as carbon source substrate significantly increased the activity of cellulose degrading enzyme and the proportion of acetate, decreased the proportion of propionate, butyrate and isobutyrate. Furthermore, the degradation ability and energy utilization efficiency of fungus in the presence of glucose were improved by means of regulating the expression of cellulose degrading enzyme gene and participating in starch and sucrose metabolism pathway, and other glycan degradation pathways, which provides a theoretical basis for the application of Orpinomyces sp. YF3 in practical production and facilitates the application of Orpinomyces sp. YF3 in the future.

Feed intake, microbial adherence and fibrolytic activity in residues of forage samples incubated in the rumen of sheep fed grass forages and/or a total mixed ration

Three male sheep were fed, throughout three experimental periods, with either only forage, only total mixed ration (TMR) or a mixed diet (TMR + forage). The rich-fibre ingredients of each diet were incubated daily in situ for three days and the ruminal pH was measured every 2 h during the last day of each experimental period. Rumen pH decreased at increased proportion of TMR in diet (P &lt; 0.05). The dry matter (DM) degradability of the grass forage was higher (P &lt; 0.05) in animals receiving only forage than in those receiving the mixed diet whereas the DM degradability of the corn silage was higher (P &lt; 0.05) in animals receiving the mixed diet than in those receiving only TMR. The level of microbial adherence in residues of grass forage was higher (P &lt; 0.05) in animals fed with only forage than in those fed with the mixed diet and, the level of microbial adherence in residue of corn silage was higher (P &lt; 0.05) in animals receiving the mixed diet than in those receiving TMR. The carboxymethylcellulase activity in residues of grass forage was higher (P &lt; 0.05) in sheep fed the mixed diet whereas not significant effect of diet type was observed for this variable in residues of corn silage. In conclusion, increased inclusion of TMR in sheep diet showed a negative impact on microbial adherence and forage degradability in situ, an effect mediated by changes in rumen pH which was not compensated by increased fibrolytic activity.

Dietary supplementation of sodium butyrate enhances lactation performance by promoting nutrient digestion and mammary gland development in dairy cows

This experiment was to evaluate the influence of sodium butyrate (SB) addition on milk production, ruminal fermentation, nutrient digestion, and the development and metabolism regulation of the mammary gland in dairy cows. Forty Holstein dairy cows averaging 710 ± 18.5 kg body weight, 72.8 ± 3.66 d in milk (DIM), and 41.4 ± 1.42 kg/d milk production were divided into four treatments blocked by DIM and milk production. Treatments were control group, low SB, medium SB, and high SB with 0, 100, 200 and 300 g/d of SB addition per cow, respectively. The study lasted for 105 d. Production of milk, milk protein and lactose quadratically increased (P < 0.05), while fat-corrected milk, energy-corrected milk and milk fat yields linearly increased (P < 0.05) with increasing SB addition. The digestibility of dietary dry matter, organic matter, and crude protein linearly increased (P < 0.05), whereas the digestibility of ether extract, neutral detergent fibre, and acid detergent fibre quadratically increased (P < 0.05). Ruminal pH quadratically decreased (P = 0.04), while total volatile fatty acids (VFA) quadratically increased (P = 0.03) with increasing SB addition. The acetic acid to propionic acid ratio increased (P = 0.03) linearly due to the unaltered acetic acid molar percentage and a linear decrease in propionic acid molar percentage. Ruminal enzymatic activity of carboxymethyl-cellulase and α-amylase, populations of total bacteria, total anaerobic fungi, total protozoa, Ruminococcus albus, R. flavefaciens, Butyrivibrio fibrisolvens, Fibrobacter succinogenes, and Ruminobacter amylophilus linearly increased (P < 0.05). Blood glucose, urea nitrogen, and non-esterified fatty acids linearly decreased (P < 0.05), while total protein concentration linearly increased (P = 0.04). Moreover, the addition of SB at 200 g/d promoted (P < 0.05) mRNA and protein expression of PPARγ, SREBF1, ACACA, FASN, SCD, CCNA2, CCND1, PCNA, Bcl-2, GPR41, and the ratios of p-Akt/Akt and p-mTOR/mTOR, but decreased (P < 0.05) mRNA and protein expressions of Bax, caspase-3, and caspase-9. The results suggest that milk production and milk fat synthesis increased with SB addition by stimulating rumen fermentation, nutrient digestion, gene and protein expressions concerned with milk fat synthesis and mammary gland development.

Functionalized iron oxide nanoparticles for covalent immobilization of cellic CTec2 cellulase: enabling enzyme reusability in cellulosic biomass conversion

A modified chemical co-precipitation method was employed to synthesize silicacoated-aminefunctionalized iron oxide nanoparticle (IONP@SiO2-NH2) which has been utilized as an anchoring surface for enzyme immobilization. Cellulase Cellic CTec2 cocktail was immobilized covalently onto IONP@SiO2-NH2 using glutaraldehyde as a linker. The morphological, structural, and compositional studies of nanoparticles were characterized by field emission scanning electron microscopy with energy dispersive X-ray (FESEM)-EDX, Fourier transform infrared (FTIR), X-ray diffraction (XRD), zeta potential, and Ultraviolet/visible (UV-Vis) spectroscopy. The FTIR, FESEM, and XRD spectra confirmed the successful immobilization of cellulase onto IONP@SiO2-NH2. The free enzymewith cellulase activity of 127 FPU/mL was optimized for the immobilization process which showed the best binding efficiency at 2% glutaraldehyde, protein loading 100 mg/g with carboxymethyl cellulase activity of 290 IU/g, and total immobilization yield of 70.8%. The immobilized cellulase exhibited enhanced stability for temperature and pH over a broader rangethan the free enzyme. The immobilized enzyme-mediated hydrolysis of 1% carboxymethyl cellulose (CMC) released 0.54 g glucose/g substrate at 24 h, showing potential for cellulosic biomass conversion. The immobilized cellulase which was investigated for reusability assay retained∼80% activity even after repeated 6 cycles of CMC hydrolysis. The results showed good reusability after magnetic separation, which is the main advantage of using this nanobiocatalyst system.

Effects of coated folic acid and coated methionine on growth performance, nutrient digestibility and rumen fermentation in Simmental bulls

In one-carbon metabolism, methionine (Met) is methyl donor and folic acid (FA) is responsible for the transfer of methyl groups. Approximately 25 % of coated methionine (CMet) and coated folic acid (CFA) released in the rumen at 24 h of incubation. Therefore, the present study investigated the effects of CMet or/and CFA on growth performance, nutrient digestibility and rumen fermentation in bulls. Forty Simmental bulls, 464 ± 15.9 kg of body weight (BW), were blocked by BW and randomly assigned to four groups in a 2 × 2 factorial arrangement of treatment, with factors being CFA 0 or 6 mg FA/kg dietary dry matter (DM) and CMet 0 or 0.66 g Met/kg dietary DM. The experiment period was 81 days long with 20 days for adaptation and 61 days for data and sample collection. Dry matter intake increased (P = 0.001) with CFA or CMet addition. The CFA × CMet interaction was noticed on average daily gain (ADG; P = 0.001) and feed conversion ratio (FCR; P = 0.020); the improvements in ADG and FCR were greater for supplementing CMet in diets with CFA than in diets without CFA. The digestibility of DM, organic matter, neutral detergent fiber and acid detergent fiber increased (P < 0.050) with CFA or CMet addition. When CMet was supplemented, crude protein digestibility increased in CFA diets and was unchanged in diets without CFA. Ruminal pH decreased (P < 0.050), but total volatile fatty acid concentration increased (P < 0.050) with CFA or CMet addition. Acetate to propionate ratio was unchanged with CFA addition, but decreased (P = 0.039) with CMet addition. Rumen ammonia-N concentration decreased (P < 0.050) for either CFA or CMet addition. The activities of carboxymethyl cellulase, protease, α-amylase and cellobiase and populations of protozoa, total bacteria, fungi, dominant cellulolytic bacteria, Ruminobacter amylophilus and Butyrivibrio fibrisolvens increased (P < 0.050) with CFA or CMet supplementation. The CFA × CMet interaction was noticed on Ruminococcus albus population (P = 0.014); when CMet was supplemented, R. albus population increased for diets without CFA and was unchanged for diets with CFA. The activities of chymotrypsin in duodenum increased (P < 0.050) with CFA or CMet supplementation, and that of amylopsin and trypsin in ileum increased (P < 0.050) for bulls receiving CFA addition. The concentrations of glucose, albumin and urea nitrogen in blood were unchanged and homocysteine decreased (P < 0.050) with CFA or CMet supplementation. The CFA × CMet interaction was noticed on blood total protein concentration (P = 0.045); when CMet was added, blood total protein concentration increased for diets without CFA and was unchanged for diets with CFA. The concentration of folate in blood increased (P = 0.001) for CFA addition. The concentration of Met increased (P = 0.002) for CMet supplementation. The results indicated that dietary CFA or/and CMet supplementation improved growth performance, nutrient digestibility and rumen fermentation, and the greatest daily gain was observed for combined addition of CFA and CMet in bulls.

Integration of agricultural residues as biomass source to saccharification bioprocess and for the production of cellulases from filamentous fungi.

The production of second-generation bioethanol has several challenges, among them finding cheap and efficient enzymes for a sustainable process. In this work, we analyzed two native fungi, Cladosporium cladosporioides and Penicillium funiculosum, as a source of cellulolytic enzyme production, and corn stover, wheat bran, chickpeas, and bean straw as a carbon source in two fermentation systems: submerged and solid fermentation. Corn stover was selected for cellulase production in both fermentation systems, because we found the highest enzymatic activities when carboxymethyl cellulase activity (CMCase) was assessed using CMC as substrate. C. cladosporioides showed the highest CMCase activity (1.6U/mL), while P. funiculosum had the highest filter paper activity (Fpase) (0.39U/mL). The ß-glucosidase activities produced by both fungi were similar in submerged fermentation using corn stover as substrate. Through in-gel zymography, three polypeptides with cellulolytic activities were identified in each fungus: with molecular weights of ~ 38, 45 and 70kDa in C. cladosporioides and ~ 21, 63 and 100kDa in P. funiculosum. The best results for saccharification (10.11g/L of reducing sugars) of diluted acid pretreated corn stover were obtained after 36h of the hydrolytic process at pH 5 and 50°C using the enzyme extract of P. funiculosum. This is the first report of cellulase identification in C. cladosporioides and the saccharification of corn stover using enzymes of this fungus. Enzymatic extracts of C. cladosporioides and P. funiculosum obtained from low-cost lignocellulosic biomass have great potential for use in the production of second-generation bioethanol.

Bioethanol Production from Lignocellulosic Biomass Using Aspergillus niger and Aspergillus flavus Hydrolysis Enzymes through Immobilized S. cerevisiae

Lignocellulose, the main component of a plant cell wall, is a potential renewable bioenergy source. It is composed of cellulose, hemicellulose, and lignin structures. Cellulose is a linear polysaccharide that is hydrolyzed chemically or enzymatically by cellulase. The addition of lignocellulosic biomass, such as wheat bran and coffee pulp, into the fermentation culture, induces the production of cellulases. Cellulose accounts for 20% of the enzyme market worldwide, demonstrating benefits in diverse applications, especially bioethanol and biogas generation. The aim is to evaluate the optimal condition for bioethanol production by previously isolated fungal species from different soil types in the eastern region of the Kingdom of Saudi Arabia. This study attempts to evaluate and optimize the culture conditions of lignocellulosic biomass under SSF using the highest cellulases-producer strains in the region: Aspergillus niger and Aspergillus flavus (GenBank Accession No. MT328516 and MT328429, respectively) to produce raw sugar that consequently is used in the next step of bioethanol production. This process has two parts: (1) hydrolyze lignocellulosic biomass to obtain raw sugar using A. niger and A. flavus that produce cellulase, and (2) produce bioethanol through the conversion of the raw sugar produced from the cellulolysis into ethanol using Saccharomyces cerevisiae. The optimal conditions under SSF were seven days of incubation, 5% glucose as a carbon source, 1% ammonium sulfate as a nitrogen source, and 80% moisture for both isolates. Biochemical characterization showed stability for the immobilized enzyme in all temperature ranges (from 20 °C to 70 °C), while the free enzyme exhibited its maximum at 20 °C of 1.14 IU/mL. CMCase production was the highest at pH 4.0 (1.26 IU/mL) for free enzyme and at pH 5.0 (2.09 IU/mL) for the immobilized form. The CMCase activity increased steadily with an increase in water level and attained a maximum of 80% moisture content. The maximum enzyme activity was with coffee pulp as a substrate of 7.37 IU/mL and 6.38 IU/mL for A. niger and A. flavus after seven days of incubation, respectively. The Carboxymethyl Cellulase (CMCase) activity in immobilized enzymes showed good storage stability under SSF for six weeks, maintaining 90% of its initial activity, while the free enzyme retained only 59% of its original activity. As a carbon source, glucose was the best inducer of CMCase activity with coffee pulp substrate (7.41 IU/mL and 6.33 IU/mL for A. niger and A. flavus, respectively). In both fungal strains, ammonium sulfate caused maximum CMCase activities with coffee pulp as substrate (7.62 IU/mL and 6.47 IU/mL for A. niger and A. flavus, respectively). Immobilized S. cerevisiae showed an increase in ethanol production compared to free cells. In the case of immobilized S. cerevisiae cells, the concentration of ethanol was increased steadily with increasing fermentation time and attained a maximum of 71.39 mg/mL (A. niger) and 11.73 mg/mL (A. flavus) after 72 h of fermentation.

Isolation and Characterization of Bacteria from Natural Hot Spring and Insights into the Thermophilic Cellulase Production.

A thermophilic cellulase-producing bacterium,Bacillus velezensisstrain MRC 5958, from Bakra natural hot Springs, India was characterized through genome sequencing. It has a genome size of 4,467,129bp and a GC content of 45.7%. A cellulase purified from its fermentation broth has a molecular weight of about 18kDa. The optimum temperature and pH for carboxymethyl cellulase activity were at 55°C and pH ~ 7.0. The enzyme is stable over a wide range of temperatures from 30°C to 70°C with maximum activity observed at 48h of incubation. The strain produces cellulase on alkali-treated sugarcane bagasse, rice straw, rice husk, rice bran, and sawdust. The sugarcane bagasse exhibited the most effective carbon source for cellulase production at (85 U/ml) followed by rice bran (68 U/ml), rice husk (60 U/ml), rice straw (48 U/ml), and sawdust (39 U/ml). Therefore, this strain can be a potential thermostable cellulase-producing candidate for converting the waste biomass into biofuel and other industrial enzymes.