Optimization and Characterization of Spirulina and Chlorella Hydrolysates for Industrial Application.

Chlorella and Spirulina are the most used microalgae mainly as powder, tablets, or capsules. However, the recent change in lifestyle of modern society encouraged the emergence of liquid food supplements. The current work evaluated the efficiency of several hydrolysis methods (ultrasound-assisted hydrolysis UAH, acid hydrolysis AH, autoclave-assisted hydrolysis AAH, and enzymatic hydrolysis EH) in order to develop liquid dietary supplements from Chlorella and Spirulina biomasses. Results showed that, EH gave the highest proteins content (78% and 31% for Spirulina and Chlorella, respectively) and also increased pigments content (4.5 mg/mL of phycocyanin and 12 µg/mL of carotenoids). Hydrolysates obtained with EH showed the highest scavenging activity (95-91%), allowing us, with the other above features, to propose this method as convenient for liquid food supplements development. Nevertheless, it has been shown that the choice of hydrolysis method depended on the vocation of the product to be prepared.

The effects of high-pressure, enzymatic, and high-pressure-assisted enzymatic treatment on the properties of soluble dietary fibers and their use in jelly prepared with grape waste extract.

This study aimed to investigate the physicochemical attributes of soluble dietary fibers (SDFs) of grape, which were isolated after enzymatic (using cellulase [0.1 MPa/60°C/30 min]), high-pressure (HP) (100 MPa/60°C/30 min), or HP-assisted enzymatic treatment (using cellulase [100 MPa/60°C/30 min]), then to evaluate textural properties, color, and microbiological load of jelly prepared using grape waste extract and either pectin or SDF types. HP-assisted enzymatic treatment increased glucose adsorption capacity by more than 50%, and the water-holding capacity of SDF more than twofold as compared to the levels measured in untreated-SDF. After treatments, glucose and galactose contents decreased, whereas fructose, mannose, xylose, arabinose, and rhamnose ratios increased. The arabinose ratio increased more than twice by the effect of HP, whereas the xylose content increased almost fivefold with HP-assisted enzymatic treatment. For the textural properties of jelly, HP-assisted enzymatic treated-SDF provided almost double values in gel strength and adhesiveness than those contributed by untreated-SDF. It was followed by HP-treated SDF jelly. The results showed that HP-assisted enzymatic treatment developed more similar outcomes with enzymatic treatment, rather than HP treatment alone. HP-assisted enzymatic hydrolysis is recommended for treating SDF for use in jelly due to its synergistic effect. PRACTICAL APPLICATION: High-pressure-assisted cellulase treatment provided the best properties to SDF for jelly. In combined treatment, impacts of cellulase treatment were more prominent than HP effects. Therefore, the use of HP assistance for enzymatic hydrolysis shortens the processing time. Moreover, the technological and functional properties (water holding, glucose adsorption capacity, and monosaccharide composition) of the combined treated-fiber can improve. In addition, the color and textural properties of the jelly prepared with this treated-fiber can be enhanced. In this way, it may be possible to obtain a good thickening agent. This material can also be an alternative to pectin.

Quantification of Xylanolytic and Cellulolytic Activities of Fungal Strains Isolated from Palmaria palmata to Enhance R-Phycoerythrin Extraction of Palmaria palmata: From Seaweed to Seaweed.

R-phycoerythrin (R-PE) can be enzymatically extracted from red seaweeds such as Palmaria palmata. This pigment has numerous applications and is notably known as an antioxidant, antitumoral or anti-inflammatory agent. Enzymes secreted by P. palmata associated fungal strains were assumed to be efficient and adapted for R-PE extraction from this macroalga. The aim of the present study was to quantify both xylanolytic and cellulolytic activities of enzymatic extracts obtained from six Palmaria palmata derived fungal strains. Degradation of P. palmata biomass by fungal enzymatic extracts was also investigated, focused on soluble protein and R-PE extraction. Enzymatic extracts were obtained by solid state fermentation. Macroalgal degradation abilities were evaluated by measuring reducing sugar release using DNS assays. Soluble proteins and R-PE recovery yields were evaluated through bicinchoninic acid and spectrophotometric assays, respectively. Various enzymatic activities were obtained according to fungal isolates up to 978 U/mL for xylanase and 50 U/mL for cellulase. Enzymatic extract allowed high degrading abilities, with four of the six fungal strains assessed exhibiting at least equal results as the commercial enzymes for the reducing sugar release. Similarly, all six strains allowed the same soluble protein extraction yield and four of them led to an improvement of R-PE extraction. R-PE extraction from P. palamata using marine fungal enzymes appeared particularly promising. To the best of our knowledge, this study is the first on the use of enzymes of P. palmata associated fungi in the degradation of its own biomass for biomolecules recovery.

High-solids enzymatic saccharification of starch-rich raw herbal biomass residues for producing high titers of glucose.

The bioresource utilization of herbal biomass residues (HBRs) has been receiving more attention. Herein, three different HBRs from Isatidis Radix (IR) and Sophorae Flavescentis Radix (SFR) and Ginseng Radix (GR) were subjected to batch and fed-batch enzymatic hydrolysis to produce high-concentration glucose. Compositional analysis showed the three HBRs had substantial starch content (26.36-63.29%) and relatively low cellulose contents (7.85-21.02%). Due to their high starch content, the combined action of cellulolytic and amylolytic enzymes resulted in greater release of glucose from the raw HBRs compared to using the individual enzyme alone. Batch enzymatic hydrolysis of 10% (w/v) raw HBRs with low loadings of cellulase (≤ 10 FPU/g substrate) and amylolytic enzymes (≤ 5.0 mg/g substrate) led to a high glucan conversion of ≥ 70%. The addition of PEG 6000 and Tween 20 did not contribute to glucose production. Furthermore, to achieve higher glucose concentrations, fed-batch enzymatic hydrolysis was conducted using a total solid loading of 30% (w/v). After 48-h of hydrolysis, glucose concentrations of 125 g/L and 92 g/L were obtained for IR and SFR residues, respectively. GR residue yielded an 83 g/L glucose concentration after 96 h of digestion. The high glucose concentrations produced from these raw HBRs indicate their potential as ideal substrate for a profitable biorefinery. Notably, the obvious advantage of using these HBRs is the elimination of the pretreatment step, which is typically required for agricultural and woody biomass in similar studies.

Pectin from plantain peels: Green recovery for transformation into reinforced packaging films.

Plantain peels as agro-waste are generated in the millions of tons per year with no profitable management strategies. On the other hand, the excessive use of plastic packaging threatens the environment and human health. This research aimed to address both problems via a green approach. High-quality pectin was recovered from plantain peels via an enzyme-assisted and ethanol-recycling process. The yield and galacturonic acid (GalA) content of the recovered low methoxy pectin was 12.43% and 25.0%, respectively, when cellulase was added at 50 U per 5 g peel powder, with a significantly higher recovery rate and purity than the pectin products extracted with no cellulase (P ≤ 0.05). The recovered pectin was further integrated and reinforced with beeswax solid-lipid nanoparticles (BSLNs) to fabricate films as a potential alternative packaging material to single-use plastics. The reinforced pectin films showed improved light barrier, water resistance, mechanical, conformational, and morphological properties. This study presents a sustainable strategy to transform plantain peels into pectin products and pectin-based packaging films with broad applications.

Comparison of Alkaline Sulfite Pretreatment and Acid Sulfite Pretreatment with Low Chemical Loading in Saccharification of Poplar.

Sulfite pretreatment is a productive process for lignin dissolution in lignocelluloses and to reduce the hydrophobicity of lignin by sulfonation, thus promoting the hydrolyzability of the substrate. Previously, sulfite pretreatment needs high dosages of chemicals and thus results in the high cost of the pretreatment and the great pressure of environmental pollution. To overcome these problems, it was crucial to research whether alkaline sulfite pretreatment (ALS) and acid sulfite pretreatment (ACS) with low chemical loading could enhance the saccharification of poplar. In this work, the results indicated that with low loading of chemicals in sulfite pretreatment, ALS pretreatment (1.6% Na2SO3 and 0.5% NaOH) at 180 °C removed more lignin, resulted in lower hydrophobicity and higher cellulase adsorption capacity of poplar than ACS pretreatment (1.6% Na2SO3 and 0.5% H2SO4) at 180 °C. A satisfying glucose yield of 84.9% and a xylose yield of 76.0% were obtained from poplar after ALS pretreatment with 1.6% Na2SO3 and 0.5% NaOH at 180 °C for 1 h using 10 FPU cellulase/g dry matter, saving sodium sulfite by 60.0% compared to the loading of sulfite in traditional sulfite pretreatment. The strategy developed in this work reduced chemical loading and cellulase loading in alkali sulfite pretreatment for the saccharification of poplar.

Biosurfactant promoted enzymatic saccharification of alkali­pretreated reed straw.

The decrease of cellulase activity and unproductive adsorption of lignin are important obstructive factors for inefficient enzymatic hydrolysis. This paper applied five different kinds of biosurfactants including rhamnolipid, sophorolipid, chitin, tea saponin, and sodium lignosulfonate in the enzymatic hydrolysis process of alkali-pretreated reed straw (RS) to enhance the saccharification efficiency. When 8 g/L sophorolipid is added, the efficiency of enzymatic hydrolysis is 91.68 %, which is 30.65 % higher than that without using any biosurfactant. The efficiency of enzymatic hydrolysis can be further increased to 99.56 % when 7.5 g/L sophorolipid and 1.5 g/L tea saponin are added together. This is because the sophorolipid, rhamnolipid, and chitin can synergistically hamper the enzymatic inactivation during enzymatic hydrolysis, while tea saponin and sodium lignosulfonate can inhibit the non-productive adsorption of lignin. This work proposed a very effective method to improve the efficiency of enzymatic hydrolysis and reduce the dosage of the enzyme by adding biosurfactants.

Highly efficient glucose production from raw non-pretreated Chinese medicinal herbal residues via the synergism of cellulase and amylolytic enzymes.

Available literature on Chinese medicinal herbal residues (CMHRs) bioconversion highlights pretreatment prior to saccharification with cellulase without considering the presence of starch constituent. Herein, four commonly found CMHRs were tested for starch content, and it was found they all contained starch with content ranging from 4.74% to 16.78%. Hydrolysis of raw CMHRs with combined cellulase and amylolytic enzymes yielded increments of 16.85% to 26.51% in 48-h glucan conversion compared to cellulase alone. Further study showed 48-h glucan conversion of raw CMHRs outperformed that pretreated by water-ethanol successive extraction, ultrasound and acid, but underperformed alkali-pretreated CMHRs. Although increasing 48-h glucan conversion in the range of 7.40% to 24.10% compared to raw CMHRs, alkaline pretreatment demonstrated low glucose recovery and incurred additional cost, making it unfavorable. Saccharification of the four raw CMHRs with combined enzymes seems like a preferred option considering the elimination of high-cost pretreatment step.

Customized optimization of lignocellulolytic enzyme cocktails for efficient conversion of pectin-rich biomass residues.

Pectin is a major component in many agricultural feedstocks. Despite the wide use in industrial production of cellulases and hemicellulases, the fungus Trichoderma reesei lacks a complete enzyme set for pectin degradation. In this study, three representative pectinolytic enzymes were expressed and screened for their abilities to improve the efficiency of T. reesei enzymes on the conversion of different agricultural residues. By replacing 5 % of the T. reesei proteins, endopolygalacturonase and pectin lyase remarkably increased the release of sugars from inferior tobacco leaves. In contrast, pectin methylesterase showed the strongest improving effect (by 31.1 %) on the hydrolysis of beetroot residue. The pectin in beetroot residue was only mildly degraded with the supplementation of pectin methylesterase, which allowed the extraction of pectin keeping the original emulsifying activity with a 51.1 % higher yield. The results provide a basis for precise optimization of lignocellulolytic enzyme systems for targeted valorization of pectin-rich agricultural residues.

Enhancing the Activity of Carboxymethyl Cellulase Enzyme Using Highly Stable Selenium Nanoparticles Biosynthesized by Bacillus paralicheniformis Y4.

The inorganic selenium is absorbed and utilized inefficiently, and the range between toxicity and demand is narrow, so the application is strictly limited. Selenium nanoparticles have higher bioactivity and biosafety properties, including increased antioxidant and anticancer properties. Thus, producing and applying eco-friendly, non-toxic selenium nanoparticles in feed additives is crucial. Bacillus paralicheniformis Y4 was investigated for its potential ability to produce selenium nanoparticles and the activity of carboxymethyl cellulases. The selenium nanoparticles were characterized using zeta potential analyses, Fourier transform infrared (FTIR) spectroscopy, and scanning electron microscopy (SEM). Additionally, evaluations of the anti-α-glucosidase activity and the antioxidant activity of the selenium nanoparticles and the ethyl acetate extracts of Y4 were conducted. B. paralicheniformis Y4 exhibited high selenite tolerance of 400 mM and the selenium nanoparticles had an average particle size of 80 nm with a zeta potential value of -35.8 mV at a pH of 7.0, suggesting that the particles are relatively stable against aggregation. After 72 h of incubation with 5 mM selenite, B. paralicheniformis Y4 was able to reduce it by 76.4%, yielding red spherical bio-derived selenium nanoparticles and increasing the carboxymethyl cellulase activity by 1.49 times to 8.96 U/mL. For the first time, this study reports that the carboxymethyl cellulase activity of Bacillus paralicheniforis was greatly enhanced by selenite. The results also indicated that B. paralicheniformis Y4 could be capable of ecologically removing selenite from contaminated sites and has great potential for producing selenium nanoparticles as feed additives to enhance the added value of agricultural products.

Effects of a combination of fibrolytic and amylolytic enzymes on ruminal enzyme activities, bacterial diversity, blood profile and milk production in dairy cows.

We hypothesised that adding a combination of fibrolytic and amylolytic enzymes to the diet of early-lactation dairy cows would improve rumen enzyme activity and bacterial diversity, promote energy metabolism, and benefit milk production in cows. Twenty multiparous early-lactation (90 ±â€¯5 d) Holstein cows with similar body conditions were randomly allocated to control (CON, n = 10) and experimental (EXP, n = 10) groups in a completely randomised single-factor design. The CON was fed only a basal total mixed ration diet, and the diet of the EXP was supplemented with a combination of fibrolytic and amylolytic enzymes at 70 g/cow/d (cellulase 3 500 CU/g, xylanase 2 000 XU/g, ß-glucanase 17 500 GU/g, and amylase 37 000 AU/g). The experiment lasted 28 days, with 21 days for adaptation and 7 days for sampling. Enzyme addition increased the activity levels of α-amylase and xylanase, and the ammonia-N concentration (P < 0.05) tended to increase the activity of ß-glucanase (P = 0.08) in rumen fluid. However, there was no significant difference in the rumen bacterial richness and diversity, phylum (richness > 0.1%) or genus (richness > 1%) composition between the CON and EXP groups (P > 0.05). A tendency of difference was found between CON and EXP (R = 0.22, P = 0.098) in principal component analysis. Ten genera showed different abundances across the CON and EXP groups (linear discriminant analysis effect size, linear discriminant analysis > 2). EXP increased the ratio of albumin to globulin and the concentrations of total cholesterol and low-density lipoprotein cholesterol (P < 0.05) and tended to increase triglycerides (P = 0.09) in blood. Milk yield, 3.5% fat-corrected milk yield and energy-corrected milk yield increased with enzyme supplementation (P < 0.05). The production levels of milk fat and lactose increased, but the percentage of solids, not fat and protein, decreased in EXP (P < 0.05). Although the DM intake was not affected, the feed efficiency tended to increase (P = 0.07) in EXP. In conclusion, dietary supplementation with a mixture of fibrolytic and amylolytic enzymes on multiparous early-lactation dairy cows increased α-amylase and xylanase activity levels in rumen fluid, enhanced milk performance and tended to improve the feed efficiency in cows.

Press water from the mechanical drying of Douglas-fir wood chips has multiple beneficial effects on lignocellulolytic fungi.

BACKGROUND: The mechanical drying of wood chips is an innovative method that improves the heating value of sawmill by-products in an energy-efficient continuous process. The liquid that comes out of the wood chips as press water (PW), however, contains a variety of undissolved as well as dissolved organic substances. The disposal of the PW as wastewater would generate additional costs due to its high organic load, offsetting the benefits in energy costs associated with the enhanced heating value of the wood chips. Our research explored if the organic load in PW could be utilized as a substrate by cellulolytic filamentous fungi. Hence, using the industrially relevant Ascomycete Trichoderma reesei RUT-C30 as well as several Basidiomycete wood-rotting fungi, we examined the potential of press water obtained from Douglas-fir wood chips to be used in the growth and enzyme production media. RESULTS: The addition of PW supernatant to liquid cultures of T. reesei RUT-C30 resulted in a significant enhancement of the endoglucanase and endoxylanase activities with a substantially shortened lag-phase. A partial replacement of Ca2+, Mg2+, K+, as well as a complete replacement of Fe2+, Mn2+, Zn2+ by supplementing PW of the liquid media was achieved without negative effects on enzyme production. Concentrations of PW above 50% showed no adverse effects regarding the achievable endoglucanase activity but affected the endoxylanase activity to some extent. Exploring the enhancing potential of several individual PW components after chemical analysis revealed that the observed lag-phase reduction of T. reesei RUT-C30 was not caused by the dissolved sugars and ions, nor the wood particles in the PW sediment, suggesting that other, so far non-identified, compounds are responsible. However, also the growth rate of several basidiomycetes was significantly enhanced by the supplementation of raw PW to the agar medium. Moreover, their cultivation in liquid cultures reduced the turbidity of the PW substantially. CONCLUSIONS: PW was identified as a suitable media supplement for lignocellulolytic fungi, including the cellulase and xylanase producer T. reesei RUT-C30 and several wood-degrading basidiomycetes. The possibility to replace several minerals, trace elements and an equal volume of fresh water in liquid media with PW and the ability of fungal mycelia to filter out the suspended solids is a promising way to combine biological wastewater treatment with value-adding biotechnological applications.

Allelopathic inhibitory effect on the growth of microcystis aeruginosa by improved ultrasonic-cellulase extract of Vallisneria.

The unique and efficient characteristics of allelopathy in submerged plants make it an environmentally friendly method to control harmful algal blooms. Increased research focus has been placed on the improved allelochemical extraction methods of submerged plants because of their cost-utility relationships. In this study, the growth inhibition effect of Vallisneria extract on Microcystis aeruginosa (M. aeruginosa) cells through the combination of enzyme and ultrasonic-assisted extraction method was analyzed. By establishing a co-cultivation experiment, the growth indicators, photosynthetic system, and oxidative stress system of M. aeruginosa were determined. The reactive oxygen species (ROS) and superoxide dismutase (SOD) activity, as well as the catalase (CAT) and Malondialdehyde (MDA) levels of algal cells were found to have increased significantly after co-cultivation, which indicated that the Vallisneria ultrasonic-cellulase extract could induce oxidative stress in Microcystis aeruginosa cells. The Vallisneria extract could promote at low concentrations and inhibit at high concentrations on the growth of Microcystis aeruginosa. The effective suppression of growth of algae cells with the extract was observed at 5 g/L (fresh weight). The results showed that the Vallisneria ultrasonic-cellulase extract had a significant inhibitory effect on M. aeruginosa, making the effective ingredients a useful reference for algae inhibitors.

Isolation and characterization of a mesophilic cellulolytic endophyte Preussia africana from Juniperus oxycedrus.

The medicinal plant Juniperus oxycedrus is less recognized for the diversity of its fungal endophytes and their potential to produce extracellular enzymes. The present study is the first report on the isolation and identification of a mesophilic endophytic strain JO-A, Preussia africana, from fresh stems of the J. oxycedrus endemic tree in the Ifrane region-Morocco, and the evaluation of its ability to produce cellulases. A one-time multi-parameter one-factor screening was optimized to select factors that enhance cellulase production in P. africana. The maximum production of both CMCase and FPase activities were 1.913 IU.mL-1 and 0.885 IU.mL-1, respectively, when the medium was supplemented with 2% w/v glucose. These remarkable titers were tenfold greater than those obtained under the initial non-optimized conditions. This mesophilic P. africana JO-A strain grows and actively produces cellulases at 37 °C demonstrating its great potential for various biotechnology applications. The cellulolytic extract showed the highest enzymatic activities at pH 5.0 and 50 °C with a half-life of 24 h at 50 °C.

Cell-Wall-Degrading Enzymes-Related Genes Originating from Rhizoctonia solani Increase Sugar Beet Root Damage in the Presence of Leuconostoc mesenteroides.

Sugar beet crown and root rot caused by Rhizoctonia solani is a major yield constraint. Root rot is highly increased when R. solani and Leuconostoc mesenteroides co-infect roots. We hypothesized that the absence of plant cell-wall-degrading enzymes in L. mesenteroides and their supply by R. solani during close contact, causes increased damage. In planta root inoculation with or without cell-wall-degrading enzymes showed greater rot when L. mesenteroides was combined with cellulase (22 mm rot), polygalacturonase (47 mm), and pectin lyase (57 mm) versus these enzymes (0-26 mm), R. solani (20 mm), and L. mesenteroides (13 mm) individually. Carbohydrate analysis revealed increased simpler carbohydrates (namely glucose + galactose, and fructose) in the infected roots versus mock control, possibly due to the degradation of complex cell wall carbohydrates. Expression of R. solani cellulase, polygalacturonase, and pectin lyase genes during root infection corroborated well with the enzyme data. Global mRNAseq analysis identified candidate genes and highly co-expressed gene modules in all three organisms that might be critical in host plant defense and pathogenesis. Targeting R. solani cell-wall-degrading enzymes in the future could be an effective strategy to mitigate root damage during its interaction with L. mesenteroides.

α-Amylase and cellulase production by novel halotolerant Bacillus sp.PM06 isolated from sugarcane pressmud.

A novel Bacillus sp.PM06 isolated from sugarcane waste pressmud was tested for extracellular α-amylase and cellulase enzyme production. The effect of different substrates, nitrogen sources, pH, and temperature on growth and extracellular enzyme production was examined. Bacillus sp.PM06 was able to grow with starch and carboxymethyl cellulose (CMC) as a sole source of carbon and ammonium chloride was found to be the best nitrogen source. Maximum enzyme production was obtained at 48 H for both α-amylase and cellulase. The optimal condition for measuring enzyme activity was found to be pH 5.5 at 50 °C for α-amylase and pH 6.4 at 60 °C for cellulase respectively. It was found that Bacillus sp.PM06 exhibited halotolerance up to 2 M Sodium chloride (NaCl) and Potassium chloride (KCl). The isolate could produce α-amylase in the presence of 2 M NaCl and 1 M KCl. However, the strain produced cellulase even in the presence of 2 M NaCl and KCl. Concomitant production of both enzymes was observed when the medium was supplemented with starch and CMC. A maximum of 31 ± 1.15 U/mL of amylase and 15 ± 1.5 U/mL of cellulase was produced in 48 H. The enzyme was partially purified by Ammonium sulphate (NH4 )2 SO4 precipitation with 2.2 and 2.3-fold purification.

[Plant biomass degradation by filamentous fungi and production of renewable chemicals: a review].

Plant biomass represents a vast resource of carbon. In China, it is estimated that 1 billion tons of biomass is available each year. The conversion of these biomass resources into bioethanol or other bio-based chemicals, if fully commercialized, may reduce at least 200 million tons of crude oil import. Therefore, bioethanol and bulk chemicals are the core components of the biomanufacturing using plant biomass as carbon sources. Since the foundation of Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences (TIB, CAS), we have proposed a strategy of "two replacements and one upgrade". Utilizing renewable carbon resources instead of non-renewable petrochemical resources to produce bulk chemicals is included in our strategy. It is a long-term effort for TIB to develop plant biomass biomanufacturing to produce renewable chemicals. Continuous and systematic research was carried out in these two fields, and significant progress has been made in the past 10 years since the foundation of TIB. Here we review the progress of TIB in this field, mainly focusing on fungal system, including the mechanism of cellulose degradation by filamentous fungi and the strategy of consolidated bioprocessing of biomass. Based on this, malic acid, fuel ethanol and other bulk chemicals were produced through one-step conversion of biomass. Besides, the commercial processes for production of bulk chemicals such as succinic and lactic acid from renewable carbon resources, which were developed by TIB, were also be discussed. These examples clearly demonstrated that bulk chemicals can be obtained from biomass instead of from petroleum. Research on plant biomass biotransformation and renewable chemicals production in TIB has provided an alternative route for the development of low-carbon bioeconomy in China, and will contribute to the goal of carbon neutralization of China.

Medium supplementation and thorough optimization to induce carboxymethyl cellulase production by Trichoderma reesei under solid state fermentation of nettle biomass.

In the present study, the production of cellulase by Trichoderma reesei under solid-state fermentation of nettle biomass was promoted through supplementation of the culture media using carbonaceous additives and comprehensive optimization of the cultivation via the Taguchi method. CMCase activities about 5.5-6.1 U/gds were obtained by fermentation of the autoclave-pretreated biomass, among various chemical and physical pretreatments. Then, several additives including Tween 80, betaine, carboxymethyl cellulose, and lactose were individually or in combination added to the culture media to induce the enzyme production. The results proved that such additives could act as either inducers or inhibitors. Furthermore, CMCase activity surprisingly increased to 14.0 U/gds by supplementing the fermentation medium with the optimal mixture of additives including 0.08 mg/gds Tween 80, 0.4 mg/gds betaine, and 0.2 mg/gds carboxymethyl cellulose. Factor screening according to Plackett-Burman design confirmed that the levels of Urea and MgSO4 among basal medium constituents as well as pH of the medium were significantly affected CMCase production. By optimizing the levels of these factors, CMCase activity of 18.8 U/gds was obtained, which was noticeably higher than that of fermentation of the raw nettle. The applied procedure can be promisingly used to convert the nettle biomass into valuable products.

Induction of cellulase production by Sr2+ in Trichoderma reesei via calcium signaling transduction.

Trichoderma reesei RUT-C30 is a well-known high-yielding cellulase-producing fungal strain that converts lignocellulose into cellulosic sugar for resource regeneration. Calcium is a ubiquitous secondary messenger that regulates growth and cellulase production in T. reesei. We serendipitously found that adding Sr2+ to the medium significantly increased cellulase activity in the T. reesei RUT-C30 strain and upregulated the expression of cellulase-related genes. Further studies showed that Sr2+ supplementation increased the cytosolic calcium concentration and activated the calcium-responsive signal transduction pathway of Ca2+-calcineurin-responsive zinc finger transcription factor 1 (CRZ1). Using the plasma membrane Ca2+ channel blocker, LaCl3, we demonstrated that Sr2+ induces cellulase production via the calcium signaling pathway. Supplementation with the corresponding concentrations of Sr2+ also inhibited colony growth. Sr2+ supplementation led to an increase in intracellular reactive oxygen species (ROS) and upregulated the transcriptional levels of intracellular superoxide dismutase (sod1) and catalase (cat1). We further demonstrated that ROS content was detrimental to cellulase production, which was alleviated by the ROS scavenger N-acetyl cysteine (NAC). This study demonstrated for the first time that Sr2+ supplementation stimulates cellulase production and upregulates cellulase genes via the calcium signaling transduction pathway. Sr2+ leads to an increase in intracellular ROS, which is detrimental to cellulase production and can be alleviated by the ROS scavenger NAC. Our results provide insights into the mechanistic study of cellulase synthesis and the discovery of novel inducers of cellulase.

Effect of Cellulase Enzyme Produced from Penicilliumchrysogenum on the Milk Production, Composition, Amino Acid, and Fatty Acid Profiles of Egyptian Buffaloes Fed a High-Forage Diet.

The experiment was conducted to study the effects of supplementing a cellulase enzymes cocktail to lactating buffaloes' diet, on the nutrient intake, nutrient digestibility, and milk production performance and composition. Twenty-four lactating Egyptian buffaloes were assigned into one of the following treatments: CON-control consisted of a total mixed ration, CENZ-the total mixed ration supplemented by a commercial source of cellulase enzyme, FENZ-the total mixed ration supplemented with cellulase enzyme cocktail produced in-farm. Supplementing the diet with the in-farm source of cellulase (FENZ) had a significantly higher impact on crude protein, neutral detergent fiber, and acid detergent fiber digestibility. However, FENZ tended to increase the EE digestibility compared to CENZ. FENZ showed significantly higher nutrient digestibility percentages compared to other groups. Supplementing the diet with cellulase enzymes (CON vs. ENZ) significantly increased the daily milk yield and the fat correct milk yield; both yields were significantly higher with FENZ than all groups. Oleic, linoleic, and linolenic acid concentration were significantly higher with cellulase enzymes supplementation (CON vs. ENZ) and the conjugated linoleic acid concentration. Supplementing fungal cellulase enzyme produced on a farm-scale has improved milk productivity, fat yield, and milk fat unsaturated fatty acids profile in lactating buffaloes.