Low-cost and efficient strategy for brown algal hydrolysis: Combination of alginate lyase and cellulase.

Brown algae are rich in biostimulants that not only stimulate the overall development and growth of plants but also have great beneficial effects on the whole soil-plant system. However, alginate, the major component of brown algae, is comparatively difficult to degrade. The cost of preparing alginate oligosaccharides (AOSs) is still too high to produce seaweed fertilizer. In this work, the marine bacterium Vibrio sp. B1Z05 is found to be capable of efficient alginate depolymerization and harbors an extended pathway for alginate metabolism. The B1Z05 extracellular cell-free supernatant exhibited great potential for AOS production at low cost, which, together with cellulase, can efficiently hydrolyze seaweed. The brown algal hydrolysis rates were significantly greater than those of the commercial alginate lyase product CE201, and the obtained seaweed extracts were rich in phytohormones. This work provides a low-cost but efficient strategy for the sustainable production of desirable AOSs and seaweed fertilizer.

Enhancing the Heterologous Expression of a Thermophilic Endoglucanase and Its Cost-Effective Production in Pichia pastoris Using Multiple Strategies.

Although Pichia pastoris was successfully used for heterologous gene expression for more than twenty years, many factors influencing protein expression remain unclear. Here, we optimized the expression of a thermophilic endoglucanase from Thermothielavioides terrestris (TtCel45A) for cost-effective production in Pichia pastoris. To achieve this, we established a multifactorial regulation strategy that involved selecting a genome-editing system, utilizing neutral loci, incorporating multiple copies of the heterologous expression cassette, and optimizing high-density fermentation for the co-production of single-cell protein (SCP). Notably, even though all neutral sites were used, there was still a slight difference in the enzymatic activity of heterologously expressed TtCel45A. Interestingly, the optimal gene copy number for the chromosomal expression of TtCel45A was found to be three, indicating limitations in translational capacity, post-translational processing, and secretion, ultimately impacting protein yields in P. pastoris. We suggest that multiple parameters might influence a kinetic competition between protein elongation and mRNA degradation. During high-density fermentation, the highest protein concentration and endoglucanase activity of TtCel45A with three copies reached 15.8 g/L and 9640 IU/mL, respectively. At the same time, the remaining SCP of P. pastoris exhibited a crude protein and amino acid content of up to 59.32% and 46.98%, respectively. These findings suggested that SCP from P. pastoris holds great promise as a sustainable and cost-effective alternative for meeting the global protein demand, while also enabling the production of thermophilic TtCel45A in a single industrial process.

Bacterial cellulase production via co-fermentation of paddy straw and Litchi waste and its stability assessment in the presence of ZnMg mixed-phase hydroxide-based nanocomposite derived from Litchi chinensis seeds.

Co-fermentation via co-cultured bacterial microorganisms to develop enzymes in solid-state fermentation (SSF) is a promising approach. This strategy is imperative in a series of sustainable and effective approaches due to superior microbial growth and the use of a combination of inexpensive feedstocks for enzyme production wherein mutually participating enzyme-producing microbial communities are employed. Moreover, the addition of nanomaterials to this technique may aid in its prominent advantage of enhancing enzyme production. This strategy may be able to decrease the overall cost of the bioprocessing to produce enzymes by further implementing biogenic, route-derived nanomaterials as catalysts. Therefore, the present study attempts to explore endoglucanase (EG) production using a bacterial coculture system by employing two different bacterial strains, namely, Bacillus subtilis and Serratia marcescens under SSF in the presence of a ZnMg hydroxide-based nanocomposite as a nanocatalyst. The nanocatalyst based on ZnMg hydroxide has been prepared via green synthesis using Litchi waste seed, while SSF for EG production has been conducted using cofermentation of litchi seed (Ls) and paddy straw (Ps) waste. Under an optimized substrate concentration ratio of 5:6 Ps:Ls and in the presence of 2.0 mg of nanocatalyst, the cocultured bacterial system produced 1.6 IU/mL of EG enzyme, which was ~1.33 fold higher as compared to the control. Additionally, the same enzyme showed its stability for 135 min in the presence of 1.0 mg of nanocatalyst at 38 °C. The nanocatalyst has been synthesized using the green method, wherein waste litchi seed is used as a reducing agent, and the nanocatalyst could be employed to improve the production and functional stability of crude enzymes. The findings of the present study may have significant application in lignocellulosic-based biorefinaries and cellulosic waste management.

Endo-Exoglucanase Synergism for Cellulose Nanofibril Production Assessment and Characterization.

A study to produce cellulose nanofibrils (CNF) from kraft cellulose pulp was conducted using a centroid simplex mixture design. The enzyme blend contains 69% endoglucanase and 31% exoglucanase. The central composite rotational design (CCRD) optimized the CNF production process by achieving a higher crystallinity index. It thus corresponded to a solid loading of 15 g/L and an enzyme loading of 0.974. Using the Segal formula, the crystallinity index (CrI) of the CNF was determined by X-ray diffraction to be 80.87%. The average diameter of the CNF prepared by enzymatic hydrolysis was 550-600 nm, while the one produced by enzymatic hydrolysis and with ultrasonic dispersion was 250-300 nm. Finally, synergistic interactions between the enzymes involved in nanocellulose production were demonstrated, with Colby factor values greater than one.

Investigation of a robust pretreatment technique based on ultrasound-assisted, cost-effective ionic liquid for enhancing saccharification and bioethanol production from wheat straw.

Application of cost-effective pretreatment of wheat straw is an important stage for massive bioethanol production. A new approach is aimed to enhance the pretreatment of wheat straw by using low-cost ionic liquid [TEA][HSO4] coupled with ultrasound irradiation. The pretreatment was conducted both at room temperature and at 130 °C with a high biomass loading rate of 20% and 20% wt water assisted by ultrasound at 100 W-24 kHz for 15 and 30 min. Wheat straw pretreated at 130 °C for 15 and 30 min had high delignification rates of 67.8% and 74.9%, respectively, and hemicellulose removal rates of 47.0% and 52.2%. Moreover, this pretreatment resulted in producing total reducing sugars of 24.5 and 32.1 mg/mL in enzymatic saccharification, respectively, which corresponds to saccharification yields of 67.7% and 79.8% with commercial cellulase enzyme CelluMax for 72 h. The ethanol generation rates of 38.9 and 42.0 g/L were attained for pretreated samples for 15 and 30 min, equivalent to the yields of 76.1% and 82.2% of the maximum theoretical yield following 48 h of fermentation. This demonstration provided a cheap and promising pretreatment technology in terms of efficiency and shortening the pretreatment time based on applying low-cost ionic liquid and efficient ultrasound pretreatment techniques, which facilitated the feasibility of this approach and could further develop the future of biorefinery.

Comparison of pre-treatments mediated by endoglucanase and TEMPO oxidation for eco-friendly low-cost energy production of cellulose nanofibrils.

Specific kinds of enzymes have been used as an eco-friendly pre-treatment for mechanical extraction of cellulose nanofibrils (CNFs) from vegetal pulps. Another well-established pre-treatment is the 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO)-mediated oxidation, which has gained considerable attention. Pre-treatments assist in fiber swelling, facilitating mechanical fibrillation, and reducing energy consumption; however, some of these methods are extremely expensive. This work aimed to evaluate the influence of enzymatic pre-treatment with endoglucanase on the energy consumption during mechanical fibrillation of cellulose pulps. Bleached pulps from Eucalyptus sp. and Pinus sp. were pre-treated with endoglucanase enzyme compared to TEMPO-meditated oxidation. Average diameters of CNFs pre-treated with enzymes were close to that found for TEMPO-oxidized nanofibrils (TOCNFs). Results showed that enzymatic pre-treatment did not significantly modify the pulp chemical and morphological characteristics with efficient stabilization of the CNFs suspension at higher supernatant turbidity. Energy consumption of pulps treated with endoglucanase enzymes was lower than that shown by pulps treated with TEMPO, reaching up to 58% of energy savings. The enzyme studied in the pulp treatment showed high efficiency in reducing energy consumption during mechanical fibrillation and production of films with high mechanical quality, being an eco-friendly option for pulp treatment.

Sustainable enzymatic treatment of organic waste in a framework of circular economy.

Enzymatic treatment of food and vegetable waste (FVW) is an eco-friendly approach for producing industrially relevant value-added products. This review describes the sources, activities and potential applications of crucial enzymes in FVW valorization. The specific roles of amylase, cellulase, xylanase, ligninase, protease, pectinase, tannase, lipase and zymase enzymes were explained. The exhaustive list of value-added products that could be produced from FVW is presented. FVW valorization through enzymatic and whole-cell enzymatic valorization was compared. The note on global firms specialized in enzyme production reiterates the economic importance of enzymatic treatment. This review provides information on choosing an efficient enzymatic FVW treatment strategy, such as nanoenzyme and cross-linked based enzyme immobilization, to make the process viable, sustainable and cheaper. Finally, the importance of life cycle assessment of enzymatic valorization of FVW was impressed to prove this approach is a better option to shift from a linear to a circular economy.

Development of an economically competitive Trichoderma-based platform for enzyme production: Bioprocess optimization, pilot plant scale-up, techno-economic analysis and life cycle assessment.

Despite decades of research and industrial applications of Trichoderma reesei, the development of industrially relevant strains for enzyme production including a low-cost and scalable bioprocess remains elusive. Herein, bioprocess optimization, pilot plant scale-up, techno-economic analysis and life-cycle assessment for enzyme production by an engineered T. reesei strain are reported. The developed bioprocess increased in âˆ¼ 2-fold protein productivity (0.39 g.L-1.h-1) and 1.6-fold FPase activity (196 FPU.L-1.h-1), reducing the fermentation in 4 days. Cultivation in a 65-L pilot plant bioreactor resulted in 54 g.L-1 protein in 7 days, highlighting the robustness and scalability of this bioprocess. Techno-economic analysis indicates an enzyme cost of âˆ¼ 3.2 USD.kg-1, which is below to the target proposed (4.24 USD.kg-1) in the NREL/TP-5100-47764 report, while life-cycle assessment shows a carbon footprint reduction of approximately 50% compared to a typical commercial enzyme. This study provides the fundamental knowledge for the design of economically competitive Trichoderma technologies for industrial use.

Melioration of Paddy Straw to produce cellulase-free xylanase and bioactives under Solid State Fermentation and deciphering its impact by Life Cycle Assessment.

Aiming towards zero waste management of Paddy straw (PS), the study offers a novel route for production of cellulase-free xylanase, using consortia of Trichoderma spp. under Solid State Fermentation (SSF) of PS valorized using nitrogen rich de-oiled neem cake (NC). Life Cycle Assessment (LCA) for enzyme production, performed using SimaPro software, depicted adverse impacts due to electricity consumption (92.84%) and use of ammonium sulphate salt (6.17%). Nonetheless, employing renewable energy and reducing salt consumption could help minimize these impacts. OHR-LCMS study of the partially purified enzyme revealed the presence of ß-xylanase and α-L-Arabinofuranosidase. Enzymatic saccharification of various substrates enhanced the release of reducing sugars (mg/g) from corn cob (137.54 ± 0.96), pine needle (41.43 ± 1), sugarcane bagasse (105.17 ± 0.7), and PS (76.66 ± 1.29), demonstrating its applicability in the biofuel domain. LC-MS, ICMPS, and EDX profiling of the residual spent unravelled the manifestation of bioactives, minerals, and silica, playing an essential role as biopesticide and biofertilizer.

Plasma Promotes Fungal Cellulase Production by Regulating the Levels of Intracellular NO and Ca2.

For the industrial-scale production of useful enzymes by microorganisms, technological development is required for overcoming a technical bottleneck represented by poor efficiency in the induction of enzyme gene expression and secretion. In this study, we evaluated the potential of a non-thermal atmospheric pressure plasma jet to improve the production efficiency of cellulolytic enzymes in Neurospora crassa, a filamentous fungus. The total activity of cellulolytic enzymes and protein concentration were significantly increased (1.1~1.2 times) in media containing Avicel 24-72 h after 2 and 5 min of plasma treatment. The mRNA levels of four cellulolytic enzymes in fungal hyphae grown in media with Avicel were significantly increased (1.3~17 times) 2-4 h after a 5 min of plasma treatment. The levels of intracellular NO and Ca2+ were increased in plasma-treated fungal hyphae grown in Avicel media after 48 h, and the removal of intracellular NO decreased the activity of cellulolytic enzymes in media and the level of vesicles in fungal hyphae. Our data suggest that plasma treatment can promote the transcription and secretion of cellulolytic enzymes into the culture media in the presence of Avicel (induction condition) by enhancing the intracellular level of NO and Ca2+.

Chitosan/Cellulose-Based Porous Nanofilm Delivering C-Phycocyanin: A Novel Platform for the Production of Cost-Effective Cultured Meat.

Cultured meat is artificial meat produced via the mass culture of cells without slaughtering livestock. In the production process of cultured meat, the mass proliferation for preparing abundant cells is a strenuous and time-consuming procedure requiring expensive and excess serum. Herein, C-phycocyanin (C-PC) extracted from blue algae was selected as a substitute for animal-derived serum and a polysaccharide film-based platform was developed to effectively deliver C-PC to myoblast while reducing the cost of cell medium. The polysaccharide platform has a sophisticated structure in which an agarose layer is capped on a porous multilayer film formed by molecular reassembly between chitosan and carboxymethylcellulose (CMC). The porous multilayer film provides an inner structure in which C-PC can be incorporated, and the agarose layer protects and stabilizes the C-PC. The completed platform was easily applied to a cell culture plate to efficiently release C-PC, thereby improving myoblast proliferation in a serum-reduced environment during long-term culture. We developed a cell sheet-based meat model using this polysaccharide platform to evaluate the improved cost-efficiency by the platform method in the mass proliferation of cells. This strategy and innovative technology can simplify the production system and secure price competitiveness to commercialize cultured meat.

Bioconversion of Renewable Plant Biomass. Second-Generation Biofuels: Raw Materials, Biomass Pretreatment, Enzymes, Processes, and Cost Analysis.

The review discusses various aspects of renewable plant biomass conversion and production of the second-generation biofuels, including the types of plant biomass, its composition and reaction ability in the enzymatic hydrolysis, and various pretreatment methods for increasing the biomass reactivity. Conversion of plant biomass into sugars requires the use of a complex of enzymes, the composition of which should be adapted to the biomass type and the pretreatment method. The efficiency of enzymatic hydrolysis can be increased by optimizing the composition of the enzymatic complex and by increasing the catalytic activity and operational stability of its constituent enzymes. The availability of active enzyme producers also plays an important role. Examples of practical implementation and scaling of processes for the production of second-generation biofuels are presented together with the cost analysis of bioethanol production.

Impact of Alkaline Pretreatment Condition on Enzymatic Hydrolysis of Sugarcane Bagasse and Pretreatment Cost.

A combined severity factor (RCSF) which is usually used to evaluate the effectiveness of hydrothermal pretreatment at above 100 °C had been developed to assess the influence of temperature, time, and alkali loading on pretreatment and enzymatic hydrolysis of lignocellulose. It is not suitable for evaluating alkaline pretreatment effectiveness at lower than 100 °C. According to the reported deducing process, this study modified the expression of [Formula: see text] as [Formula: see text] which is easier and more reasonable to assess the effectiveness of alkaline pretreatment. It showed that RCSF exhibited linear trend with lignin removal, and quadratic curve relation with enzymatic hydrolysis efficiency (EHE) at the same temperature. The EHE of alkali-treated SCB could attain the maximum value at lower RCSF, which indicated that it was not necessary to continuously enhance strength of alkaline pretreatment for improving EHE. Within a certain temperature range, the alkali loading was more important than temperature and time to influence pretreatment effectiveness and EHE. Furthermore, the contribution of temperature, time, and alkali loading to pretreatment cost which was seldom concerned was investigated in this work. The alkali loading contributed more than 70% to the pretreatment cost. This study laid the foundation of further optimizing alkaline pretreatment to reduce cost for its practical application.

Assessment and evaluation of cellulase production using ragi (Eleusine coracana) husk as a substrate from thermo-acidophilic Aspergillus fumigatus JCM 10253.

The cellulase production by filamentous fungi Aspergillus fumigatus JCM 10253 was carried out using agro-industrial waste ragi husk as a substrate in the microbial fermentation. The effect of the process parameters such as temperature, substrate concentration, pH, and incubation process time and their interdependence was studied using response surface methodology. The optimum cellulase activities were obtained at 50 °C under the conditions with 1-2% of substrate concentration at pH 2-4 for the incubation period of 7-8 days. The maximum carboxymethyl cellulase (CMCase) and ß-glucosidase activities with optimized process variables were 95.2 IU/mL and 0.174 IU/mL, respectively. The morphological characterization of fungus by scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR) revealed the presence of secondary protein structures. Furthermore, this study demonstrated that the application of ragi husk could be a promising feedstock for value-added industrial products. The thermo-acidophilic nature of isolated strain Aspergillus fumigatus JCM 10253 possessed a significant potential for higher titer of cellulase production that could be further employed for lignocellulosic bioethanol production.

[Fermentation of cellulase with multiple types of Salvia miltiorrhiza residues and other solid wastes from Chinese materia medica industrialization].

The solid wastes of Chinese materia dedica industrialization represented by Salvia miltiorrhiza residues have a strong small-molecule bio-recalcitrance in the process of high-value utilization of biotransformation. Highly tolerant strains were bred to break bio-recalcitrance of Salvia miltiorrhiza residues and produce high-value added cellulose, which has a significant significance for recycling and industrial utilization of solid waste. In this study, a strain of fungus, Penicillium expansum SZ13, was found with small-molecule antibacterial substance tanshinone contained in Salvia miltiorrhiza residues by a biological method. The optimal enzyme production process and peak period of SZ13 were determined. It was found that SZ13 could maintain peak enzyme production for 5 days by degrading residues under the conditions of temperature 35 ℃, rotation speed 180 r·min~(-1), 5% of residues addition, and 5% seed solution addition. Meanwhile, the ability of SZ13 to degrade the enzyme production of multiple types of residues was explored. The results showed a high enzyme activity and stable enzyme production of SZ13 in the process of degrading residues. SZ13 could efficiently utilize various types of Chinese medicine residues, such as Salvia miltiorrhiza residues, to realize the high-value utilization of cellulose in multiple types of residues.

Low-Cost Cellulase-Hemicellulase Mixture Secreted by Trichoderma harzianum EM0925 with Complete Saccharification Efficacy of Lignocellulose.

Fermentable sugars are important intermediate products in the conversion of lignocellulosic biomass to biofuels and other value-added bio-products. The main bottlenecks limiting the production of fermentable sugars from lignocellulosic biomass are the high cost and the low saccharification efficiency of degradation enzymes. Herein, we report the secretome of Trichoderma harzianum EM0925 under induction of lignocellulose. Numerously and quantitatively balanced cellulases and hemicellulases, especially high levels of glycosidases, could be secreted by T. harzianum EM0925. Compared with the commercial enzyme preparations, the T. harzianum EM0925 enzyme cocktail presented significantly higher lignocellulolytic enzyme activities and hydrolysis efficiency against lignocellulosic biomass. Moreover, 100% yields of glucose and xylose were obtained simultaneously from ultrafine grinding and alkali pretreated corn stover. These findings demonstrate a natural cellulases and hemicellulases mixture for complete conversion of biomass polysaccharide, suggesting T. harzianum EM0925 enzymes have great potential for industrial applications.

An assessment of the genomics, comparative genomics and cellulose degradation potential of Mucilaginibacter polytrichastri strain RG4-7.

In this study, whole genome sequencing and comparative genomic analyses were performed for Mucilaginibacter polytrichastri RG4-7 and its carboxymethyl cellulose degradation potential was assessed. The results showed that the genome of strain RG4-7 was 5.84 Mb and contained 5019 predicted genes, in which a high proportion of strain-specific genes were related to carbohydrate metabolism. The carboxymethyl cellulose (CMC) degradation and cellulase activity tests revealed the strong cellulose degradation ability, CMCase and ß-glucosidase activity in strain RG4-7. Real-time RT-PCR testing of most cellulose degradation related glycoside hydrolase (GH) families showed that GH9 (OKS85969), GH1 (OKS85832), GH3 (OKS89331 and OKS85615) were significantly up-regulated when strain RG4-7 was inoculated with CMC-Na, which suggested that GH9, GH1 and GH3 might determine its cellulose degradation ability. Certainly, further research need to be done to elucidate cellulose degradation mechanisms in strain RG4-7 in order to develop its industrial application value in lignocellulosic biomass degradation and waste management.

Microbial dynamics for lignocellulosic waste bioconversion and its importance with modern circular economy, challenges and future perspectives.

An extensive use of microbial dynamics for utilizing the lignocellulosic wastes has been attributed to their efficiency in bioenergy and bioproducts development as a cost effective high nutritional value. The integration of lignocellulosic waste into the circular economy can scaleup the sustainable bioproducts and bioenergy development. In this review paper, the aim is to describe the existing research efforts on organic lignocellulosic waste, cellulase producing microbes, their potential enzyme, modern circular economy with associated challenges and future perspectives. Presently, it has been reviewed that microbial cellulases have provided treasure bioproducts visions into industrial bioproducts marvels unveiled through lignocellulosic waste cutting-edge microbial explorations. Furthermore, the review focused on new insights of the growing circular economy of lignocellulosic waste used for many bioproducts and bioenergy dealings and explored the emergent lignocellulosic biorefinery approaches which could then be applied to review industrial-scale sustainable economic models for upgraded bioproducts and other production associated problems.

GeneHunt for rapid domain-specific annotation of glycoside hydrolases.

The identification of glycoside hydrolases (GHs) for efficient polysaccharide deconstruction is essential for the development of biofuels. Here, we investigate the potential of sequential HMM-profile identification for the rapid and precise identification of the multi-domain architecture of GHs from various datasets. First, as a validation, we successfully reannotated >98% of the biochemically characterized enzymes listed on the CAZy database. Next, we analyzed the 43 million non-redundant sequences from the M5nr data and identified 322,068 unique GHs. Finally, we searched 129 assembled metagenomes retrieved from MG-RAST for environmental GHs and identified 160,790 additional enzymes. Although most identified sequences corresponded to single domain enzymes, many contained several domains, including known accessory domains and some domains never identified in association with GH. Several sequences displayed multiple catalytic domains and few of these potential multi-activity proteins combined potentially synergistic domains. Finally, we produced and confirmed the biochemical activities of a GH5-GH10 cellulase-xylanase and a GH11-CE4 xylanase-esterase. Globally, this "gene to enzyme pipeline" provides a rationale for mining large datasets in order to identify new catalysts combining unique properties for the efficient deconstruction of polysaccharides.

Raw oil palm frond leaves as cost-effective substrate for cellulase and xylanase productions by Trichoderma asperellum UC1 under solid-state fermentation.

Production of cellulases and xylanase by a novel Trichoderma asperellum UC1 (GenBank accession no. MF774876) under solid state fermentation (SSF) of raw oil palm frond leaves (OPFL) was optimized. Under optimum fermentation parameters (30 °C, 60-80% moisture content, 2.5 × 106 spores/g inoculum size) maximum CMCase, FPase, ß-glucosidase and xylanase activity were recorded at 136.16 IU/g, 26.03 U/g, 130.09 IU/g and 255.01 U/g, respectively. Cellulases and xylanase were produced between a broad pH range of pH 6.0-12.0. The enzyme complex that comprised of four endo-ß-1,4-xylanases and endoglucanases, alongside exoglucanase and ß-glucosidase showed thermophilic and acidophilic characteristics at 50-60 °C and pH 3.0-4.0, respectively. Glucose (16.87 mg/g) and fructose (18.09 mg/g) were among the dominant sugar products from the in situ hydrolysis of OPFL, aside from cellobiose (105.92 mg/g) and xylose (1.08 mg/g). Thermal and pH stability tests revealed that enzymes CMCase, FPase, ß-glucosidase and xylanase retained 50% residual activities for up to 15.18, 4.06, 17.47 and 15.16 h of incubation at 60 °C, as well as 64.59, 25.14, 68.59 and 19.20 h at pH 4.0, respectively. Based on the findings, it appeared that the unique polymeric structure of raw OPFL favored cellulases and xylanase productions.