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.

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.

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.

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.

Cellulolytic enzyme production from agricultural residues for biofuel purpose on circular economy approach.

This study evaluated the production of cellulolytic enzymes from different agricultural residues. The crude enzyme extract produced was characterized and applied for saccharification of some agricultural residues. Maximum cellulolytic activities were obtained using soybean hulls. All enzymatic activities were highly stable at 40 °C at a pH range of 4.5-5.5. For stability at low temperatures, the enzyme extract was stored at freezing temperature and cooling for about 290 days without major loss of activity. The Km values found for total cellulase (FPase), endoglucanase (CMCase), and xylanase were 19.73 mg ml-1, 0.65 mg ml-1, and 22.64 mg ml-1, respectively, and Vmax values were 0.82 mol min-1 mg-1, 0.62 mol min-1 mg-1, and 104.17 mol min-1 mg-1 to cellulose, carboxymethyl cellulose, and xylan, respectively. In the saccharification tests, the total amount of total reducing sugars (TRS) released from 1 g of soybean hulls catalyzed by the enzymes present in the crude enzyme extract was 0.16 g g-1 dry substrate.

Assessment of Antioxidant and Antimicrobial Properties of Lignin from Corn Stover Residue Pretreated with Low-Moisture Anhydrous Ammonia and Enzymatic Hydrolysis Process.

Lignin accounts for 15-35% of dry biomass materials. Therefore, developing value-added co-products from lignin residues is increasingly important to improve the economic viability of biofuel production from biomass resources. The main objective of this work was to study the lignin extracts from corn stover residue obtained from a new and improved process for bioethanol production. Extraction conditions that favored high lignin yield were optimized, and antioxidant and antimicrobial activities of the resulting lignin were investigated. Potential estrogenic toxicity of lignin extracts was also evaluated. The corn stover was pretreated by low-moisture anhydrous ammonia (LMAA) and then subjected to enzymatic hydrolysis using cellulase and hemicellulase. The residues were then added with sodium hydroxide and extracted for different temperatures and times for enhancing lignin yield and the bioactivities. The optimal extraction conditions using 4% (w/v) sodium hydroxide were determined to be 50 °C, 120 min, and 1:8 (w:v), the ratio between corn stover solids and extracting liquid. Under the optimal condition, 33.92 g of lignin yield per 100 g of corn stover residue was obtained. Furthermore, the extracts produced using these conditions showed the highest antioxidant activity by the hydrophilic oxygen radical absorbance capacity (ORAC) assay. The extracts also displayed significant antimicrobial activities against Listeria innocua. Minimal estrogenic impacts were observed for all lignin extracts when tested using the MCF-7 cell proliferation assay. Thus, the lignin extracts could be used for antioxidant and antimicrobial applications, and improve the value of the co-products from the biomass-based biorefinery.

Characterization of a thermophilic cellulase from Geobacillus sp. HTA426, an efficient cellulase-producer on alkali pretreated of lignocellulosic biomass.

A themophilic cellulase-producing bacterium was isolated from a hot spring district and identified as Geobacillus sp. HTA426. The cellulase enzyme produced by the Geobacillus sp. HTA426 was purified through ammonium sulfate precipitation and ion exchange chromatography, with the recovery yield and fold purification of 10.14% and 5.12, respectively. The purified cellulase has a molecular weight of 40 kDa. The optimum temperature and pH for carboxymethyl cellulase (CMCase) activity of the purified cellulase were 60°C and pH 7.0, respectively. The enzyme was also stable over a wide temperature range of 50°C to 70°C after 5 h of incubation. Moreover, the strain HTA426 was able to grow and produce cellulase on alkali-treated sugarcane bagasse, rice straw and water hyacinth as carbon sources. Enzymatic hydrolysis of sugarcane bagasse, which was regarded as the most effective carbon source for cellulase production (CMCase activity = 103.67 U/mL), followed by rice straw (74.70 U/mL) and water hyacinth (51.10 U/mL). This strain producing an efficient thermostable cellulose is a potential candidate for developing a more efficient and cost-effective process for converting lignocellulosic biomass into biofuel and other industrial process.

Cost-effective production of cellulose hydrolysing enzymes from Trichoderma sp. RCK65 under SSF and its evaluation in saccharification of cellulosic substrates.

Trichoderma sp. is a potential cellulase producing mesophilic fungi which grow under mild acidic condition. In this study, growth and nutritional conditions were manipulated for the maximum and cost-effective production of cellulase using lab strain Trichoderma sp. RCK65 and checked for its efficiency in hydrolysis of Prosopis juliflora (a woody substrate). Preliminary studies suggested that when 48 h old secondary fungal culture (20 % v/w) was inoculated in wheat bran moistened with mineral salt solution (pH 4.5 and 1:3 solid to moisture ratio), incubated at 30 °C and after 72 h, it produced maximum cellulase (CMCase 145 U/gds, FPase 38 U/gds and ß-glucosidase 105 U/gds). However, using statistical approach a S:L ratio (1:1) was surprisingly found to be optimum that improved cellulase that is CMCase activity by 6.21 %, FPase activity by 23.68 % and ß-glucosidase activity by 37.28 %. The estimated cost of crude enzyme (Rs. 5.311/1000 FPase units) seems to be economically feasible which may be due to high enzyme titre, less cultivation time and low media cost. Moreover, when the crude enzyme was used to saccharify pretreated Prosopis juliflora (a woody substrate), it resulted up to 83 % (w/w) saccharification.

Cost evaluation of cellulase enzyme for industrial-scale cellulosic ethanol production based on rigorous Aspen Plus modeling.

Cost reduction on cellulase enzyme usage has been the central effort in the commercialization of fuel ethanol production from lignocellulose biomass. Therefore, establishing an accurate evaluation method on cellulase enzyme cost is crucially important to support the health development of the future biorefinery industry. Currently, the cellulase cost evaluation methods were complicated and various controversial or even conflict results were presented. To give a reliable evaluation on this important topic, a rigorous analysis based on the Aspen Plus flowsheet simulation in the commercial scale ethanol plant was proposed in this study. The minimum ethanol selling price (MESP) was used as the indicator to show the impacts of varying enzyme supply modes, enzyme prices, process parameters, as well as enzyme loading on the enzyme cost. The results reveal that the enzyme cost drives the cellulosic ethanol price below the minimum profit point when the enzyme is purchased from the current industrial enzyme market. An innovative production of cellulase enzyme such as on-site enzyme production should be explored and tested in the industrial scale to yield an economically sound enzyme supply for the future cellulosic ethanol production.

Advances in improving the performance of cellulase in ionic liquids for lignocellulose biorefinery.

Ionic liquids (ILs) have been considered as a class of promising solvents that can dissolve lignocellulosic biomass and then provide enzymatic hydrolyzable holocellulose. However, most of available cellulases are completely or partially inactivated in the presence of even low concentrations of ILs. To more fully exploit the benefits of ILs to lignocellulose biorefinery, it is critical to improve the compatibility between cellulase and ILs. Various attempts have been made to screen natural IL-tolerant cellulases from different microhabitats. Several physical and chemical methods for stabilizing cellulases in ILs were also developed. Moreover, recent advances in protein engineering have greatly facilitated the rational engineering of cellulases by site-directed mutagenesis for the IL stability. This review is aimed to provide the first detailed overview of the current advances in improving the performance of cellulase in non-natural IL environments. New ideas from the most representative progresses and technical challenges will be summarized and discussed.

Co-solvent pretreatment reduces costly enzyme requirements for high sugar and ethanol yields from lignocellulosic biomass.

We introduce a new pretreatment called co-solvent-enhanced lignocellulosic fractionation (CELF) to reduce enzyme costs dramatically for high sugar yields from hemicellulose and cellulose, which is essential for the low-cost conversion of biomass to fuels. CELF employs THF miscible with aqueous dilute acid to obtain up to 95 % theoretical yield of glucose, xylose, and arabinose from corn stover even if coupled with enzymatic hydrolysis at only 2 mgenzyme gglucan (-1) . The unusually high saccharification with such low enzyme loadings can be attributed to a very high lignin removal, which is supported by compositional analysis, fractal kinetic modeling, and SEM imaging. Subsequently, nearly pure lignin product can be precipitated by the evaporation of volatile THF for recovery and recycling. Simultaneous saccharification and fermentation of CELF-pretreated solids with low enzyme loadings and Saccharomyces cerevisiae produced twice as much ethanol as that from dilute-acid-pretreated solids if both were optimized for corn stover.

Manufacturing economics of plant-made biologics: case studies in therapeutic and industrial enzymes.

Production of recombinant biologics in plants has received considerable attention as an alternative platform to traditional microbial and animal cell culture. Industrially relevant features of plant systems include proper eukaryotic protein processing, inherent safety due to lack of adventitious agents, more facile scalability, faster production (transient systems), and potentially lower costs. Lower manufacturing cost has been widely claimed as an intuitive feature of the platform by the plant-made biologics community, even though cost information resides within a few private companies and studies accurately documenting such an advantage have been lacking. We present two technoeconomic case studies representing plant-made enzymes for diverse applications: human butyrylcholinesterase produced indoors for use as a medical countermeasure and cellulases produced in the field for the conversion of cellulosic biomass into ethanol as a fuel extender. Production economics were modeled based on results reported with the latest-generation expression technologies on Nicotiana host plants. We evaluated process unit operations and calculated bulk active and per-dose or per-unit costs using SuperPro Designer modeling software. Our analyses indicate that substantial cost advantages over alternative platforms can be achieved with plant systems, but these advantages are molecule/product-specific and depend on the relative cost-efficiencies of alternative sources of the same product.

Thermobifida fusca cellulases exhibit limited surface diffusion on bacterial micro-crystalline cellulose.

Elucidation of cellulase-cellulose interactions is key to modeling biomass deconstruction and in understanding the processes that lead to cellulase inactivation. Here, fluorescence recovery after photobleaching and single molecule tracking (SMT) experiments are used to assess the surface diffusion of Thermobifida fusca cellulases on bacterial micro-crystalline cellulose. Our results show that cellulases exhibit limited surface diffusion when bound to crystalline cellulose and that a large fraction of the cellulases remain immobile even at temperatures optimal for catalysis. A comparison of our experimental results to Monte Carlo (MC) simulations, which use published diffusion coefficients to model cellulase displacements, shows that even those enzymes that are mobile on the cellulose surface exhibit significantly slower diffusive motions than previously reported. In addition, it is observed that the enzymes that show significant displacements exhibit complex, non-steady surface motions, which suggest that cellulose-bound cellulases exist in molecular states with different diffusive characteristics. These results challenge the notion that cellulases can freely diffuse over cellulose surfaces without catalyzing bond cleavage.

Optimizing the enzyme loading and incubation time in enzymatic hydrolysis of lignocellulosic substrates.

A mathematical model for costing enzymatic hydrolysis of lignocellulosics is presented. This model is based on three variable parameters describing substrate characteristics and three unit costs for substrate, enzymes and incubation. The model is used to minimize the cost of fermentable sugars, as intermediate products on the route to ethanol or other biorefinery products, by calculating optimized values of enzyme loading and incubation time. This approach allows comparisons between substrates, with processing conditions optimized independently for each substrate. Steam-exploded pine wood was hydrolyzed in order to test the theoretical relationship between sugar yield and processing conditions.

Scaled-up separation of cellobiohydrolase1 from a cellulase mixture by ion-exchange chromatography.

Enzymatic hydrolysis of cellulose often involves cellulases produced by Trichoderma reesei, of which cellobiohydrolase1 (CBH1) is the most abundant (about 60% of total cellulases) and plays an important role in the hydrolysis of crystalline cellulose. A method for separating sufficient quantities from the bulk cellulase cocktail is highly desirable for many studies, such as those that aim to characterize binding and hydrolysis kinetics of CBH1. In this work, CBH1 was separated from other Spezyme CP cellulases by ion-exchange chromatography using an efficient modification of a smaller scale process. The ion-exchange column was connected to a vacuum manifold system to provide a steady flow through parallel columns and thus achieve scale-up for enzyme separation. With five 5-mL columns running in parallel, about 55 mg of CBH1 was separated from 145 mg of Spezyme CP in a single separation. Step elution was used to replace the continuous gradient used at smaller scale. The purified CBH1 was collected in the fraction eluted with a buffer containing 0.33 M salt and showed comparable purity and activity as the enzyme purified by a fast protein liquid chromatography system. The stability of separated CBH1 was studied for up to 2 days and good thermal stability was observed. Separated CBH1 also showed both high adsorption to bacterial microcrystalline cellulose with ~4 µmol/g maximum adsorption and a K(a) of 5.55 ± 2.34 µM(-1) , and good hydrolytic activity based on atomic force microscopy observations that show a reduction in fiber height.

Bamboo saccharification through cellulose solvent-based biomass pretreatment followed by enzymatic hydrolysis at ultra-low cellulase loadings.

The modified cellulose solvent- (concentrated phosphoric acid) and organic solvent- (95% ethanol) based lignocellulose fractionation (COSLIF) was applied to a naturally-dry moso bamboo sample. The biomass dissolution conditions were 50 degrees C, 1 atm for 60 min. Glucan digestibility was 88.2% at an ultra-low cellulase loading of one filter paper unit per gram of glucan. The overall glucose and xylose yields were 86.0% and 82.6%, respectively. COSLIF efficiently destructed bamboo's fibril structure, resulting in a approximately 33-fold increase in cellulose accessibility to cellulase (CAC) from 0.27 to 9.14 m(2) per gram of biomass. Cost analysis indicated that a 15-fold decrease in use of costly cellulase would be of importance to decrease overall costs of biomass saccharification when cellulase costs are higher than $0.15 per gallon of cellulosic ethanol.

Development of activity-based cost functions for cellulase, invertase, and other enzymes.

As enzyme chemistry plays an increasingly important role in the chemical industry, cost analysis of these enzymes becomes a necessity. In this paper, we examine the aspects that affect the cost of enzymes based upon enzyme activity. The basis for this study stems from a previously developed objective function that quantifies the tradeoffs in enzyme purification via the foam fractionation process (Cherry et al., Braz J Chem Eng 17:233-238, 2000). A generalized cost function is developed from our results that could be used to aid in both industrial and lab scale chemical processing. The generalized cost function shows several nonobvious results that could lead to significant savings. Additionally, the parameters involved in the operation and scaling up of enzyme processing could be optimized to minimize costs. We show that there are typically three regimes in the enzyme cost analysis function: the low activity prelinear region, the moderate activity linear region, and high activity power-law region. The overall form of the cost analysis function appears to robustly fit the power law form.

An evaluation of spherical designs for molecular-like surfaces.

The use of spherical harmonics in the molecular sciences is widespread. They have been employed with success in, for instance, the crystallographic fast rotation function, small-angle scattering particle reconstruction, molecular surface visualisation, protein-protein docking, active site analysis and protein function prediction. The calculation of spherical harmonic expansion coefficients requires integration over the full sphere and can be a computationally cumbersome and also numerically sensitive (with respect to the integration weights) procedure. It is shown here how the use of spherical t-designs and pre-computed near-equal weight integration layouts can significantly reduce the computational effort in the determination of spherical harmonic expansion coefficients for molecular surfaces, thus giving rise to a robust and highly efficient algorithm for the construction of molecular-like objects.

Enzyme recovery and recycling following hydrolysis of ammonia fiber explosion-treated corn stover.

Both cellulase and cellobiase can be effectively recovered from hydrolyzed biomass using an ultrafiltration recovery method. Recovery of cellulase ranged from 60 to 66.6% and for cellobiase from 76.4 to 88%. Economic analysis shows that cost savings gained by enzyme recycling are sensitive to enzyme pricing and loading. At the demonstrated recovery of 60% and current loading of 15 Filter paper units of cellulase/g of glucan, enzyme recycling is expected to generate a cost savings of approx 15%. If recovery efficiency can be improved to 70%, the savings will increase to >25%, and at 90% recovery the savings will be 50%.

Effect of reduction in yeast and enzyme concentrations in a simultaneous- saccharification-and-fermentation-based bioethanol process: technical and economic evaluation.

The ethanol production cost in a simultaneous saccharification and fermentation-based bioethanol process is influenced by the requirements for yeast production and for enzymes. The main objective of this study was to evaluate--technically and economically--the influence of these two factors on the production cost. A base case with 5 g/L of baker's yeast and an initial concentration of water-insoluble solids of 5% resulted in an experimental yield of 85%. When these data were implemented in Aspen Plus, yeast was assumed to be produced from sugars in the hydrolysate, reducing the overall ethanol yield to 69%. The ethanol production cost was 4.80 SEK/L (2.34 US$/gal). When adapted yeast was used at 2 g/L, an experimental yield of 74% was achieved and the estimated ethanol production cost was the same as in the base case. A 50% reduction in enzyme addition resulted in an increased production cost, to 5.06 SEK/L (2.47 US$/gal) owing to reduced ethanol yield.