Enzyme Cocktail Research Articles

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

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

Enzymatic digestibility of lignocellulosic wood biomass: Effect of enzyme treatment in supercritical carbon dioxide and biomass pretreatment

Energy and resource intensive mechanical and chemical pretreatment along with the use of hazardous chemicals are major bottlenecks in widespread lignocellulosic biomass utilization. Herein, the study investigated different pretreatment methods on spruce wood namely supercritical CO2 (scCO2) pretreatment, ultrasound-assisted alkaline pretreatment, and acetosolv pulping-alkaline hydrogen peroxide bleaching, to enhance the enzymatic digestibility of wood using optimized enzyme cocktail. Also, the effect of scCO2 pretreatment on enzyme cocktail was investigated after optimizing the concentration and temperature of cellulolytic enzymes. The impact of scCO2 and ultrasound-assisted alkaline pretreatments of wood were insignificant for the enzymatic digestibility, and acetosolv pulping-alkaline hydrogen peroxide bleaching was the most effective pretreatment that showed the release of total reducing sugar yield (TRS) of ∼95.0 wt% of total hydrolyzable sugars (THS) in enzymatic hydrolysis. The optimized enzyme cocktail showed higher yield than individual enzymes with degree of synergism 1.34 among the enzymes, and scCO2 pretreatment of cocktail for 0.5–1.0 h at 10.0–22.0 MPa and 38.0–54.0 °C had insignificant effect on the enzyme's primary and global secondary structure of cocktail and its activity.

Process optimization for saccharification and fermentation of the Organic Fraction of Municipal Solid Waste (OFMSW) to maximize ethanol production performance

The Organic Fraction of Municipal Solid Waste (OFMSW) contains paper and cardboard, an attractive cellulosic feedstock for bioethanol production. Waste paper/cardboard (WPC) was subjected to mild acid treatment in solids loadings from 20 to 27.5 %. Different doses of commercial enzyme cocktail and various adjuvants facilitating enzymatic saccharification were tested. Enzyme dosing of 3.75 FPU/g fiber with very slow fed-batch saccharification of 27.5 % (w/v) solids from model lignin-free paper pulp resulted in 12.6 % (w/v) total sugar after 74 h. Use of cheap non-catalytic waste protein adjuvants including Soymeal, Protisyr, Feather meal strongly improved enzymatic saccharification and reduced the overall process cost by reducing commercial enzyme dosing. After fermentation with the industrial yeast strain BMD44 (Cellusec® 1.0) an ethanol titer of 5.77 % (v/v) was obtained using a reduced enzyme loading of 2.5 FPU/g of actual industrial WPC fiber. These results show the potential of economically profitable second-generation bioethanol production with WPC fiber.

305 Dietary Carbohydrase Mixture Supplementation and Fiber Levels on Ileal Digestibility of Nutrients in Diets for Gestating Sows

Abstract Supplementation of multi-carbohydrase enzyme cocktails can increase nutrient digestibility of fibrous diets in grower pigs, particularly acting on dietary non-starch polysaccharides (NSP). Although NSP degrading enzymes are extensively used in sow diets, there is little data characterizing the NSP enzyme effects. Our previous work demonstrated a 10% increase in nutrient digestibility in diets fed to gestating sows. The objective of this study was to evaluate the effect of a carbohydrase mixture (CM) and dietary fiber level on standardized ileal digestibility (SID) of protein and amino acids (AA) and apparent ileal digestibility (AID) of NSP in gestation diets fed to pregnant sows. Ileal cannulas were placed in 12 gestating sows (Parity 0 to 2) who were allowed to recover for 1 week, then assigned to either a high (HF; 17.5% NDF) or low fiber (LF; 10% NDF) diet with one of 3 levels of CM supplementation (‘0’, ‘0.08’, and ‘0.10’%) or a nitrogen-free diet to determine ileal endogenous AA losses. Diets were corn-soybean meal based with soyhulls and corn DDGS as fiber sources and formulated to reflect industry-relevant diets. Thus, oil was included at 3% in HF to maintain similar formulated metabolizable energy content. Sows were fed once daily at 2.2 kg/d. Ileal digesta samples were collected for 12 h on 2 successive days following a 7-day adaptation period. At the end of digesta collection, sows were assigned a different diet in a new adaptation/digesta collection period. There were 5 collection periods for a total of 8 observations/diet. Digesta was analyzed for crude protein, amino acids, NSP, and titanium as the indigestible marker to determine AID and SID. There was no interaction between diet fiber level and CM supplementation. Carbohydrase mixture supplementation to gestating sow diets did not impact SID of crude protein and AA regardless of dietary fiber level. The SID of His, Ile, Lys, Phe, Thr, Trp, and Val were 3 to 6% lower (P < 0.09) in HF than LF independent of CM. There was no impact of fiber level on the average SID of amino acids in gestating sow diets which was 83.5%. Supplementation of CM did not impact AID of NSP components, but sows fed HF had greater AID of arabinose (LF: 26.5% vs HF: 40.6%), xylose (LF: 3.5% vs HF: 40.9%), and total NSP (LF: 25.9% vs HF: 40.0%) compared with sows fed LF (P < 0.05). Inclusion of CM in high fibrous gestating sow diets did not increase the SID of AA or AID of NSP, but a high fiber gestation diet reduced SID of AA. As such, differences in SID of AA should be considered when formulating high fibrous diets for gestating sows.

Effect of alkaline and steam pre-treatment on saccharification of corn cob and production of cellullase from fungal consortium

Lignocellulosic biomass as fossil fuel alternative comes with its challenges such as its inherent stability and recalcitrance. The use of commercial cellulase in the removal of lignin comes with high cost. This research seeks to answer questions on how alkaline and steam pre-treatment improve cellulose/glucose liberation from lignocellulosic biomass (corn cobs) using a consortium of <i>pichia kudriavzevii</i> and <i>cyberlindnera fabianii.</i><br /> In the current study, the effectiveness of steam and sodium hydroxide (NaOH) pre-treatment for reducing corn cob structure was examined, and the pre-treated biomass afterwards was exposed to the hydrolyzing activity of a consortium enzyme cocktail that was custom-formulated. The results of an analysis of composition showed that while alkaline pre-treated corn cob (APC) had 1.2% lignin, 75.8% cellulose, and 10.9% hemicellulose, steam pre-treated corn cob (SPC) had 2.5% lignin, 67.2% cellulose, and 25% hemicellulose. Lignin was eliminated from the biomass of corn cobs using both steam and NaOH pre-treatment. The hydrolyzing effect of the holocellulolytic enzyme cocktail, prepared with two multifunctional enzymes, was applied to the alkaline and steam pre-treated samples. This hydrolyzed SPCs more effectively than APC feedstocks, revealing that steam was a more effective pre-treatment attaining a remarkable 8.33 U/mg endoglucanase, 5.56 U/mg exoglycanase and 8.97U/mg beta-glucosidase levels (event 1) and glucose peak concentration of 0.433 mol/mL at 48 hours (event 2); according to a thorough examination of cellulase capacity and glucose levels.<br /> Overall, the consortium enzyme cocktail effectively hydrolyzed agricultural feedstocks that had undergone alkaline pre-treatment, making it a desirable option for usage in the bioconversion procedure in the biorefinery sector. This study demonstrates an effective technique for turning agricultural waste (corn cob) into high-value products through effective and practical chemical pre-processing.

Comparing DNA isolation methods for forest trees: quality, plastic footprint, and time-efficiency

BackgroundGenetic and genomic studies are seeing an increase in sample sizes together with a wider range of species investigated in response to environmental change concerns. In turn, these changes may come with challenges including the time and difficulty to isolate nucleic acids (DNA or RNA), the sequencing cost and environmental impacts of the growing amount of plastic waste generated in the process. Pseudotsuga menziesii var. menziesii (Mirbel) Franco (PM), Tsuga heterophylla (Raf.) Sarg. (TH) and Thuja plicata Donn ex D.Don (TP) are conifer species found in diverse woodlands both as natives and naturalized exotics. Our study was carried out whilst investigating their genetics to understand their population structure and potential for adaptation.ResultsIn the present study, we compared two different DNA isolation methods, i.e., spin-column DNeasy plant mini kit (QIAGEN), and temperature-driven enzymatic cocktail Plant DNA Extraction (MicroGEM). The quantity of recovered DNA and the quality of DNA were assessed along with the plastic footprint and time needed for three tree species. Both methods were optimised and proven to provide enough DNA for each studied species. The yield of DNA for each method depended on the species: QIAGEN showed higher yield in P. menziesii and T. heterophylla, while T. plicata recovered similar amount of DNA for both methods. The DNA quality was investigated using DNA barcoding techniques by confirming species identity and species discrimination. No difference was detected in the PCR amplification of the two barcoding loci, (rbcL and trnH-psbA), and the recovered sequences between DNA isolation methods. Measurement of the plastic use and the processing time per sample indicated that MicroGEM had a 52.64% lower plastic footprint and was 51.8% faster than QIAGEN.ConclusionsQIAGEN gave higher yields in two of the species although both methods showed similar quality results across all species. However, MicroGEM was clearly advantageous to decrease the plastic footprint and improve the time efficiency. Overall, MicroGEM recovers sufficient and reliable DNA to perform common downstream analyses such as PCR and sequencing. Our findings illustrate the benefits of research and efforts towards developing more sustainable methods and techniques to reduce the environmental footprint of molecular analyses.

Intensification of corn fiber saccharification using a tailor made enzymatic cocktail

The transition from an economic model based on resource extraction to a more sustainable and circular economy requires the development of innovative methods to unlock the potential of raw materials such as lignocellulosic biomasses. Corn fiber differs from more traditional lignocellulosic biomasses due to its high starch content, which provides additional carbohydrates for fermentation-based biomanufacturing processes. Due to its unique chemical composition, this study focused on the development of a tailor made enzymatic cocktail for corn fiber saccharification into monosaccharides. Three commercially available hydrolytic enzymes (Cellic® CTec2, Pentopan® Mono BG, and Termamyl® 300 L) were combined to hydrolyze the polysaccharide structure of the three main carbohydrate fractions of corn fiber (cellulose, hemicellulose and starch, respectively). Prior to saccharification, corn fiber was submitted to a mild hydrothermal pretreatment (30 min at 100 °C). Then, two experimental designs were used to render an enzymatic cocktail capable of providing efficient release of monosaccharides. Using 60 FPU/g DM of Cellic® CTec2 and 4.62 U/g DM of Termamyl® 300 L, without addition of Pentopan® Mono BG, resulted in the highest efficiencies for glucose and xylose release (66% and 30%, respectively). While higher enzyme dosages could enhance the saccharification efficiency, adding more enzymes would have a more pronounced effect on the overall process costs rather than in increasing the efficiency for monosaccharides release. The results revealed that the recalcitrance of corn fiber poses a problem for its full enzymatic degradation. This fact combined with the unique chemical composition of this material, justify the need for developing a tailor made enzymatic cocktail for its degradation. However, attention should also be given to the pretreatment step to reduce even more the recalcitrance of corn fiber and improve the performance of the tailored cocktail, as a consequence.

Integrated biorefinery for xylooligosaccharides, pectin, and bioenergy production from orange waste

AbstractOrange waste is one of the most underutilized biowastes. It accounts for 50% of the fruit weight after juice extraction, presenting an environmental problem due to inappropriate disposal. Industrial waste exploitation is a sustainable way to obtain bioproducts and bioenergy. However, the efficiency of chemical treatments and enzymatic cocktails is still a bottleneck in these processes. One potential route to reduce costs is to design consecutive extraction and hydrolysis stages to yield more products from different fractions of the same feedstock, reducing the use of time and bulk materials. This study assessed the economic performance of an orange processing waste (OPW) biorefinery for xylooligosaccharides (XOS), pectin, and bioenergy, involving three aspects: (1) experimental procedures, (2) a simulation approach, and (3) optimization tools. The optimization of hydrothermal treatment for pectin extraction decreased material costs by more than 60% and preserved the xylan content for xylooligosaccharide production by enzymatic hydrolysis. This study analyzed different biorefinery capacities, and solid waste exploitation for heat and steam generation, improving sustainability. The production cost per kg of high‐purity pectin and xylooligosaccharides is 3.67 $ with an efficient solvent recovery and self‐sufficiency for standard power usage. These results proved the economic feasibility and low environmental impact of the OPW biorefinery.

Effect of exogenous fibrolytic enzymes cocktail incorporation on in vitro degradation and fermentation characteristics of oats straw based total mixed ration

The study was conducted to evaluate the effects of increasing doses: 0 (control), 0.20, 0.40, 0.60, 0.80 and 1.00 % DM of an exogenous fibrolytic enzymes (EFE) cocktail preparation on in vitro gas production (GP), nutrient degradability and fermentation characteristics of oats straw based Total Mixed Ration (TMR) with 60:40 roughage to concentrate ratio using sheep rumen liquor. The chemical composition of all the feed ingredients used for preparation of the experimental TMR (containing 16.63% crude protein and 90.51% organic matter) were within the normal ranges. Increasing the incorporation level of enzyme cocktail linearly as well as quadratically increased net GP, metabolisable energy content, short chain fatty acid concentrations and microbial crude protein production up to 0.60% DM level (L3) with no additional improvement at further higher levels. There were significant improvements in degradability of dry matter, organic matter and neutral detergent fibre up to the enzyme dose of 0.60% DM (L3) with constant values thereafter. Fermentation characteristics response to varying incorporation doses of EFE cocktail also revealed improvements up to 0.60% DM level (L3) with no effect on non-protein nitrogen contents. It is recommended that EFE cocktail incorporation dose of 0.60% DM to be used for efficient utilisation of oats straw based complete feed; however, this requires further testing by in vivo studies.

Development of tailored bioprocess for pretreatment and saccharification of corn stalk into bioethanol using hydrolytic enzymes cocktail and fermentative yeasts

Agriculture waste residue (corn stalk), a rich source of reducing sugars explored in the present study for the production of bioethanol using hydrolytic enzymes and fermenting yeast cocktails. Corn stalk pulp was biologically pretreated using hydrolytic enzymes cocktail of ligninase, cellulase and xylanase produced from Bacillus sp. PHS-05, Bacillus subtilis CP-S66 and Bacillus safensis XP-S7 with 2.34 ± 0.28 U/ml, 6.89 ± 0.36 U/ml and 11.9 ± 0.22 U/ml enzyme activities respectively. This biological pretreatment of corn stalk pulp resulted into 51.41 ± 0.34 % removal of lignin and 77.43 ± 2.44 % extraction of reducing sugar (C5 & C6). The changes occur in corn stalk pulp after enzymatic saccharification was confirmed by Fourier-Transform Infrared Spectroscopy (FTIR). Corn stalk sugar hydrolysate was co-fermented into 0.77 ± 0.06 g/g bioethanol with yield of 26.6 ± 0.46 g/kg of biomass, using Kluyveromyces marxianus MTCC 1498 and Saccharomyces cerevisiae. Further, purity (94.27 %) and volumetric productivity (0.20 ± 0.04 g/L/h) of bioethanol was confirmed using Gas Chromatography-Mass Spectrometry (GC-MS). Present findings provide valuable insight to obtain second generation biofuels to solve energy crisis, and environmental problems besides boosting socioeconomic prosperity through sustainable management of agro-residues.

Agroindustrial and food processing residues valorization for solid-state fermentation processes: A case for optimizing the co-production of hydrolytic enzymes

In the pursuit of sustainability, managing agro-industrial and food processing residues (AFR) efficiently is crucial. This study proposes a systematic approach to convert AFR into valuable products via solid-state fermentation (SSF). Using fungal enzyme production as a case study, this adaptable methodology suits any SSF bioprocess. Initially, AFR's physicochemical properties were evaluated to assess their feasible use as carbon sources and solid matrices for SSF. Then, five strains were screened for their capability to produce enzymes (Xylanase, X; pectinase, P; cellulase, C). Apple pomace (AP) and brewery spent grain (BSG) with Aspergillus sp. (strain G5) were selected. Subsequent steps involved a two-phase statistical approach, identifying critical factors and optimizing them. Process conditions were screened using a Plackett-Burman design, narrowing critical variables to three (BSG/AP, pH, humidity). Response Surface Methodology (Central Composite Design) further optimized these factors for co-synthesis of X, P, and C. The humidity had the most significant effect on the three responses. The optimum conditions depended on each enzyme and were further validated to maximize either X, P or C. The obtained extracts were used for pectin extraction from orange peels. The extract containing primarily xylanase (X = 582.39, P = 22.86, C = 26.10 U mL−1) showed major pectin yield recovery (12.33 ± 0.53%) and it was obtained using the optimal settings of BSG/AP (81/19), humidity (50.40%), and pH (4.58). The findings will enable adjusting process conditions to obtain enzymatic cocktails with a tailored composition for specific applications.

A new paradigm in lignocellulolytic enzyme cocktail optimization: Free from expert-level prior knowledge and experimental datasets

Effectively pairing diverse lignocellulolytic enzyme cocktails with intricately structured lignocellulosic substrates is an enduring challenge for science and technology. To date, extensive trial-and-error remains the primary approach and no deep-learning methods were developed to address it due to limited experimental data and incomplete expert-level knowledge of enzyme-cocktail-substrate structure-dynamics-function relationships. Here, a novel model is developed to tackle this issue in efficient, cost-effective, and high-throughput manners. It needs no pre-labeled datasets, instead utilizing simple features, eliminating the reliance on expert-level prior knowledge of reaction mechanisms. Experimentally optimal combinations were found within predicted ranges of tailor-made combinations with precision of 91.98%, covering 80.00% of overall top-100. Practical tests demonstrated its effectiveness in narrowing down potential optimal combinations, speeding up targeted screening, and enabling efficient degradation of lignocellulosic biomass. The method has good applications in artificial proteins biosynthesis from low-value lignocellulosic straw, providing alternative solutions for biomass biorefining challenges in complex enzyme-cocktail-substrate interactions.

Enhancing thermophilic anaerobic digestion of municipal sludge: An investigation

Thermophilic anaerobic digestion (TAD), although less explored as opposed to mesophilic AD is a viable strategy to reduce the volume of municipal sludge and generate renewable energy in the form of biogas simultaneously. In this study, biochemical methane potential was tested to compare three different pretreatments i.e., ultrasonication, biochar (black spruce wood and bark, California almond hull), enzyme (cocktail of endoglucanase, β-glucosidase, xylanase) for their ability to enhance TAD of municipal sludge. In addition to conventional parameters such as chemical oxygen demand (COD), volatile solids (VS), and biogas, the respiratory value and the pathogen removal were also evaluated. Ultrasonication for 20 min was found to be the best pretreatment in terms of higher COD removal, biogas generation and VS reduction. This is possible due to the increase in soluble fraction of organic matter after ultrasonication which facilitated effective hydrolysis and reduction in start-up time. However, pathogen removal was mostly facilitated by AD and the contribution of pretreatment was minimal.

Enzyme cocktail with hyperactive lipase through solid-state fermentation by the novel strain Penicillium sp. Y-21

Lipase is a kind of industrial enzyme preparation with various catalytic abilities and is widely used in food, energy, medicine and other fields. To increase lipase and enzyme cocktail activity through solid-state fermentation, the novel strain Penicillium sp. Y-21 was obtained through ethyl methanesulfonate (EMS) mutation from the novel strain Y, which was isolated from soils. Solid-state fermentation by strain Y-21 using agricultural byproducts was carried out in tray bioreactors. The optimum culture composition for enzyme cocktail fermentation was soybean meal 20 g, 3% (w/w) glucose, 1% (w/w) peptone, 5% (w/w) lard, 0.04% (w/w) CaCl2, 0.04% (w/w) FeCl3, 28 °C for 72 h. The enzyme cocktail produced by strain Y-21 is a kind of multienzyme complex, containing xylanase, glucanase, acidic protease, pectinase, cellulase and lipase, and their enzymatic activities (unit: U g−1) were 8000, 6000, 8000, 2000, 3000 and 120, respectively. During the fermentation process, the lipase coding genes pel, pha, and p12 were also studied and amplified from the RNA of Penicillium sp. Y-21 by RT-PCR. The results showed that the pel gene played an important role in enzyme production. Afterwards, an enzyme cocktail can be added to chicken feed as an additive, which improves animal growth and feed efficiency.

Enhancement of rice husks saccharification through hydrolase preparation assisted by lytic polysaccharide monooxygenase

Rice husk is an abundant agricultural waste generated from rice production, but its application is limited. Considering its complex components, the rice husk was hydrolyzed by different enzymes to enhance its saccharification. In this study, saccharification of the rice husk by cellulase, xylosidase, and xylanase was first investigated. The synergistic effect of LPMO on the above hydrolases and different enzyme combinations in the saccharification process was then explored. Thereafter, the formulation of the enzyme cocktail and the degradation conditions were optimized to obtain the highest saccharification efficiency. The results showed that the optimum enzyme cocktail consists of Celluclast 1.5 L (83.3 mg/g substrate), the key enzymes in the saccharification process, worked with BpXyl (20 mg/g substrate), BpXyn11 (24 mg/g substrate), and R17L/N25G (4 mg/g substrate). The highest reducing sugar concentration (1.19 mg/mL) was obtained at pH 6.0 and 60 ℃ for 24 h. Fourier transform infrared spectroscopy and scanning electron microscopy were employed to characterize the structural changes in the rice husk after degradation. The results showed that the key chemical bonds in cellulose and hemicellulose were broken. This study illuminated the concept of saccharifying lignocellulose from rice husk using LPMO synergistically assisted combined-hydrolase including cellulase, xylosidase, and xylanase, and provided a theoretical basis for lignocellulose biodegradation.

Immobilization of β-glucosidase on polyethersulfone membrane for cellobiose hydrolysis

Glucosidase is one of the enzyme cocktail that is used for converting lignocellulose to glucose. The significance of glucosidase is that it converts the cellobiose to glucose and subsequently avoid product inhibition caused by cellobiose. In this work, β-glucosidase was immobilized on polyethersulfone membrane via glutardehyde crosslinking for the hydrolysis of cellobiose. Based on the peaks of FTIR spectra of the membrane, enzyme was adsorbed based on the absorbance between 3100 and 3500 cm−1. The percentage of enzyme adsorption in the membrane was 6.8 % based on the initial protein solution. Kinetic parameters of β-glucosidase of immobilized on membrane have been studied and compared with free enzyme used. The Km values for the free and immobilized gained were 166.137 g/L and 36.176 g/L respectively. The immobilized enzyme showed lower Km indicating higher affinity of substrates than free enzyme. The Vmax value obtained was 60.606 g/L h for immobilized enzyme and 136.986 g/L h for free enzyme probably due to limited access of substrates towards the immobilized enzymes. The immobilized enzyme can be reused for at least seven cycles.

MicroMagnify: A Multiplexed Expansion Microscopy Method for Pathogens and Infected Tissues.

Super-resolution optical imaging tools are crucial in microbiology to understand the complex structures and behavior of microorganisms such as bacteria, fungi, and viruses. However, the capabilities of these tools, particularly when it comes to imaging pathogens and infected tissues, remain limited. MicroMagnify (µMagnify) is developed, a nanoscale multiplexed imaging method for pathogens and infected tissues that are derived from an expansion microscopy technique with a universal biomolecular anchor. The combination of heat denaturation and enzyme cocktails essential is found for robust cell wall digestion and expansion of microbial cells and infected tissues without distortion. µMagnify efficiently retains biomolecules suitable for high-plex fluorescence imaging with nanoscale precision. It demonstrates up to eightfold expansion with µMagnify on a broad range of pathogen-containing specimens, including bacterial and fungal biofilms, infected culture cells, fungus-infected mouse tone, and formalin-fixed paraffin-embedded human cornea infected by various pathogens. Additionally, an associated virtual reality tool is developed to facilitate the visualization and navigation of complex 3D images generated by this method in an immersive environment allowing collaborative exploration among researchers worldwide. µMagnify is a valuable imaging platform for studying how microbes interact with their host systems and enables the development of new diagnosis strategies against infectious diseases.

Enzymatic valorization of cellulosic and hemicellulosic-based biomasses via the production of antioxidant water-soluble hydrolyzate of maize stalks and the green bio-deinking of mixed office waste paper

AbstractBio-valorization of various biomasses provides a sustainable promising approach for the eco-friendly production of variable value-added products. Herein, the current study devoted to the enzymatic valorization of two widely available biomasses, namely, maize stalks and waste paper. The cellulytic and hemicellulytic-rich cocktail was produced through the fermentation of rice straw by a locally isolated fungal strain Aspergillus terreus. The potential applicability of the produced cocktail for the enzymatic hydrolysis of the polysaccharide constituents of maize stalks was evaluated under various strategies. The reported results indicated that the microwave pretreatment of the biomass yielding a water-soluble hydrolyzate rich in cellobiose and xylobiose, sustained by thin layer (TLC) and high-performance liquid chromatographic (HPLC) measurements, in addition to phenolic compounds. Moreover, the enzymatic hydrolysis of the extracted hemicellulosic fraction from maize stalks was rich in xylooligosaccharides and phenolic compounds higher than that released from the hydrolysis of commercial xylan. The estimated antioxidant activity of the resulted hydrolyzate was also monitored by the scavenging of 1,1-diphenyl-2-picrylhydrazyl free radical spectrophotometrically at 515 nm. Moreover, the potential applicability of the produced enzymatic cocktail was examined for the bio-deinking of waste paper. The physical, chemical, and surface morphological characteristics of the treated paper sample was compared to a blank one regarding the whiteness index, ash content, scanning electron microscopy (SEM), energy dispersive X-ray (EDX), and Fourier transform infrared spectroscopy (FTIR). On the base of the estimated results, the produced enzymatic cocktail possessed efficient dislodgement ability for the printed ink from the paper surface.

Anaerobic Fermentation of Mixed Fruits Peel Waste for Functional Enzymes Production Employing Palm Sugar and Molasses as The Carbon Source

The daily abundant generation of fruit peel waste potentially triggers environmental problems if no appropriate management is performed. Besides, fruit peel waste can be a valuable source for functional enzyme production. This study aims to investigate the use of molasses and palm sugar as sugar source during anaerobic fermentation of banana and papaya peel waste to produce functional enzymes. The fermentation was subjected to biomass at various banana peels to papaya peels mass ratio for 3 months. The feed consisted of sugar:biomass:water, and their mass ratio was kept constant at 1:3:10. Both brown functional enzyme cocktails obtained from the fermentation using palm sugar and molasses as carbon source were acidic with pH of 3.7 and 4.4, respectively. The amylase, protease, and lipase activities of the functional enzyme cocktails was maximum when the biomass mixture contained four portions of banana peel and one portion of papaya peel. In addition, molasses was found to be the better sugar source than palm sugar for producing functional enzymes from aerobic fermentation peel fruits comprises banana and papaya peels. This study proved that fruit peel waste can be converted to valuable functional enzymes as one of the solutions of fruit peel waste management Keywords: anaerobic fermentation; carbon source; enzyme activity; functional enzyme; organic waste

Simultaneous saccharification and butanediol production from unpretreated lignocellulosic biomass using an enzymatic cocktail of a newly constructed bacterial consortium

Bioconversion lignocellulosic biomass (LCB) to 2,3-butanediol (2,3-BDO) is a sustainable and energy-intensive practice, but its economic feasibility has been hindered by costly pretreatment and the cumbersome fermentation process. Herein, the hindgut metagenome of Cyrtotrachelus buqueti is assembled and sequenced to construct bacterial consortia for efficient LCB degradation. One of these consortia, named artificial synthetic bacteria community II (ASCII), is effective in degrading unpretreated LCB, achieving degradation rates of 85.33% for cellulose, 77.43% for hemicellulose, and 57.63% for lignin. The secretory protein extracted from ASCII is used for simultaneous saccharification and fermentation (SSF) from unpretreated LCB. Furthermore, fed-batch SSF resulted in 40.62 g/L of 2,3-BDO with a productivity of 0.42 g/L/h reached by Klebsiella sp. CQHG35. This study provides a new approach for the construction of bacterial consortia and their use in SSF for the production of 2,3-BDO from unpretreated LCB, representing a significant breakthrough in this field.