Cellulolytic Enzyme Cocktails Research Articles

Monitoring corn stover processing by the fungus Ustilago maydis.

A key aspect of sustainable bioeconomy is the recirculation of renewable, agricultural waste streams as substrates for microbial production of high-value compounds. One approach is the bioconversion of corn stover, an abundant maize crop byproduct, using the fungal maize pathogen Ustilagomaydis. U.maydis is already used as a unicellular biocatalyst in the production of several industrially-relevant compounds using plant biomass hydrolysates. In this study, we demonstrate that U. maydis can grow using untreated corn stover as its sole carbon source. We developed a small-scale bioreactor platform to investigate U.maydis processing of corn stover, combining online monitoring of fungal growth and metabolic activity profiles with biochemical analyses of the pre- and post-fermentation residues. Our results reveal that U. maydis primarily utilizes soluble sugars i.e., glucose, sucrose and fructose present in corn stover, with only limited exploitation of the abundant lignocellulosic carbohydrates. Thus, we further explored the biotechnological potential of enhancing U.maydis´ lignocellulosic utilization. Additive performance improvements of up to 120% were achieved when using a maize mutant with increased biomass digestibility, co-fermentation with a commercial cellulolytic enzyme cocktail, and exploiting engineered fungal strains expressing diverse lignocellulose-degrading enzymes. This work represents a key step towards scaling up the production of sustainable compounds from corn stover using U. maydis and provides a tool for the detailed monitoring of the fungal processing of plant biomass substrates.

Enzymatic hydrolysis of waste streams originating from wastewater treatment plants

BackgroundAchieving climate neutrality is a goal that calls for action in all sectors. The requirements for improving waste management and reducing carbon emissions from the energy sector present an opportunity for wastewater treatment plants (WWTPs) to introduce sustainable waste treatment practices. A common biotechnological approach for waste valorization is the production of sugars from lignocellulosic waste biomass via biological hydrolysis. WWTPs produce waste streams such as sewage sludge and screenings which have not yet been fully explored as feedstocks for sugar production yet are promising because of their carbohydrate content and the lack of lignin structures. This study aims to explore the enzymatic hydrolysis of various waste streams originating from WWTPs by using a laboratory-made and a commercial cellulolytic enzyme cocktail for the production of sugars. Additionally, the impact of lipid and protein recovery from sewage sludge prior to the hydrolysis was assessed.ResultsTreatment with a laboratory-made enzyme cocktail produced by Irpex lacteus (IL) produced 31.2 mg sugar per g dry wastewater screenings. A commercial enzyme formulation released 101 mg sugar per g dry screenings, corresponding to 90% degree of saccharification. There was an increase in sugar levels for all sewage substrates during the hydrolysis with IL enzyme. Lipid and protein recovery from primary and secondary sludge prior to the hydrolysis with IL enzyme was not advantageous in terms of sugar production.ConclusionsThe laboratory-made fungal IL enzyme showed its versatility and possible application beyond the typical lignocellulosic biomass. Wastewater screenings are well suited for valorization through sugar production by enzymatic hydrolysis. Saccharification of screenings represents a viable strategy to divert this waste stream from landfill and achieve the waste treatment and renewable energy targets set by the European Union. The investigation of lipid and protein recovery from sewage sludge showed the challenges of integrating resource recovery and saccharification processes.

Conversion of industrially relevant lignocellulosic biomass into monomers: By customization of cellulolytic enzyme cocktail using simplex-lattice mixture designing (SLMD)

The use of differently pre-treated lignocellulosic biomass (LCB), which contains varying levels of cellulose, hemicellulose and lignin content, is profitable during biofuel production but also require unique enzymatic preparations to convert these differently pre-treated LCB into their monomeric constituents. To address this question, this study employed simplex-lattice mixture design strategy to customize lignocellulolytic enzymatic cocktails for hydrolysis of different pre-treated substrates, obtained from PRAJ and IOCL Industries. In-house recombinant fungal auxiliary/accessory enzymes were evaluated to work in synergy with Cellic®CTec3. The mixtures of recombinant proteins derived from Scytalidium thermophilum CM-4T with Cellic®CTec3 were found to optimally hydrolyse diluted acid pre-treated rice straw slurries obtained from PRAJ and IOCL Industry using Simplex-lattice mixture designing (SLMD) and showed 70.39 % and 84.46 % saccharification efficiency, respectively.

Light Exposure of Lignin Affects the Saccharification Efficiency of LPMO-Containing Cellulolytic Enzyme Cocktails

Light Exposure of Lignin Affects the Saccharification Efficiency of LPMO-Containing Cellulolytic Enzyme Cocktails

Exploring the use of supercritical carbon dioxide in enzymatic hydrolysis of cellulosic substrate

Enzymes are critical components for production of fermentable sugars in lignocellulosic biorefineries. Their activity could be influenced by various reaction parameters. Herein, a formulated cellulolytic enzyme cocktail was immobilized into mesoporous aerogel material and used under high pressure environment of supercritical CO2 (scCO2). The cocktail was immobilized at different enzyme loads (36.0 to 144.0 mg enzyme/g aerogel) using sol-gel entrapment method and scCO2 drying. The immobilized cocktail at 144.0 mg/g aerogel enzyme load retained a residual yield of >50.0 % after fourth cycle of reuse compared to first cycle under atmospheric pressure. Sugar yield by the cocktail aerogel was below 10 wt% in second cycle under scCO2 due to enzyme leaching. Additionally, surface area and porous volume of reused aerogels was decreased over the reuse cycles. The study suggests that scCO2 could be employed for aerogel preparation after optimizing the immobilization process for pure enzymes rather than crude enzymes.

Tailoring a cellulolytic enzyme cocktail for efficient hydrolysis of mildly pretreated lignocellulosic biomass

Commercially available cellulase cocktails frequently demonstrate high efficiency in hydrolyzing easily digestible pretreated biomass, which often lacks hemicellulose and/or lignin fractions. However, the challenge arises with enzymatic hydrolysis of mildly pretreated lignocellulosic biomasses, which contain cellulose, hemicellulose and lignin in high proportions. This study aimed to address this question by evaluating the supplementation of a commercial cellulolytic cocktail with accessory hemicellulases and two additives (H2O2 and Tween® 80). Statistical optimization methods were employed to enhance the release of glucose and xylose from mildly pretreated sugarcane bagasse. The optimized supplement composition resulted in the production of 304 and 124 mg g−1 DM of glucose and xylose, respectively, significantly increasing glucose release by 84% and xylose release by 94% compared to using only the cellulolytic cocktail. This enhancement might be attributed to a coordinated hemicellulases action degrading hemicellulose, creating more space for cellulase activity, potentially boosted by the presence of H2O2 and Tween® 80. However, the addition of different concentrations of H2O2 in combination with hemicellulase and Tween® 80 did not result a significant difference on sugar release, which could be attributed to the limited range of concentrations studied (5 to 65 µM). The results obtained in this study using the mix of three supplements were also compared to the addition of only hemicellulase and only Tween® 80 to the cellulolytic cocktail. A significant increase in glucose release of 39% and 41%, respectively, was observed when using the optimized combination. For xylose, the increase was 38% and 41%, respectively. This study underscores the substantial potential in optimizing enzyme cocktails for the hydrolysis of mildly pretreated lignocellulosic biomass by using enzymes and additive combinations tailored to the specific biomass composition.

Immobilization of Cellulolytic Enzymes in Accurel® MP1000

Cellulases are a class of enzymes of great industrial interest that present several strategic applications. However, the high cost of enzyme production, coupled with the instabilities and complexities of proteins required for hydrolytic processes, still limits their use in several protocols. Therefore, enzyme immobilization may be an essential tool to overcome these issues. The present work aimed to evaluate the immobilization of cellulolytic enzymes of the commercial enzyme cocktail Celluclast® 1.5 L in comparison to the cellulolytic enzyme cocktail produced from the wild strain Trichoderma harzianum I14-12 in Accurel® MP1000. Among the variables studied were temperature at 40 °C, ionic strength of 50 mM, and 72 h of immobilization, with 15 m·L −1 of proteins generated biocatalysts with high immobilization efficiencies (87% for ACC-Celluclast biocatalyst and 95% for ACC-ThI1412 biocatalyst), high retention of activity, and specific activities in the support for CMCase (DNS method), FPase (filter paper method) and β-glucosidase (p-nitrophenyl-β-D-glucopyranoside method). Presenting a lower protein concentration (0.32 m·L−1) than the commercial Celluclast® 1.5 L preparation (45 m·L−1), the ACC-ThI1412-derived immobilized biocatalyst showed thermal stability at temperatures higher than 60 °C, maintaining more than 90% of the residual activities of FPase, CMCase, and β-glucosidase. In contrast, the commercial-free enzyme presented a maximum catalytic activity at only 40 °C. Moreover, the difference in molecular weight between the component enzymes of the extract was responsible for different hydrophobic and lodging interactions of proteins on the support, generating a robust and competitive biocatalyst.

Deciphering the efficient cellulose degradation by the thermophilic fungus Myceliophthora thermophila focused on the synergistic action of glycoside hydrolases and lytic polysaccharide monooxygenases

The thermophilic fungus Myceliophthora thermophila as an efficient decomposer secretes various glycoside hydrolases and auxiliary oxidation enzymes to deconstruct cellulose. However, the core enzymes critical for efficient cellulose degradation and their interactions with other cellulolytic enzymes remain unclear. Herein, the transcriptomic analysis of M. thermophila grown on Avicel exhibited that cellulases from GH5_5, GH6 and GH7, and lytic polysaccharide monooxygenases (LPMOs) from AA9 contributed to cellulose degradation. Moreover, the peptide mass fingerprinting analysis of major extracellular proteins and corresponding gene-knockout strains studies revealed that MtCel7A and MtCel5A were the core cellulolytic enzymes. Furthermore, synergistic experiments found that hydrolytic efficiencies of MtCel7A and MtCel5A were both improved by mixture C1/C4 oxidizing MtLPMO9H, but inhibited by C1 oxidizing MtLPMO9E and C4 oxidizing MtLPMO9J respectively. These results demonstrated the potential application of C1/C4 oxidizing LPMOs for future designing novel cellulolytic enzyme cocktails on the efficient conversion of cellulose into biofuels and biochemicals.

A sustainable process for co-production of xylooligosaccharides and ethanol from alkali treated sugarcane bagasse: A strategy towards waste management

Present study aims at sustainable utilization of sugarcane bagasse (SCB) for production of valuable prebiotic xylooligosaccharides (XOS) along with second generation ethanol. Fractionation of SCB into hemicellulose rich liquid fraction and cellulose rich solid residue was achieved using alkaline treatment. Carbohydrate rich precipitate obtained from liquid fraction was utilized for XOS production using inhouse produced endoxylanase. XOS production from SCB xylan was optimized by employing response surface methodology. Under optimized conditions, maximum XOS yield was 227.72 mg/g of carbohydrate rich precipitates. The solid residue obtained after alkaline pretreatment was used for ethanol fermentation by prehydrolysis and simultaneous saccharification and fermentation (P-SSF) process using cellulolytic enzyme cocktail and Saccharomyces cerevisiae SM1. Maximum ethanol concentration, productivity and yield were 79.76 ± 0.16 g/L, 0.83 g/L/h and 69.38%, respectively by employing P-SSF process. Based on the experimental data it can be predicted that bioconversion of 100 g raw SCB can yield 6.26 g of XOS (DP 2-DP 5), 15.95 g ethanol and 1.44 g of xylitol. Present investigation reports an integrated process for effective bioconversion of SCB into value added products by maximum utilization of cellulosic and hemicellulosic fractions simultaneously using indigenously produced fungal enzymes.

Enzyme-assisted extraction of fucoidan from Kjellmaniella crassifolia based on kinetic study of enzymatic hydrolysis of algal cellulose

Fucoidan extraction from Kjellmaniella crassifolia was enhanced through β-glucosidase fortified cellulase treatment. Kinetic study of the hydrolysis of isolated seaweed cellulose by commercial enzymes was carried out, and cellulolytic enzyme cocktail was optimized according to the degradation kinetics using response surface methodology for fucoidan extraction. Cellulose (9.88 wt%, d.b.) found in the seaweed was highly crystalline (CrI: 83.24 %). β-glucosidase apparently improved the affinity of the cellulolytic enzymes for the cellulose by eliminating cellobiose feedback inhibition. Treating the seaweed with 30 FPU cellulase supplemented with 286.64 CBU β-glucosidase per gram cellulose, followed by conventional ethanol precipitation, the fucoidan extraction yield based on seaweed feedstock dry matter reached 4.74 %. The total sugar content and sulfate content of the product reached 76.68 % and 22.83 %, respectively, with fucose accounting for 62.46 % of the total sugar. The product extracted under optimal conditions exhibited apparently concentration-dependent antioxidant activity, with IC50 values for DPPH, Hydroxyl and ABTS radical scavenging reaching 2.43 mg/mL, 1.02 mg/mL and 1.12 mg/mL, respectively. The results showed that the proposed enzymatic treatment based on cellulose hydrolysis kinetics effectively disrupted the cell wall and facilitated the release and extraction of fucoidan from the seaweed.

Defining the Frontiers of Synergism between Cellulolytic Enzymes for Improved Hydrolysis of Lignocellulosic Feedstocks

Lignocellulose has economic potential as a bio-resource for the production of value-added products (VAPs) and biofuels. The commercialization of biofuels and VAPs requires efficient enzyme cocktail activities that can lower their costs. However, the basis of the synergism between enzymes that compose cellulolytic enzyme cocktails for depolymerizing lignocellulose is not understood. This review aims to address the degree of synergism (DS) thresholds between the cellulolytic enzymes and how this can be used in the formulation of effective cellulolytic enzyme cocktails. DS is a powerful tool that distinguishes between enzymes’ synergism and anti-synergism during the hydrolysis of biomass. It has been established that cellulases, or cellulases and lytic polysaccharide monooxygenases (LPMOs), always synergize during cellulose hydrolysis. However, recent evidence suggests that this is not always the case, as synergism depends on the specific mechanism of action of each enzyme in the combination. Additionally, expansins, nonenzymatic proteins responsible for loosening cell wall fibers, seem to also synergize with cellulases during biomass depolymerization. This review highlighted the following four key factors linked to DS: (1) a DS threshold at which the enzymes synergize and produce a higher product yield than their theoretical sum, (2) a DS threshold at which the enzymes display synergism, but not a higher product yield, (3) a DS threshold at which enzymes do not synergize, and (4) a DS threshold that displays anti-synergy. This review deconvolutes the DS concept for cellulolytic enzymes, to postulate an experimental design approach for achieving higher synergism and cellulose conversion yields.

The effects of chemical and structural factors on the enzymatic saccharification of Eucalyptus sp. samples pre-treated by various technologies

Lignocellulosic biomass is an important renewable resource to produce biofuels, due to its abundance and high polysaccharide content. Bioconversion is generally achieved in two stages: (i) pre-treatment, to reduce biomass recalcitrance, and (ii) enzymatic cellulose hydrolysis into fermentable sugars. However, the key structural and chemical features of biomass which hinder enzymatic hydrolysis remains challenging to evaluate. In this study, we explored the effect of eight different pre-treatments on the enzymatic hydrolysis of a Eucalyptus sp. – a potential feedstock for biofuel production. The analytical data collected covered the mass fractions of cellulose, hemicellulose and lignin, the syringyl: guaiacyl (S/G) ratio of lignin, functional group determination, crystallinity index (CrI) and accessibility of cellulose. Enzymatic hydrolysis was then performed using an in-house optimised cellulolytic enzyme cocktail (either alone or supplemented with xylanase or laccase) to elucidate the key factors of biomass recalcitrance which influence its enzymatic hydrolysis. Enzymatic saccharification was improved by changes in Eucalyptus induced by steam explosion pre-treatment, such as substantial removal of hemicelluloses, increased cellulose accessibility, and disruption of cell wall architecture and exposure of fibres. Partial removal of lignin, increased S/G lignin subunit ratio and lowered cellulose crystallinity exhibited no significant positive effects on biomass enzymatic hydrolyzability. Thus, this study provides insight into important chemical and structural features related to biomass recalcitrance arising from various pre-treatment methods, which can ultimately be used as a platform for the development of more efficient conversion technologies for novel and competitive bio-refineries.

Optimization of Solid State Fermentation of Mixed-Culture ofPenicilliumConsortium for Cellulase and Lytic Polysaccharide Monooxygenases Production

In order to produce higher titre of cellulolytic enzyme cocktails, present study deals with fourPenicilliumconsortium mixed-culture and to investigate their feasibility of producing higher cellulase and auxiliary enzymes via solid state fermentation (SSF). Among different lignocellulosic waste, rice straw was found to be the most suitable substrate. In addition, thePenicilliumconsortium in a mixture ratio of 1:1:1:1 which exhibited higher enzyme activity than the monoculture. Nitrogen sources of tryptone had significant influences on cellulase and lytic polysaccharide monooxygenases (LPMOs) production. The highest cellulase and LPMO activities were 16.65 ± 1.83 U/g and 33.87 ± 0.45 U/g, respectively. The SSF process condition used to obtain these activities were at 30 °C during 5 days. These results show that the optimized studied of mixed-cultivation system withPenicilliumconsortium have potential to be exploiting a complex consortium for the enrichment of cellulolytic enzyme cocktails for bioethanol processes.

Proteomic Profiling of the Secretome of Trichoderma reesei.

Trichoderma reesei (T. reesei) is the workhorse for the production of industrial cellulolytic enzyme cocktails for cellulose hydrolysis. However, the current industrial process using enzyme cocktails is not efficient enough for the cost-effective generation of cellulosic sugar. Here, we describe a protocol for the application of a state-of-the-art LC-MS/MS-based proteomics method for studying the T. reesei secretome. A protein-free minimal chemically defined cell culture medium must be used for a successful secretome analysis. A lignocellulose substrate can be added to this minimal medium to stimulate the fungal secretion of enzymes specific to that substrate. The secretory proteins in the conditioned medium can be purified for quantitative proteomics profiling. T. reesei secretes several hundred enzymes including cellulases, hemicellulases, pectinases, proteases, oxidoreductases, and many putative proteins when it is stimulated with lignocellulose. By combining an understanding of the basic biomass hydrolytic mechanisms with the discovery of novel enzymes, more effective enzyme cocktails can be designed for a sustainable biochemical-based biorefinery.

Promoting and Impeding Effects of Lytic Polysaccharide Monooxygenases on Glycoside Hydrolase Activity

Lytic polysaccharide monooxygenases (LPMOs) have attracted attention due to their ability to boost cellulolytic enzyme cocktails for application in biorefineries. However, the interplay between LPMOs and individual glycoside hydrolases remains poorly understood. We investigated how the activity of two cellobiohydrolases (Cel7A and Cel6A) and an endoglucanase (Cel7B) from Trichoderma reesei were affected by a C1-oxidizing LPMO from Thielavia terrestris (TtAA9). We quantified products from a mixture of LPMO and glycoside hydrolase and estimated separate contributions of products by each of the enzymes. Hereby, we assessed if an observed synergy reflected a promotion of the activity of hydrolase, LPMO, or both. We consistently found that TtAA9 affected the investigated hydrolases differently. It strongly impeded the turnover of the reducing end cellobiohydrolase, TrCel7A, moderately promoted the turnover of the nonreducing end cellobiohydrolase TrCel6A, and promoted the turnover of the endoglucanase, TrCel7B up to 5-fold. The promoting effect on the endoglucanase increased with hydrolysis extent, indicating that the promoting effect became more important as the recalcitrance of the substrate increased. Experiments with mixtures containing multiple glycoside hydrolases suggested that the LPMO primarily promoted the activity of the endoglucanase, whereas promotion of TrCel6A was secondary.

Identification of an intracellular β-glucosidase in Aspergillus niger with transglycosylation activity.

Aspergillus niger is featured with its copious amount of extracellular β-glucosidase which is generally used to balance the cellulolytic enzyme cocktails for lignocellulose saccharification. However, whether or not A. niger produces any intracellular β-glucosidase remains obscure. In this study, we analyzed a total of fifteen putative β-glucosidase genes (bgls) in A. niger CBS 513.88 genome and the five of them were predicted as intracellular bgls due to the lack of signal peptide of extracellular proteins. After further characterization of these five genes through a Saccharomyces cerevisiae in vivo system, only An03g03740 (designated bgl1B) was confirmed to be a β-glucosidase gene. Western blot and mass spectrometry analysis confirmed BGL1B protein localization inside the cell. BGL1B exhibited the maximal activity at 40 °C and pH 5.6. The Km for p-nitrophenyl-β-D-glucopyranoside and Ki for glucose were 0.233 ± 0.058 mM and 119.8 ± 4.35 mM, respectively. BGL1B showed a strong transglycosylation activity while hydrolyzing cellodextrins with sophorose, laminaribiose, and cellotriose formed from cellobiose, and sophorose and laminaribiose formed from cellotriose. The confirmation of the intracellular β-glucosidase BGL1B in A. niger further extends our understanding of how A. niger utilizes lignocellulose. KEY POINTS: • Identification of putative genes revealed a novel β-glucosidase in Aspergillus niger. • Newly identified β-glucosidase BGL1B was an intracellular enzyme of A. niger. • BGL1B exhibited a strong transglycosylation activity.

The use of lytic polysaccharide monooxygenases in anaerobic digestion of lignocellulosic materials

BackgroundThe recent discovery that LPMOs can work under anaerobic conditions when supplied with low amounts H2O2 opens the possibility of using LPMOs as enzyme aids in biogas reactors to increase methane yields from lignocellulosic materials. We have explored this possibility by studying anaerobic digestion of various lignocellulosic materials: Avicel, milled spruce and birch wood, and a lignin-rich hydrolysis residue from steam-exploded birch. The digestions were added LPMOs and various cellulolytic enzyme cocktails and were carried out with or without addition of H2O2.ResultsIn several cases, enzyme addition had a beneficial effect on methane production, which was partly due to components present in the enzyme preparations. It was possible to detect LPMO activity during the initial phases of the anaerobic digestions of Avicel, and in some cases LPMO activity could be correlated with improved methane production from lignocellulosic materials. However, a positive effect on methane production was only seen when LPMOs were added together with cellulases, and never upon addition of LPMOs only. Generally, the experimental outcomes showed substrate-dependent variations in process efficiency and the importance of LPMOs and added H2O2. These differences could relate to variations in the type and content of lignin, which again will affect the activity of the LPMO, the fate of the added H2O2 and the generation of potentially damaging reactive-oxygen species. The observed effects showed that the interplay between cellulases and LPMOs is important for the overall efficiency of the process.ConclusionThis study shows that it may be possible to harness the power of LPMOs in anaerobic digestion processes and improve biogas production, but also highlight the complexity of the reaction systems at hand. One complicating factor was that the enzymes themselves and other organic components in the enzyme preparations acted as substrates for biogas production, meaning that good control reactions were essential to detect effects caused by enzyme activity. As also observed during regular aerobic enzymatic digestion of lignocellulosic biomass, the type and contents of lignin in the substrates likely plays a major role in determining the impact of LPMOs and of cellulolytic enzymes in general. More work is needed to unravel the interplay between LPMOs, O2, H2O2, and the multitude of redox-active components found in anaerobic bioreactors degrading lignocellulosic substrates.

Miscanthus straw as substrate for biosuccinic acid production: Focusing on pretreatment and downstream processing

The main aim of this study was to optimize pretreatment strategies of Miscanthus × giganteus for biosuccinic acid production. A successful pretreatment with organosolv method (80% w/w of glycerol, 1.25% of H2SO4), prevented sugars conversion to furfurals and organic acids, and thereby resulted in high sugar recovery (glucan > 98%, xylan > 91%) and biomass delignification (60%). Pretreated biomass was subjected to hydrolysis with various cellulolytic enzyme cocktails (Viscozyme® L, Carezyme 1000L®, β-Glucanase, Cellic® CTec2, Cellic® HTec2). The most effective enzymes mixture composed of Cellic® CTec2 (10% w/w), β-Glucanase (5% w/w) and Cellic® HTec2 (1% w/w) resulted in high glucose (93.1%) and xylose (69.2%) yields after glycerol-based pretreatment. Succinic acid yield of 75–82% was obtained after hydrolysates fermentation, using Actinobacillus succinogenes 130Z. Finally a successful downstream concept for succinic acid purification was proposed. The succinic acid recovery with high purity (>98%) was developed.

Optimization of microwave assisted enzymatic extraction of steviol glycosides and phenolic compounds from Stevia leaf

Stevia (Stevia rebaudiana Bertoni) has gained great interests due to its non-caloric and health related properties with its steviol glycosides and phytochemical constituents. Currently, it is being utilized as a natural sweetener in many food formulations such as soft drinks, jams, dairy products, chocolates, etc. Green extraction techniques using non-hazardous solvents and yield-enhancing strategies have been subject matters of various disciplines such as food technology, chemistry and pharmacology. Stevia powders are industrially produced by aqueous-extraction with further purification and drying steps. There has been no any report handling combined microwave and enzyme assisted technique on the extraction of steviol glycosides and antioxidant compounds to date. The objective of this study was to determine optimum processing parameters of microwave assisted enzymatic extraction of steviol glycosides and phenolic compounds by investigating the effects of independent variables. The plant was collected from a stevia garden and its leaves were detached from flower part, dried and milled into powder form before commencement of the treatments. Operations were performed using a microwave extraction system equipped with a digital control system for irradiation time, temperature and microwave power. Viscozyme L which is a widely used cellulolytic enzyme cocktail was used in this study. The effects of enzyme concentration (1.2 - 60 FBG unit g-1), temperature (25-60?C) and time (1 - 20 min) were evaluated using a five-level rotatable central composite design by response surface methodology (RSM). The validities of models were assessed by ANOVA outputs which were determination coefficient (R2), lack of fit and Fisher's test value (F-value). The maximized yields of stevioside, rebaudioside A and total phenolic compounds were determined as 62.5, 25.6 and 20.7 mg g-1, respectively at optimum processing conditions which were estimated as 10.9 FBG unit g-1, 53?C and 16 min.

Hydrolyzed corn stover as a promising feedstock for 2‐phenylethanol production by nonconventional yeast

AbstractBACKGROUNDPlant biomass rich in lignocelluloses may be applicable for the production of highly valuable bioproducts. In this study, the use of hydrolyzed corn stover as a carbon source for yeast producing 2‐phenylethanol (2‐PE) is investigated. 2‐PE is an alcohol with a rosy scent, commonly used in the food and cosmetic industries as an aroma and preservative. The growing demand for this compound motivates the development of new methods of production.RESULTSThe corn stover biomass was first pretreated with an alkaline solution of H2O2 and secondly hydrolysed with a cellulolytic enzyme cocktail to cause the saccharification of polycarbohydrates and the release of fermentable monosugars. This resulted in saccharification yields of up to 88.1 ± 7.6% and 92.5 ± 9.1% related to cellulose (glucan) and hemicellulose (arabinoxylan) content, respectively. Next, the obtained hydrolyzates were used as a feedstock for 2‐PE production by two nonconventional yeasts, Metschnikowia chrysoperlae WUT25 and Pichia fermentans WUT36. The highest 2‐PE titer of 3.67 g L−1 was attained with the WUT36 strain. Finally, 2‐PE was extracted from the fermentation broth with ethyl acetate (100% recovery) and purified by distillation to 97.27 ± 0.08%.CONCLUSIONThis is the first report demonstrating the promising and complete route of 2‐PE production by yeast in a medium based on the hydrolyzate of corn stover. © 2018 Society of Chemical Industry