Optimized biotransformation of acid-treated water melon peel hydrolyzate into ethanol

Today, global focus of research is to explore the solution of energy crisis and environmental pollution. Like other agricultural countries, bulk quantities of watermelon peels (WMP) are disposed-off in environment as waste in Pakistan and appropriate management of this waste is the need of hour to save environment from pollution. The work emphasizes the role of ethanologenic yeasts to utilize significant sugars present in WMP for low-cost bioethanol fermentation. Dilute hydrochloric acid hydrolysis of WMP was carried out on optimized conditions employing RSM (response surface methodology) following central composite design (CCD). This experimental design is based on optimization of ethanologenesis involving some key independent parameters such as WMP hydrolysate and synthetic media ratio (X1), incubation temperature (X2) and incubation temperature (X3) for maximal ethanol yield exploiting standard (Saccharomyces cerevisiae K7) as well as experimental (Metchnikowia cibodasensisY34) yeasts. The results revealed that maximal ethanol yields obtained from S. cerevisiae K7 was 0.36±0.02 g/g of reducing sugars whereas M. cibodasensisY34, yielded 0.40±0.01 g ethanol/g of reducing sugars. The yeast isolate M. cibodasensisY34 appeared as promising ethanologen and embodies prospective potential for fermentative valorization of WMP-to-bioethanol.

Fermentation of Dairy-Relevant Sugars by Saccharomyces, Kluyveromyces, and Brettanomyces: An Exploratory Study with Implications for the Utilization of Acid Whey, Part I

Acid whey from Greek-style yogurt (YAW) is an underutilized byproduct and a challenge for the dairy industry. One alternative is the fermentation of YAW by yeasts such as Saccharomyces, Brettanomyces, and Kluyveromyces spp., to produce new styles of fermented beverages. Previous research in our group suggested that the sugar profiles of the dairy coproducts impacted the fermentation profiles produced by B. claussenii. The present work aims to describe the fermentation of dairy sugars by S. cerevisiae, K. marxianus, and B. claussenii, under conditions comparable to those of YAW. For this purpose, four preparations of yeast nitrogen base, each containing 40 g/L of either lactose (LAC), glucose (GLU), galactose (GAL), or a 1:1 mixture of glucose and galactose (GLU:GAL), all at pH 4.20, were used as fermentation media. The fermentation was performed independently by each organism at 25 °C under anoxic conditions, while density, pH, cell count, ethanol, and organic acids were monitored. Non-linear modeling was used to characterize density curves, and Analysis of Variance and Tukey’s Honest Significant Difference tests were used to compare fermentation products. K. marxianus and S. cerevisiae displayed rapid sugar consumption with consistent ethanol yields in all media, as opposed to B. claussenii, which showed more variable results. The latter organism exhibited what appears to be a selective glucose fermentation in GLU:GAL, which will be explored in the future. These results provide a deeper understanding of dairy sugar utilization by relevant yeasts, allowing for future work to optimize fermentations to improve value-added beverage and ingredient production from YAW.

AI-based forecasting of ethanol fermentation using yeast morphological data

Several industries require getting information of products as soon as possible during fermentation. However, the trade-off between sensing speed and data quantity presents challenges for forecasting fermentation product yields. In this study, we tried to develop AI models to forecast ethanol yields in yeast fermentation cultures, using cell morphological data. Our platform involves the quick acquisition of yeast morphological images using a non-staining protocol, extraction of high-dimensional morphological data using image processing software, and forecasting of ethanol yields via supervised machine learning. We found that the neural network algorithm produced the best performance, which had a coefficient of determination of > 0.9 even at 30 and 60 min in the future. The model was validated using test data collected using the CalMorph-PC(10) system, which enables rapid image acquisition within 10 min. AI-based forecasting of product yields based on cell morphology will facilitate the management and stable production of desired biocommodities.

Biotechnological Approaches to Lowering the Ethanol Yield during Wine Fermentation

One of the most prominent consequences of global climate warming for the wine industry is a clear increase of the sugar content in grapes, and thus the alcohol level in wines. Among the several approaches to address this important issue, this review focuses on biotechnological solutions, mostly relying on the selection and improvement of wine yeast strains for reduced ethanol yields. Other possibilities are also presented. Researchers are resorting to both S. cerevisiae and alternative wine yeast species for the lowering of alcohol yields. In addition to the use of selected strains under more or less standard fermentation conditions, aerobic fermentation is increasingly being explored for this purpose. Genetic improvement is also playing a role in the development of biotechnological tools to counter the increase in the wine alcohol levels. The use of recombinant wine yeasts is restricted to research, but its contribution to the advancement of the field is still relevant. Furthermore, genetic improvement by non-GMO approaches is providing some interesting results, and will probably result in the development of commercial yeast strains with a lower alcohol yield in the near future. The optimization of fermentation processes using natural isolates is, anyway, the most probable source of advancement in the short term for the production of wines with lower alcohol contents.

Effective fermentation of sugarcane bagasse whole slurries using robust xylose-capable Saccharomyces cerevisiae

The pre-treatment of lignocellulose material toward cellulosic bioethanol production releases microbial inhibitors that severely limit the fermentation ability of Saccharomyces cerevisiae. This study evaluated to what degree robust xylose capable strains may improve the fermentability of non-detoxified sugarcane bagasse (SCB) slurries derived from steam explosion (StEX), and further compared this to slurries derived from ammonia fibre expansion (AFEX) pre-treatment. Initial screening in separate hydrolyses and co-fermentation processes using StEx-SCB hydrolysates identified S. cerevisiae TP-1 and CelluXTM4 with higher xylose consumption (≥ 88%) and ethanol concentrations (≥ 50 g/L). Subsequent fermentations compared StEx and AFEX pre-treated SCB material under industrially relevant fed-batch pre-hydrolysis simultaneous saccharification and co-fermentation (PSSCF) conditions, which resulted in only 3 g/L differences in ethanol titres for StEx and AFEX PSSCF fermentations. The study achieved non-detoxified whole-slurry co-fermentation using StEx pre-treated SCB, with higher ethanol yields than previously reported, by utilising robust xylose-capable strains.

EFFECT OF CO2-ADDED STEAM EXPLOSION ON OIL PALM EMPTY FRUIT BUNCH FOR BIOETHANOL PRODUCTION

This study aimed to investigate the effect of adding CO2 as an impregnation agent in steam explosion on oil palm empty fruit bunch (EFB) for bioethanol production. The influence of this treatment on the characteristics of EFB, enzymatic hydrolysis, and fermentation of EFB was evaluated in this investigation. CO2-added steam explosion was conducted varying the CO2 impregnation time (0, 30, 60 min). The results showed that the addition of CO2 in steam explosion increased the surface area, pore area, and pore volume of EFB. Furthermore, this treatment enabled obtaining yields of glucose and ethanol of 84.14% and 56.01%, respectively, for 60 min CO2 impregnation time. These results were higher than the glucose and ethanol yields of the sample treated by conventional steam explosion, which reached 58.12% and 41.37%, respectively. The findings illustrate the possibility of applying CO2-added steam explosion (CO2SE) for increasing the efficiency of biomass conversion.

Coordinately Express Hemicellulolytic Enzymes in Kluyveromyces Marxianus to Improve the Saccharification and Ethanol Production from Corncobs

BackgroundHemicelluloses act as one factor contributing to the recalcitrance of lignocelluloses that prevent cellulases to degrade the cellulose efficiently even in low quantities. Supplement of hemicellulases can enhance performance of commercial cellulases in the enzymatic hydrolyses of lignocellulose. Kluyveromyce marxianu is an attractive yeast for cellulosic ethanol fermentation, as well as a promising host for heterologous protein production, since it has remarkable thermotolerance, high growth rate, and broad substrate spectrum etc. In this study, we attempted to coordinately express multiple hemicellulases in K. marxianus through a 2A-mediated ribosomes skipping to self-cleave polyproteins, and investigated their capabilities for saccharification and ethanol production from corncobs.ResultsTwo polycistronic genes IMPX and IMPαX were constructed to test the self-cleavage of P2A sequence from Foot and Mouth Disease virus (FMDV) in K. marxianus. The IMPX gene consisted of a β-mannanase gene M330 (without the stop codon), a P2A sequence and a β-xylanase gene Xyn-CDBFV in turn, while in the IMPαX gene there was an additional α-factor signal sequence in frame with the N-terminus of Xyn-CDBFV. The extracellular β-mannanase activities of IMPX and IMPαX strains were 21.34 and 15.50 U/mL repectively. By contrast, the IMPαX strain secreted 136.17 U/mLof the β-xylanase, which was much higher than that of IMPX strain 42.07 U/mL. Based on these, two recombinant strains, the IXαR and IMPαXPαR, were constructed to coordinately and secretorily express two xylantic enzymes a β-D-xylosidase RuXyn1 and Xyn-CDBFV, or three hemicellulolytic enzymes including M330, Xyn-CDBFV and RuXyn1. In fed-batch fermentations, extracellular activities of β-xylanase and β-xylosidase in the IMPαX strain were 1664.2 and 0.90 U/mL, while productions of secretory β-mannanase, β-xylanase, and β-xylosidase in the IMPαXPαR strain were 159.8, 2210.5, and 1.25 U/ml of respectively. Hemicellulolytic enzymes of these two strains enhanced the yields of both glucose and xylose from diluted acid pretreated (DAP) corncobs when acted synergistically with commercial cellulases. In hybrid saccharification and fermentation (HSF) of DAP corncobs, hemicellulases of the IMPαXPαR strain increased the ethanol yields by 8.7% at 144 h. When using aqueous ammonia pretreated (AAP) corncobs as HSF feedstocks, the IMPαXPαR strain increased both ethanol and xylose yields, which were about 12.7% and 18.2% more than that of the control at 120 h. Our results indicated that coordinately expression of hemicellulolytic enzymes in K. marxianus could promote the saccharification and ethanol production from corncobs.ConclusionsThe FMDV P2A sequence showed high efficiency in self-cleavage of polyproteins in K. marxianus, and could be used for secretory expression of multiple enzymes in present of their own signal sequences. The IMPαXPαR strain that coexpressed three hemicellulolytic enzymes improved the saccharification and ethanol production from corncobs, and could be used as a promising strain for ethanol production from lignocelluloses.

Rational engineering of Saccharomyces cerevisiae towards improved tolerance to multiple inhibitors in lignocellulose fermentations

Background The fermentation of lignocellulose hydrolysates to ethanol requires robust xylose-capable Saccharomycescerevisiae strains able to operate in the presence of microbial inhibitory stresses. This study aimed at developing industrial S.cerevisiae strains with enhanced tolerance towards pretreatment-derived microbial inhibitors, by identifying novel gene combinations that confer resistance to multiple inhibitors (thus cumulative inhibitor resistance phenotype) with minimum impact on the xylose fermentation ability. The strategy consisted of multiple sequential delta-integrations of double-gene cassettes containing one gene conferring broad inhibitor tolerance (ARI1, PAD1 or TAL1) coupled with an inhibitor-specific gene (ADH6, FDH1 or ICT1). The performances of the transformants were compared with the parental strain in terms of biomass growth, ethanol yields and productivity, as well as detoxification capacities in a synthetic inhibitor cocktail, sugarcane bagasse hydrolysate as well as hardwood spent sulphite liquor. Results The first and second round of delta-integrated transformants exhibited a trade-off between biomass and ethanol yield. Transformants showed increased inhibitor resistance phenotypes relative to parental controls specifically in fermentations with concentrated spent sulphite liquors at 40% and 80% v/v concentrations in 2% SC media. Unexpectedly, the xylose fermentation capacity of the transformants was reduced compared to the parental control, but certain combinations of genes had a minor impact (e.g. TAL1 + FDH1). The TAL1 + ICT1 combination negatively impacted on both biomass growth and ethanol yield, which could be linked to the ICT1 protein increasing transformant susceptibility to weak acids and temperature due to cell membrane changes. Conclusions The integration of the selected genes was proven to increase tolerance to pretreatment inhibitors in synthetic or industrial hydrolysates, but they were limited to the fermentation of glucose. However, some gene combination sequences had a reduced impact on xylose conversion.

Production of cellulosic ethanol from alkali treated wheat straw using P-SSF process and bioconversion of hemicellulosic fraction into high value products

Background: Lignocellulosic biomass is an attractive resource for production of ethanol because of its abundance and lower cost. The economics of lignocellulosic ethanol production can be improved by enhancing the ethanol titres along with utilisation of waste generated during bioconversion process. Objective: The present study was aimed at development of a bioconversion process for production high concentration of ethanol from alkali treated cellulose rich wheat straw (WS) and utilization of unused hemicellulosic fraction into value added products. Methods: WS was subjected to microwave assisted alkali (MAA) treatment. Scanning electron microscopy and Fourier transform infrared spectroscopy were used to analyse structural changes in untreated and pretreated WS. Bioethanol production from pretreated WS was carried out by pre hydrolysis and simultaneous saccharification and fermentation (P-SSF) process using newly isolated Saccharomyces cerevisisae SM1. Liquid fraction generated during pretreatment was utilised for xylooligosaccharides (XOS) production using indigenously produced endoxylanase. Results: MAA treatment of WS was successful in enriching cellulose content of WS by solubilizing hemicellulose and lignin. Ethanol fermentation by P-SSF method lead to high concentration of ethanol (42.10±1.15 g/L) in 48 h. Ethanol productivity and yield were, 0.88 g/L/h and 69.14%, respectively. It can be predicted that 7.143 tons of raw WS may be required to produce 1 ton of ethanol and for additional revenue 191.93 kg xylitol and 263.58 kg XOS (DP2 - DP5) can also be produced simultaneously. Conclusion: The study has demonstrated the feasibility of a bio-refinery process for production of value added compounds in addition to high ethanol yields.

Mushroom-mediated delignification of agricultural wastes for bio-ethanol production

Biological pretreatment is a cost-effective method of delignifying lignocellulosic biomass, making it less recalcitrant to hydrolysis into fermentable sugars. In this study, selected agricultural wastes were pretreated with mushrooms (Lentinus squarrosulus and Pleurotus ostreatus) to delignify them for bioethanol production. The substrates were supplemented with 0.2 % CaCO3, inoculated with 12 % (w/w) L. squarrosulus and Pleurotus ostreatus spawns and incubated at 25 oC for 21 days. The highest lignin removal and highest bioethanol yield of 77.45 % and 13.98 % were obtained from bean husks pretreated with L. squarrosulus. Similarly, 64.29 % and 60.92 % lignin were removed from the Pleurotus ostreatus-pretreated banana leaves and sawdust, respectively, while 12.08 % and 13.05 % bio-ethanol yields were recorded, respectively. These findings demonstrate that affordable and straightforward mushroom delignification of abundant and cheap biomass can improve hydrolysis outcomes, thus easing bioethanol production.