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.

A Hydrotrope Pretreatment for Stabilized Lignin Extraction and High Titer Ethanol Production

The biomass pretreatment strategies using organic acids facilitate lignin removal and enhance the enzymatic digestion of cellulose. However, lignin always suffers a severe and irreversible condensation. The newly generated C-C bonds dramatically affect its further upgrading. In this study, we used a recyclable hydrotrope (p-Toluenessulfonic acid, p-TsOH) to dissolve lignin under mild condition and stabilized lignin with a quenching agent (formaldehyde, FA) during extraction, achieving both value-added lignin extraction and efficient enzymatic saccharification of cellulose. Approximately 63.7% of lignin was dissolved by 80% (wt. %) p-TsOH with 1.5% FA addition at 80 o C, 30 min. The obtained lignin was characterized by FTIR spectroscopy, TGA, 2D HSQC NMR spectroscopy, and GPC. The results indicated that the extracted lignin exhibited excellent properties, such as light color, a low molecular weight (Mw, 5371 g/mol), and a narrow polydispersity (Mw/Mn, 1.63). The pretreated substrate was converted to ethanol via a quasi-simultaneous saccharification and fermentation process (Q-SSF). After fermentation of 60 h, the ethanol concentration reached 38.7±3.3 g/L which was equivalent to a theoretical ethanol yield of 82.9±2.2% based on the glucan content, while the residual glucose concentration was only 4.69±1.4 g/L. In short, this pretreatment strategy protected lignin to form new C-C linkages and improved the enzymatic saccharification of glucan for high-titer ethanol production.

Effect of Vinasse Recycling on Effluent Reduction from Distilleries: Case of Metehara Distillery, Ethiopia

This study was conducted at the ethanol plant of Metehara sugar factory, at a laboratory scale, to assess the effect of recycling vinasse into the fermentation process on effluent reduction. Vinasse is an effluent produced from distilleries. The experimental design included vinasse concentrations at 4 dilution rates (0 (control), 20, 35, 50, and 65% of process water) with 2 replicates and 6 responses, as follows: ethanol yield, fermentation efficiency, residual sugar concentration, cell count, cell viability, and calcium oxide content. In this study, the actual operational parameters of the ethanol plant were maintained during the experiment. The result of the experiment indicates that, with up to 20% vinasse recycling, there was no influential impact on the ethanol yield, the fermentation efficiency, the residual sugar concentration, or the calcium oxide content, attributable to the recycling, as compared to the control. Above 20% vinasse recycling, ethanol yield and fermentation efficiency decreased sharply from those of the control. In addition, with 20% vinasse recycling put into practice, the amount of vinasse generated will be reduced by about 19.5% and about 114.2 tons of water will be saved per day. Moreover, the excess amount of vinasse produced by the distillery, which is beyond the handling capacity of bio-compost plant of the distillery, will reduce from 105 to 36.8 tons per day. Therefore, it is possible to recycle vinasse into the fermenter up to 20% on dilution water of Metehara distillery, without causing any impacts on the distillery’s performance.

Yeasts with Fermentative Potential Associated with Fruits of Camu-Camu (Myrciaria dubia, Kunth) from North of Brazilian Amazon

Considering the high biotechnological potential of yeasts associated to edible fruits, a screening for these microorganisms, capable of alcoholic fermentation, was performed in ripe fruits of camu-camu (Myrciaria dubia, Kunth). The fruits were collected from north of Brazilian Amazon, in the floodplain of the Cauamé River. Yeasts were isolated, and fermentation capability was evaluated using Durham tubes. Quantitative assays were performed to calculate ethanol yield (g g−1), specific growth rate (h−1), and ethanol productivity (g L−1·h−1). Taxonomic identification was performed by ribosomal gene nucleotide sequence analysis by alignment using BLASTN. A total of fifteen yeast colonies were isolated, and three of them presented promising ability to ferment glucose to ethanol. These isolates were identified as Candida orthopsilosis, Pichia kudriavzevii, and Meyerozyma caribbica. When cultured in broth containing 180 g·L−1 of glucose, M. caribbica CC003 reached 91.7 percent of the maximum theoretical ethanol concentration (84.4 g·L−1), presenting an ethanol yield and productivity of 0.4688 g·g−1 and 0.781 g·L−1·h−1, respectively. These results indicate a promising potential of this isolate for bioprocess applications. This paper is a rare report of C. orthopsilosis with endophytic habit because most of the references indicate it as a human pathogen. Besides this, M. caribbica is a promising fermenter for alcoholic beverages due to its osmotolerance and high ethanol yield. This is the first paper reporting endophytic yeasts associated with fruits of Myrciaria dubia.

Revitalizing the ethanologenic bacterium Zymomonas mobilis for sugar reduction in high-sugar-content fruits and commercial products

The excessive consumption of sugars can cause health issues. Different strategies have been developed to reduce sugars in the diets. However, sugars in fruits and commercial products may be difficult to reduce, limiting their usage among certain populations of people. Zymomonas mobilis is a generally recognized as safe (GRAS) probiotic bacterium with the capability to produce levan-type prebiotics, and thrives in high-sugar environments with unique characteristics to be developed for lignocellulosic biofuel and biochemical production. In this study, the sugar reduction capabilities of Z. mobilis ZM4 were examined using two fruits of pear and persimmon and three high-sugar-content commercial products of two pear pastes (PPs) and one Chinese traditional wine (CTW). Our results demonstrated that Z. mobilis ZM4 can utilize sugars in fruits with about 20 g/L ethanol and less than 5 g/L sorbitol produced within 22 h using pears, and about 45 g/L ethanol and 30 g/L sorbitol produced within 34 h using persimmons. When PPs made from pears were used, Z. mobilis can utilize nearly all glucose (ca. 60 g/L) and most fructose (110 g/L) within 100 h with 40 ~ 60 g/L ethanol and more than 20 g/L sorbitol produced resulting in a final sorbitol concentration above 80 g/L. In the high-sugar-content alcoholic Chinese traditional wine, which contains mostly glucose and ethanol, Z. mobilis can reduce nearly all sugars with about 30 g/L ethanol produced, resulting in a final ethanol above 90 g/L. The ethanol yield and percentage yield of Z. mobilis in 50 ~ 60% CTW were 0.44 ~ 0.50 g/g and 86 ~ 97%, respectively, which are close to its theoretical yields—especially in 60% CTW. Although the ethanol yield and percentage yield in PPs were lower than those in CTW, they were similar to those in fruits of pears and persimmons with an ethanol yield around 0.30 ~ 0.37 g/g and ethanol percentage yield around 60 ~ 72%, which could be due to the formation of sorbitol and/or levan in the presence of both glucose and fructose. Our study also compared the fermentation performance of the classical ethanologenic yeast Saccharomyces cerevisiae BY4743 to Z. mobilis, with results suggesting that Z. mobilis ZM4 had better performance than that of yeast S. cerevisiae BY4743 given a higher sugar conversion rate and ethanol yield for sugar reduction. This work thus laid a foundation for utilizing the advantages of Z. mobilis in the food industry to reduce sugar concentrations or potentially produce alcoholic prebiotic beverages. Graphical Abstract

Coordinately express hemicellulolytic enzymes in Kluyveromyces marxianus to improve the saccharification and ethanol production from corncobs

Background Hemicellulose acts as one factor contributing to the recalcitrance of lignocellulose that prevents cellulases to degrade the cellulose efficiently even in low quantities. Supplement of hemicellulases can enhance the performance of commercial cellulases in the enzymatic hydrolyses of lignocellulose. Kluyveromyce marxianus 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 ribosome skipping to self-cleave polyproteins, and investigated their capabilities for saccharification and ethanol production from corncobs. Results Two polycistronic genes IMPX and IMPαX were constructed to test the self-cleavage of P2A sequence from the 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. 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 the IMPX and IMPαX strains were 21.34 and 15.50 U/mL, respectively, but the extracellular β-xylanase activity of IMPαX strain was much higher than that of the IMPX strain, which was 136.17 and 42.07 U/mL, respectively. Subsequently, two recombinant strains, the IXPαR and IMPαXPαR, were constructed to coordinately and secretorily express two xylantic enzymes, Xyn-CDBFV and β-D-xylosidase RuXyn1, or three hemicellulolytic enzymes including M330, Xyn-CDBFV and RuXyn1. In fed-batch fermentation, extracellular activities of β-xylanase and β-xylosidase in the IXPαR strain were 1664.2 and 0.90 U/mL. Similarly, the IMPαXPαR strain secreted the three enzymes, β-mannanase, β-xylanase, and β-xylosidase, with the activities of 159.8, 2210.5, and 1.25 U/mL, respectively. Hemicellulolases of both strains enhanced the yields of 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 yield by 8.7% at 144 h compared with the control. However, both ethanol and xylose yields were increased by 12.7 and 18.2%, respectively, at 120 h in HSF of aqueous ammonia pretreated (AAP) corncobs with this strain. Our results indicated that coordinate expression of hemicellulolytic enzymes in K. marxianus promoted the saccharification and ethanol production from corncobs. Conclusions The 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 the presence of their signal sequences. The IMPαXPαR strain 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.

Effect of nitrogen fertilization on growth, yield and ethanol production of sweet sorghum (Sorghum bicolor (L. Moench)

Field trials were carried out to test the effect of nitrogen fertilization on the growth, stalks and juice yield, sugar content and ethanol production of sweet sorghum. The experiments were carried out on winter 2013 in the Domonstration Farm of the Faculty of Agriculture, University of Khartoum, Shambat and Experimental Farm of the Soba Research Station for Reclamation of Saline and Sodic soils. The treatments comprised the addition of nitrogen fertilizer (urea) at three levels (0, 43 and 86 Kg N ha-1) and two sweet sorghum genotypes (Baladi and RSSS.11), arranged in randomized complete block design replicated 4 times. Results indicated that there were significant (P ≥ 0.05) differences between genotypes in stem diameter at Soba location, stalk height and leaves area at Shambat location, number of green leaves per plant, stalk juice and ethanol yield in both locations. With the genotype RSSS.11 generally superior over genotype Baladi. Nitrogen fertilization increased significantly (P ≥ 0.05) number of green leaves per plant and leaves area at Shambat location and plant height and juice and ethanol yield in both locations. With the Soba location recorded the highest values over Shambat location and 86 kg N ha-1 recorded the highest ethanol yield (255.6l h-1).

Production of Bioethanol—A Review of Factors Affecting Ethanol Yield

Fossil fuels are a major contributor to climate change, and as the demand for energy production increases, alternative sources (e.g., renewables) are becoming more attractive. Biofuels such as bioethanol reduce reliance on fossil fuels and can be compatible with the existing fleet of internal combustion engines. Incorporation of biofuels can reduce internal combustion engine (ICE) fleet carbon dioxide emissions. Bioethanol is typically produced via microbial fermentation of fermentable sugars, such as glucose, to ethanol. Traditional feedstocks (e.g., first-generation feedstock) include cereal grains, sugar cane, and sugar beets. However, due to concerns regarding food sustainability, lignocellulosic (second-generation) and algal biomass (third-generation) feedstocks have been investigated. Ethanol yield from fermentation is dependent on a multitude of factors. This review compares bioethanol production from a range of feedstocks, and elaborates on available technologies, including fermentation practices. The importance of maintaining nutrient homeostasis of yeast is also examined. The purpose of this review is to provide industrial producers and policy makers insight into available technologies, yields of bioethanol achieved by current manufacturing practices, and goals for future innovation.

Application of electric potential improves ethanol production from xylose by active sludge

Background Low-cost raw materials such as lignocellulosic materials have been utilized in second-generation ethanol production process. However, the sequential and slow conversion of xylose into target products remains one of the main challenges for realizing efficient industrial lignocellulosic biorefinery. Results By applying different constant potentials to different microbial electrolysis cells with xylose as the sole carbon source, we analyzed the output of metabolites, microbial community structures, electron flow, and carbon flow in the process of xylose electro-fermentation by domesticated activated sludge. The bioreactors produced currents when applying positive potentials. The peak currents of the + 0.242 V, + 0.542 V and + 0.842 V reactors were 0.96 × 10–6 A, 3.36 × 10–6 A and 6.43 × 10–6 A, respectively. The application of potentials promoted the xylose consumption, and the maximum consumption rate in the + 0.542 V reactor was 95.5%, which was 34.8 times that of the reactor without applied potential. The potential application also promoted the production of ethanol and acetate. The maximum ethanol yield (0.652 mol mol−1 xylose) was obtained in the + 0.842 V reactor. The maximum acetate concentration (1,874 µmol L−1) was observed in the + 0.842 V reactor. The optimal potential for ethanol production was + 0.842 V with the maximum ethanol yield and energy saving. The application of positive potential caused the microorganisms to carry out ethanol fermentation, and the application of negative potential forced the microorganisms to carry out acetic fermentation. The potential application changed the diversity and community structure of microorganisms in the reactors, and the two most significantly changed families were Paenibacillaceae and Bacillaceae. Conclusion The constructed microbial electrolysis cells with different potentials obtained better production yield and selectivity compared with the reactor without applied potential. Our work provides strategies for the subsequent fermentation processes with different needs.

Experimental Whisky Fermentations: Influence of Wort Pretreatments

In addition to ethanol yield, the production of flavour congeners during fermentation is a major consideration for Scotch whisky producers. Experimental whisky fermentations can provide useful information to the industry, and this is the focus of this paper. This study investigated the impact of wort pretreatments (boiled, autoclaved, filtered) on fermentation performance and flavour development in Scotch whisky distillates as an alternative to freezing wort for storage. Our study showed that no significant sensorial differences were detected in low wines (first distillates), while the chemical compositions showed clear changes in increased levels of esters and higher alcohols in boiled and autoclaved wort. In contrast, filtered wort comprised overall lower levels of congeners. Regarding alcohol yield, all three pretreatments resulted in decreased yields. In practice, the pretreatment of wort prior to fermentation requires additional process operations, while freezing requires large storage units. The pretreatments adopted in this study significantly influence the composition of the malt wort used for experimental whisky fermentations, and this results in a poorer fermentation performance compared with untreated wort. We recommend the use of fresh or frozen wort as the best options for small-scale fermentation trials.