Fermentation Efficiency Research Articles

Bioethanol Production from Ammonia Pretreated Rice Straw

Rice straw is produced in large quantity throughout the world. Rice straw is a leading feedstock for bioethanol production. Diluted ammonia pretreatment for one week at room temperature was found to be effective pretreatment. This pretreated rice straw was acid hydrolyzed and subsequently fermented with Saccharomyces cerevisiae CP11 strain. 1.5% ammonia pretreatment at room temperature for one week resulted 82.4% delignification and 78.49% of acid hydrolysis. Acid hydrolysate was fermented with maximum ethanol concentration 5.70 % with an ethanol yield of 0.46g/g and fermentation efficiency of 90.6%. Diluted ammonia pretreatment at higher temperature has reduced delignification, saccharification and fermentation efficiency with more phenols and furfurals.

Natural Variation in Diauxic Shift between Patagonian Saccharomyces eubayanus Strains.

The study of natural variation can untap novel alleles with immense value for biotechnological applications. Saccharomyces eubayanus Patagonian isolates exhibit differences in the diauxic shift between glucose and maltose, representing a suitable model to study their natural genetic variation for novel strains for brewing. However, little is known about the genetic variants and chromatin regulators responsible for these differences. Here, we show how genome-wide chromatin accessibility and gene expression differences underlie distinct diauxic shift profiles in S. eubayanus. We identified two strains with a rapid diauxic shift between glucose and maltose (CL467.1 and CBS12357) and one strain with a remarkably low fermentation efficiency and longer lag phase during diauxic shift (QC18). This is associated in the QC18 strain with lower transcriptional activity and chromatin accessibility of specific genes of maltose metabolism and higher expression levels of glucose transporters. These differences are governed by the HAP complex, which differentially regulates gene expression depending on the genetic background. We found in the QC18 strain a contrasting phenotype to those phenotypes described in S. cerevisiae, where hap4Δ, hap5Δ, and cin5Δ knockouts significantly improved the QC18 growth rate in the glucose-maltose shift. The most profound effects were found between CIN5 allelic variants, suggesting that Cin5p could strongly activate a repressor of the diauxic shift in the QC18 strain but not necessarily in the other strains. The differences between strains could originate from the tree host from which the strains were obtained, which might determine the sugar source preference and the brewing potential of the strain. IMPORTANCE The diauxic shift has been studied in budding yeast under laboratory conditions; however, few studies have addressed the diauxic shift between carbon sources under fermentative conditions. Here, we study the transcriptional and chromatin structure differences that explain the natural variation in fermentative capacity and efficiency during diauxic shift of natural isolates of S. eubayanus. Our results show how natural genetic variants in transcription factors impact sugar consumption preferences between strains. These variants have different effects depending on the genetic background, with a contrasting phenotype to those phenotypes previously described in S. cerevisiae. Our study shows how relatively simple genetic/molecular modifications/editing in the lab can facilitate the study of natural variations of microorganisms for the brewing industry.

Efficient caproate production from ethanol and acetate in open culture system through reinforcement of chain elongation process

The open culture system exhibits several advantages in bioconversion of complex substrates into caproate, yet it is urgent to develop an advanced approach to overcome the low fermentative efficiency. In this study, three reinforcement techniques were employed to reinforce the chain elongation in the open culture system, including bioaugmentation, pretreatment of anaerobic sludge (AS) and addition of cell carrier. Results showed that the caproate production was improved to 48.9 mmol/L using bioaugmentation technology. With removal of methanogen by alkaline-thermal pretreatment, the caproate production increased to 94.8 mmol/L. The highest caproate production of 130.5 mmol/L was obtained with addition of cell carrier, which was explained by the formation of biofilm. The genus Clostridium_sensu_stricto capable of medium chain fatty acids production dominated the microbiome of the AS. This study provides an insight that efficient caproate production can be achieved in the open culture system by reinforcing the pathway of chain elongation.

Survival and Acidification Potential of Lactobacillus Plantarum MNC 21 Stored in Air-Dried Sorghum Flours

Increased commercialization of indigenous fermented foods requires availability of affordable starter cultures. The starters should also maintain functionality when stored at ambient conditions, especially where erratic power supply makes constant refrigeration unachievable. This study evaluated the survival of Lactobacillus plantarum MNC 21 starter culture air-dried (at 25 or 30°C) in sorghum flour and stored at 25°C for 30 days. Two sorghum varieties (malted and un-malted) were used. To determine their fermentation efficiency during storage, sterile sorghum malt slurries were inoculated with the dried culture and fermented at 30°C for 24 h. Acidification potential was determined at 5 days intervals by measuring microbial counts, pH and titratable acidity. Microbial concentrations dropped from 8-9 log cfu/g on day 0 to 1 log cfu/g on day 30. Sorghum variety and whether it was malted or un-malted did not affect culture survival. Culture dried at 25°C had better survival during the first 10 days (8-9 log cfu/g) than that dried at 30°C (8 log cfu/g) but survival between days 20-30 was similar (1-4 log cfu/g). The acidification potential (ability to reduce pH to ≤4.5) decreased with storage time: 4 h (day 0), 24 h (day 15), > 24 h (day 20) to no acidification (days 25-30). Air drying of starter cultures in sorghum flours coupled with storage at ambient temperatures could be adopted as a short-term preservation method. This low-cost technology is suitable for processors in developing countries where maintenance of a cold chain is hampered by unreliable electricity supply.

Substrate concentration: A more serious consideration than the amount of 5-hydroxymethylfurfural in acid-catalyzed hydrolysis during bioethanol production from starch biomass

5-hydroxymethylfurfural (5-HMF) yield during bioethanol production from starch was determined using spectrophotometry and chromatography. Increasing acid concentration and time favored 5-HMF production with HCl while yield decreased after 45-minute hydrolysis time for HNO3 and H2SO4 hydrolyzed samples. Impacts of glucose (substrate) concentration and produced 5-HMF on bioethanol yield were studied with different sulphuric acid concentrations and different α-amylase and amyloglucosidase activities. A central composite rotational design was utilized to determine the conditions of hydrolysis for optimum glucose production. The results showed that maximum glucose yield occurred at 0.5 M acid concentration and 45-minute hydrolysis time, while maximum yield was achieved at 120 and 280 units of α-amylase and amyloglucosidase activities respectively. It was shown that 5-HMF did not exhibit much inhibition on ethanol yield at low acid concentrations but became pronounced at higher acid concentrations, while high glucose concentrations had a pronounced negative effect on ethanol yield and fermentation efficiency.

Provision of High-Quality Molasses Blocks to Improve Productivity and Address Greenhouse Gas Emissions from Smallholder Cattle and Buffalo: Studies from Lao PDR.

Large ruminant production in developing countries is inefficient with low growth rates and likely high greenhouse gas emissions per unit of meat or milk produced. Trials conducted in Lao PDR from 2017 to 2020, studied ad libitum supplementation for 12 weeks with 20 kg high-quality molasses nutrient blocks (Four Seasons Pty Ltd., Brisbane, Australia), that were either non-medicated; fenbendazole-medicated (Panacur100®, Coopers Australia, 5 g/kg); triclabendazole-medicated (Fasinex®, Novartis Australia, 5 g/kg or 10 g/kg, respectively); or formulated with urea (8% or 10% urea, respectively). Average daily gains were determined for access to all molasses blocks and compared with access to control blocks, no supplementation, or previously determined free-grazing baseline average daily gains (55-84 g in cattle; 92-106 g in buffalo). Productivity was significantly improved following access to all molasses blocks. Average daily gains following access to 8% urea and control blocks were calculated for three age cohorts of cattle: young calves <8 m (238-298 g), growing cattle (143-214 g) and lactating cows (179-191 g). Modelling using IPCC Inventory software model V 2.69 of published data demonstrated a conservative net abatement of 350 kg CO2e was achievable over a 200-day feeding period. An additional trial of Emissions control blocks (n = 200) distributed to farmers (n = 60) and two educational institutions were conducted. Consumption rates (156 g/day) and farmer and institutional acceptance of these blocks were similar to our published findings with other molasses blocks, confirming all formulations of blocks improved animal productivity and body condition score, with healthier animals that were easier to manage. Modelling of changes in greenhouse gas emissions intensity identified an abatement of 470 kg CO2e per Emissions control blocks consumed, delivering a total project emissions abatement of 94 t CO2e. Provision of high-quality molasses blocks significantly improved smallholder large ruminant productivity and addition of greenhouse gas reducing agents is likely to achieve impressive abatement of greenhouse gas emissions due to improved efficiency of rumen fermentation and productivity.

Sustainable Strategies for the Conversion of Lignocellulosic Materials into Biohydrogen: Challenges and Solutions toward Carbon Neutrality

The present review mainly discusses advanced pretreatment techniques for converting lignocellulosic biomass into hydrogen. The focus of this review is also to acquire knowledge concerning lignocellulosic biomass pretreatment processes and their impact on the efficiency of biohydrogen fermentation. The deconstruction of lignocellulosic biomass is presented using various pretreatment techniques albeit with several advantages and disadvantages, particularly about the interference due to the generated inhibitory compounds is toxic to microbes used for fermentation. The use of an appropriate pretreatment process can make the recalcitrant lignocellulosic biomass substrates amenable for further microbial fermentation to produce hydrogen. Although till date there is no ideal pretreatment step available to develop a cost-effective process for conversion of lignocellulosic materials into fermentable sugars, nanotechnology seem to be a more sustainable approach as compared to the traditional processes.

Large Semi-Membrane Covered Composting System Improves the Spatial Homogeneity and Efficiency of Fermentation

Homogenous spatial distribution of fermentation characteristics, local anaerobic conditions, and large amounts of greenhouse gas (GHGs) emissions are common problems in large-scale aerobic composting systems. The aim of this study was to examine the effects of a semi-membrane covering on the spatial homogeneity and efficiency of fermentation in aerobic composting systems. In the covered group, the pile was covered with a semi-membrane, while in the non-covered group (control group), the pile was uncovered. The covered group entered the high-temperature period earlier and the spatial gradient difference in the group was smaller compared with the non-covered group. The moisture content loss ratio (5.91%) in the covered group was slower than that in the non-covered group (10.78%), and the covered group had a more homogeneous spatial distribution of water. The degradation rate of organic matter in the non-covered group (11.39%) was faster than that in the covered group (10.21%). The final germination index in the covered group (85.82%) was higher than that of the non-covered group (82.79%) and the spatial gradient difference in the covered group was smaller. Compared with the non-covered group, the oxygen consumption rate in the covered group was higher. The GHG emissions (by 30.36%) and power consumption in the covered group were reduced more significantly. The spatial microbial diversity of the non-covered group was greater compared with the covered group. This work shows that aerobic compost covered with a semi-membrane can improve the space homogeneity and efficiency of fermentation.

Bio-refinery of xylose processing wastes for green polymalic acid production and l-malic acid recovery by engineered Aureobasidium pullulans in a non-waste-disposal system

Commercial carboxylic acid fermentation is susceptible to CaSO4 solid waste pollution owing to the heavy use of neutralizer CaCO3. Polymalic acid (PMA), a biodegradable polyester generated from the fungus Aureobasidium pullulans, has attracted attention in the bioplastic and biomedical fields and is easily hydrolyzed to release the monomer l-malic acid (L-MA). This study evaluated the feasibility of using waste xylose-containing mother liquor (WXML) for efficient PMA fermentation under low pH control and L-MA recovery in a non-waste-disposal system. Herein, the multi-adaptive evolution of A. pullulans under high concentrations of WXML, coupled with low pH, was demonstrated. Subsequently, an engineered calcineurin signaling pathway overexpressing the exogenous cnb gene from Beauveria bassiana was employed to further regulate cell tolerance. A mutant strain AE-59 was obtained, and a comparable PMA titer of 49.47 ± 0.48 g/L and a yield of 0.33 g/g at low pH control with Na2CO3 in a 5-L fermenter were achieved. Finally, the direct-aqueous-phase-crystal method was employed to obtain 78.55 % of the total L-MA (with a purity of 95.4 %) from the acidic downstream broth using a low dosage of sulfuric acid for acid hydrolysis. This result showed the feasibility of L-MA production in the low-pH control mode with Na2CO3 without solid waste CaSO4. In conclusion, this study overcomes the major obstacles of calcium waste pollution for future industrial fermentative L-MA production and provides a green and sustainable route from lignocellulosic biorefinery for the production of future biopolymers and carboxylic acids.

Cysteamine Supplementation In Vitro Remarkably Promoted Rumen Fermentation Efficiency towards Propionate Production via Prevotella Enrichment and Enhancing Antioxidant Capacity.

Cysteamine (CS) is a vital antioxidant product and nutritional regulator that improves the productive performance of animals. A 2 × 4 factorial in vitro experiment was performed to determine the effect of the CS supplementation levels of 0, 20, 40, and 60 mg/g, based on substrate weight, on the ruminal fermentation, antioxidant capacity, and microorganisms of a high-forage substrate (HF, forage:corn meal = 7:3) in the Statistical Analysis System Institute. After 48 h of incubation, the in vitro dry matter disappearance and gas production in the LF group were higher when compared with a low-forage substrate (LF, forge hay:corn meal = 3:7), which was analyzed via the use of the MIXED procedure of the HF group, and these increased linearly with the increasing CS supplementation (p < 0.01). With regard to rumen fermentation, the pH and acetate were lower in the LF group compared to the HF group (p < 0.01). However, the ammonia N, microbial crude protein, total volatile fatty acids (VFA), and propionate in the LF group were greater than those in the HF group (p < 0.05). With the CS supplementation increasing, the pH, ammonia N, acetate, and A:P decreased linearly, while the microbial crude protein, total VFA, and propionate increased linearly (p < 0.01). Greater antioxidant capacity was observed in the LF group, and the increasing CS supplementation linearly increased the superoxide dismutase, catalase, glutathione peroxidase, total antioxidant capacity, glutathione, and glutathione reductase, while it decreased the malondialdehyde (p < 0.05). No difference occurred in the ruminal bacteria alpha diversity with the increasing CS supplementation, but it was higher in the LF group than in the HF group (p < 0.01). Based on the rumen bacterial community, a higher proportion of Bacteroidota, instead of Firmicutes, was in the LF group than in the HF group. Furthermore, increasing the CS supplementation linearly increased the relative abundance of Prevotella, norank_f_F082, and Prevotellaceae_UCG-001 under the two substrates (p < 0.05). Prevotella, norank_f_F082, and Prevotellaceae_UCG-001 were positively correlated with gas production, rumen fermentation, and antioxidant capacity in a Spearman correlation analysis (r > 0.31, p < 0.05). Overall, a CS supplementation of not less than 20 mg/g based on substrate weight enhanced the rumen fermentation and rumen antioxidant capacity of the fermentation system, and it guided the rumen fermentation towards glucogenic propionate by enriching the Prevotella in Bacteroidetes.

Chemolithoautotrophic reduction of CO2 to acetic acid in gas and gas-electro fermentation systems: Enrichment, microbial dynamics, and sustainability assessment

Microbial-mediated CO2 reduction was evaluated in gas and gas-electro fermentation systems with and without applied potential by selective enriched biocatalyst. Selective enrichment conditions with treated culture (Heat-shock, Acid-shock and 2-Bromoethano sulphonate (BESA)) showed at least one fold increment in acetatic acid production over the control, suggesting the influence of selective enrichment on CO2 conversion. A maximum of 0.84 g/L acetic acid was observed in the Heat+CO2 condition in gas fermentation (GF) stage with 31 mg.L−1.h−1 inorganic carbon fixation rate (ICFR) and this best condition from GF experiments was evaluated in a single-chambered microbial electrochemical system (MES; Gas-electro fermentation) under different applied potentials (-0.4 and -0.6 V vs Ag/AgCl [3.5 M KCl]). The higher acetic acid titre of 1.02 g/L was obtained with -0.6 V applied potential. Higher charge transfer rates (52% faradaic efficiency), and lower corrosion and resistances (Rct -0.4 ohm) improved the CO2 fermentation efficiencies. An abundance of Proteobacteria, Firmicutes, and Actinobacteria, in the enriched biocatalyst, represented the substantial shift of microbiome towards the CO2 feeding microbes on continuous experimental operation. The beta diversity (PCoA) also showed selective enrichment of C1 consumers majorly in the pre-treated conditions. The life cycle assessment (LCA) depicted the hotspots of the process and emphasized the adoption of renewable energy to achieve low-carbon footprints.

An energy optimization model comparing the use of sugarcane bagasse for power or ethanol production

This work addresses the energy balance of producing ethanol from sugarcane bagasse and using lignin from the bagasse as a source of electrical power through Rankine cycle and oxy-combustion. On ethanol 2 G (second generation) proposed plant, lignin is obtained from the concentration and filtration of vinasse which yields a fuel with good calorific value to be used for power generation. The energy balance for 2 G ethanol production including electric power is calculated based on parameters of a typical operational sugarcane plant, coupled with a 2 G plant whose production was assessed by means of global chemical reactions and conversion coefficients obtained from literature. These reactions permit to calculate ethanol production and electricity generation in terms of a virtually infinite number of scenarios characterized by two controlled variables (burning bagasse and straw mass flow rates) and several uncontrolled variables (biomass composition, cellulose, hemicelluloses and lignin yields, fermentation efficiencies, etc.). Results reveal that when the option for carbon capture and storage is used, net power become available when all tops and leaves plus 10 t/h of bagasse are burned. In this condition availability of ethanol is by and large unaffected, yielding a production about 60 m3/h. The application of carbon capture and storage produces a significant impact on power balance that comes from oxy-combustion use (air split unity) and CO2 compression. Another interesting result concerns biomass composition: for both bagasse and straw, higher lignin contents favor simultaneously 2 G ethanol and electricity.

Construction and metabolic analysis of acetone-butanol-ethanol fermentation using mixed acetic acid and lactic acid in wastewater

To reuse organic acids in wastewater, efficient acetone-butanol-ethanol (ABE) fermentation was constructed using mixed acetic acid and lactic acid. The mixed acids were co-consumed with glucose, producing higher butanol concentration (6.96 g/L) and butanol yield (0.494 C-mol/C-mol) than using each single acid (6.22 g/L and 0.432 C-mol/C-mol). This result was verified by 13 C tracer experiment that addition of lactic acid improved carbon distribution to butanol instead of acetone , while acetic acid was readily consumed than lactic acid at initial fermentation stage. Subsequently, pH-stat fed batch fermentation at low pH of 5.5 further improved acid consumptions (6.34 g/L acetic acid and 9.06 g/L lactic acid) and solvent productions (12.4 g/L butanol and 6.50 g/L acetone). Finally, ABE fermentation using wastewater of lactic acid fermentation was performed without inhibition. Thus, efficient ABE fermentation can be implemented using mixed acetic acid and lactic acid, which is superior to using single acid. • This is the first study using mixed acetate and lactate for ABE fermentation. • 13 C experiment verified the metabolic flux to butanol was improved by the acids. • pH and glucose played an important role in acid consumption and solvent production. • Fermentation from lactic acid fermentation broth was performed without inhibition.

Fine-Tuning of Carbon Flux and Artificial Promoters in Bacillus subtilis Enables High-Level Biosynthesis of d-Allulose.

d-Allulose is an attractive rare sugar that can be used as a low-calorie sweetener with significant health benefits. To meet the increasing market demands, it is necessary to develop an efficient and extensive microbial fermentation platform for the synthesis of d-allulose. Here, we applied a comprehensive systematic engineering strategy in Bacillus subtilis WB600 by introducing d-allulose 3-epimerase (DAEase), combined with the deactivation of fruA, levDEFG, and gmuE, to balance the metabolic network for the efficient production of d-allulose. This resulting strain initially produced 3.24 g/L of d-allulose with a yield of 0.93 g of d-allulose/g d-fructose. We further screened and obtained a suitable dual promoter combination and performed fine-tuning of its spacer region. After 64 h of fed-batch fermentation, the optimized engineered B. subtilis produced d-allulose at titers of 74.2 g/L with a yield of 0.93 g/g and a conversion rate of 27.6%. This d-allulose production strain is a promising platform for the industrial production of rare sugar.

Utilization of Mao (Antidesma thwaitesianum Muell. Arg.) Pomace Meal to Substitute Rice Bran on Feed Utilization and Rumen Fermentation in Tropical Beef Cattle.

This experiment was conducted to investigate the effects of replacing rice bran with mao pomace meal on feed intake, digestibility, and rumen fermentation in beef cattle. Four crossbred (50% Brahman × 50% Thai native) beef cattle with an initial body weight of 195 ± 13 kg and 16 months of age were used in a 4 × 4 Latin square design. The dietary treatments included four levels of RB replacement with mao pomace meal at 0, 33, 67, and 100% in concentrate diets. Rice straw was used as a roughage source, fed ad libitum. Replacement of mao pomace meal with rice bran did not affect (p > 0.05) the intakes of concentrate, rice straw, and total dry matter intake. Ether extract intake decreased linearly when increasing the levels of mao pomace meal (p < 0.01). The experimental diets had no effect (p > 0.05) on the digestibility of fiber and crude protein, while dry matter, organic matter, and ether extract digestibility decreased linearly in the group of mao pomace meal replacing rice bran (p < 0.05). Increasing levels of mao pomace meal in concentrate diets did not alter rumen pH, ammonia−nitrogen, or total volatile fatty acid concentration (p > 0.05). The proportion of propionate increased linearly (p < 0.05), whereas acetate and the acetate to propionate ratio decreased linearly (p < 0.05) when replacing rice bran with mao pomace meal. Moreover, the proportion of propionate was greatest, while acetate was lowest when mao pomace meal was included at 100% in the concentrate diet. In conclusion, the replacement of rice bran with mao pomace meal in a diet could enhance the efficiency of rumen fermentation. Nonetheless, it reduced the digestion of nutrients in tropical beef cattle.

Effects of different milk feeding levels and frequencies on performance of Holstein heifers during weaning and first lactation

In the present study, the effects of milk feeding amount and feeding frequency (FF) on performance, blood metabolites, rumen fermentation, and performance of dairy heifers during the first artificial insemination (AI) and first lactation were investigated. The treatment group consisted of 48 female Holstein heifer calves (12/treatment) distributed in a 2 × 2 factorial arrangement with milk feeding [high = 8 L/d, HL or medium = 6 L/d, ML] and feeding frequency [2 × or 3 ×]. All calves were fed on a step-up/step-down method, weaned from milk at 56 d of age, and observed until 70 d of age. Heifer calves were kept uniform from 70 d of age until the first lactation to evaluate the long-term effects of the treatments. We observed no FF effect and no interaction between the level and FF on feed intake, average daily gain (ADG), feed efficiency (FE), body weight (BW), structural growth, blood metabolites, and rumen fermentation parameters. Calves fed HL milk had higher total dry matter intake (DMI), BW, and ADG during the pre- and post-weaning periods. During the preweaning period, HL-fed calves had higher serum insulin (trend) and glucose concentrations compared to ML-fed calves. Lower age at first AI was observed in calves fed HL rather than ML regardless of FF. Weight at first AI, milk yield (305 d), and energy-corrected milk (ECM-305 d) in the first lactation showed a trend toward higher values in HL-fed calves compared to ML-fed calves. Preweaning performance and calf maturity can be positively affected by feeding high amounts of milk at both FF.

Influence of oak chips addition on the physicochemical properties of beer

The paper discusses the influence of the oak chips addition on physicochemical properties of beer. The research material consisted of wort and beer after the fermentation, brewed in the English porter style. Medium toasted oak chips, from two varieties of an oak tree (French and American), were used. Variants without the addition of oak chips were used as control samples. The research was conducted for three periods: after 18 days, 2, and 3 months. The samples containing oak chips were characterized by higher fermentation efficiency, compared to beers without the addition of chips. Titratable acidity increased with aging time in beers containing the chips. Alcohol content decreased after 3 months in all samples. Increased amounts of oak chips and aging time, resulted in greater free amino nitrogen utilization. Prolonging the storage time of the beer with the addition of oak chips changed the profile of volatile components in the beverages.

An engineered non-oxidative glycolytic bypass based on Calvin-cycle enzymes enables anaerobic co-fermentation of glucose and sorbitol by Saccharomyces cerevisiae

BackgroundSaccharomyces cerevisiae is intensively used for industrial ethanol production. Its native fermentation pathway enables a maximum product yield of 2 mol of ethanol per mole of glucose. Based on conservation laws, supply of additional electrons could support even higher ethanol yields. However, this option is disallowed by the configuration of the native yeast metabolic network. To explore metabolic engineering strategies for eliminating this constraint, we studied alcoholic fermentation of sorbitol. Sorbitol cannot be fermented anaerobically by S. cerevisiae because its oxidation to pyruvate via glycolysis yields one more NADH than conversion of glucose. To enable re-oxidation of this additional NADH by alcoholic fermentation, sorbitol metabolism was studied in S. cerevisiae strains that functionally express heterologous genes for ribulose-1,5-bisphosphate carboxylase (RuBisCO) and phosphoribulokinase (PRK). Together with the yeast non-oxidative pentose-phosphate pathway, these Calvin-cycle enzymes enable a bypass of the oxidative reaction in yeast glycolysis.ResultsConsistent with earlier reports, overproduction of the native sorbitol transporter Hxt15 and the NAD+-dependent sorbitol dehydrogenase Sor2 enabled aerobic, but not anaerobic growth of S. cerevisiae on sorbitol. In anaerobic, slow-growing chemostat cultures on glucose–sorbitol mixtures, functional expression of PRK-RuBisCO pathway genes enabled a 12-fold higher rate of sorbitol co-consumption than observed in a sorbitol-consuming reference strain. Consistent with the high Km for CO2 of the bacterial RuBisCO that was introduced in the engineered yeast strains, sorbitol consumption and increased ethanol formation depended on enrichment of the inlet gas with CO2. Prolonged chemostat cultivation on glucose–sorbitol mixtures led to loss of sorbitol co-fermentation. Whole-genome resequencing after prolonged cultivation suggested a trade-off between glucose-utilization and efficient fermentation of sorbitol via the PRK-RuBisCO pathway.ConclusionsCombination of the native sorbitol assimilation pathway of S. cerevisiae and an engineered PRK-RuBisCO pathway enabled RuBisCO-dependent, anaerobic co-fermentation of sorbitol and glucose. This study demonstrates the potential for increasing the flexibility of redox-cofactor metabolism in anaerobic S. cerevisiae cultures and, thereby, to extend substrate range and improve product yields in anaerobic yeast-based processes by enabling entry of additional electrons.

Valorization of Pineapple Leaves Waste for the Production of Bioethanol.

Being a lignocellulose-rich biomass, pineapple leaves waste (PL) could be a potential raw material for the production of biofuel, biochemicals, and other value-added products. The main aim of this study was to investigate the potential of pineapple leaves in the sustainable production of bioethanol via stepwise saccharification and fermentation. For this purpose, PL was subjected to hydrothermal pretreatment in a high-pressure reactor at 150 °C for 20 min without any catalyst, resulting in a maximum reducing sugar yield of 38.1 g/L in the liquid fraction after solid-liquid separation of the pretreated hydrolysate. Inhibitors (phenolics, furans) and oligomers production were also monitored during the pretreatment in the liquid fraction of pretreated PL. Enzymatic hydrolysis (EH) of both pretreated biomass slurry and cellulose-rich solid fraction maintained at a solid loading (dry basis) of 5% wt. was performed at 50 °C and 150 rpm using commercial cellulase at an enzyme dose of 10 FPU/gds. EH resulted in a glucose yield of 13.7 and 18.4 g/L from pretreated slurry and solid fractions, respectively. Fermentation of the sugar syrup obtained by EH of pretreated slurry and the solid fraction was performed at 30 °C for 72 h using Saccharomyces cerevisiae WLP300, resulting in significant ethanol production with more than 91% fermentation efficiency. This study reveals the potential of pineapple leaves waste for biorefinery application, and the role of inhibitors in the overall efficiency of the process when using whole biomass slurry as a substrate.

The characterisation of Wickerhamomyces anomalus M15, a highly tolerant yeast for bioethanol production using seaweed derived medium.

Advanced generation biofuels have potential for replacing fossil fuels as society moves forward into a net-zero carbon future. Marine biomass is a promising source of fermentable sugars for fermentative bioethanol production; however the medium derived from seaweed hydrolysis contains various inhibitors, such as salts that affected ethanol fermentation efficiency. In this study the stress tolerance of a marine yeast, Wickerhamomyces anomalus M15 was characterised. Specific growth rate analysis results showed that Wickerhamomyces anomalus M15 could tolerate up to 600 g/L glucose, 150 g/L xylose and 250 g/L ethanol, respectively. Using simulated concentrated seaweed hydrolysates, W. anomalus M15’s bioethanol production potential using macroalgae derived feedstocks was assessed, in which 5.8, 45.0, and 19.9 g/L ethanol was produced from brown (Laminaria digitata), green (Ulva linza) and red seaweed (Porphyra umbilicalis) based media. The fermentation of actual Ulva spp. hydrolysate harvested from United Kingdom shores resulted in a relatively low ethanol concentration (15.5 g/L) due to challenges that arose from concentrating the seaweed hydrolysate. However, fed-batch fermentation using simulated concentrated green seaweed hydrolysate achieved a concentration of 73 g/L ethanol in fermentations using both seawater and reverse osmosis water. Further fermentations conducted with an adaptive strain W. anomalus M15-500A showed improved bioethanol production of 92.7 g/L ethanol from 200 g/L glucose and reduced lag time from 93 h to 24 h in fermentation with an initial glucose concentration of 500 g/L. The results indicated that strains W. anomalus M15 and W. anomalus M15-500A have great potential for industrial bioethanol production using marine biomass derived feedstocks. It also suggested that if a concentrated high sugar content seaweed hydrolysate could be obtained, the bioethanol concentration could achieve 90 g/L or above, exceeding the minimum industrial production threshold.