Saccharomyces cerevisiae Cells Lacking the Zinc Vacuolar Transporter Zrt3 Display Improved Ethanol Productivity in Lignocellulosic Hydrolysates

Yeast-based bioethanol production from lignocellulosic hydrolysates (LH) is an attractive and sustainable alternative for biofuel production. However, the presence of acetic acid (AA) in LH is still a major problem. Indeed, above certain concentrations, AA inhibits yeast fermentation and triggers a regulated cell death (RCD) process mediated by the mitochondria and vacuole. Understanding the mechanisms involved in AA-induced RCD (AA-RCD) may thus help select robust fermentative yeast strains, providing novel insights to improve lignocellulosic ethanol (LE) production. Herein, we hypothesized that zinc vacuolar transporters are involved in vacuole-mediated AA-RCD, since zinc enhances ethanol production and zinc-dependent catalase and superoxide dismutase protect from AA-RCD. In this work, zinc limitation sensitized wild-type cells to AA-RCD, while zinc supplementation resulted in a small protective effect. Cells lacking the vacuolar zinc transporter Zrt3 were highly resistant to AA-RCD, exhibiting reduced vacuolar dysfunction. Moreover, zrt3Δ cells displayed higher ethanol productivity than their wild-type counterparts, both when cultivated in rich medium with AA (0.29 g L−1 h−1 versus 0.11 g L−1 h−1) and in an LH (0.73 g L−1 h−1 versus 0.55 g L−1 h−1). Overall, the deletion of ZRT3 emerges as a promising strategy to increase strain robustness in LE industrial production.

Bioethanol Production from Cellulose-Rich Corncob Residue by the Thermotolerant Saccharomyces cerevisiae TC-5

This study aimed to select thermotolerant yeast for bioethanol production from cellulose-rich corncob (CRC) residue. An effective yeast strain was identified as Saccharomyces cerevisiae TC-5. Bioethanol production from CRC residue via separate hydrolysis and fermentation (SHF), simultaneous saccharification and fermentation (SSF), and prehydrolysis-SSF (pre-SSF) using this strain were examined at 35–42 °C compared with the use of commercial S. cerevisiae. Temperatures up to 40 °C did not affect ethanol production by TC-5. The ethanol concentration obtained via the commercial S. cerevisiae decreased with increasing temperatures. The highest bioethanol concentrations obtained via SHF, SSF, and pre-SSF at 35–40 °C of strain TC-5 were not significantly different (20.13–21.64 g/L). The SSF process, with the highest ethanol productivity (0.291 g/L/h), was chosen to study the effect of solid loading at 40 °C. A CRC level of 12.5% (w/v) via fed-batch SSF resulted in the highest ethanol concentrations of 38.23 g/L. Thereafter, bioethanol production via fed-batch SSF with 12.5% (w/v) CRC was performed in 5-L bioreactor. The maximum ethanol concentration and ethanol productivity values were 31.96 g/L and 0.222 g/L/h, respectively. The thermotolerant S. cerevisiae TC-5 is promising yeast for bioethanol production under elevated temperatures via SSF and the use of second-generation substrates.

Optimization of Yeast, Sugar and Nutrient Concentrations for High Ethanol Production Rate Using Industrial Sugar Beet Molasses and Response Surface Methodology

Among the various agro-industrial by-products, sugar beet molasses produced by sugar refineries appear as a potential feedstock for ethanol production through yeast fermentation. A response surface methodology (RSM) was developed to better understand the effect of three process parameters (concentration of nutrient, yeast and initial sugar) on the ethanol productivity using diluted sugar beet molasses and Saccharomyces cerevisiae yeast. The first set of experiments performed at lab-scale indicated that the addition of 4 g/L of nutrient combined with a minimum of 0.2 g/L of yeast as well as a sugar concentration lower than 225 g/L was required to achieve high ethanol productivities (<15 g/L/d). The optimization allowed to considerably reduce the amount of yeast initially introduced in the fermentation substrate while still maximizing both ethanol productivity and yield process responses. Finally, scale-up assays were carried out in 7.5 and 100 L bioreactors using the optimal conditions: 150 g/L of initial sugar concentration, 0.27 g/L of yeast and 4 g/L of nutrient. Within 48 h of incubation, up to 65 g/L of ethanol were produced for both scales, corresponding to an average ethanol yield and sugar utilization rate of 82% and 85%, respectively. The results obtained in this study highlight the use of sugar beet molasses as a low-cost food residue for the sustainable production of bioethanol.

Comparison of performances of different fungal laccases in delignification and detoxification of alkali-pretreated corncob for bioethanol production

The performance of the alkaline fungal laccase PIE5 (pH 8.5) in the delignification and detoxification of alkali-pretreated corncob to produce bioethanol was evaluated and compared with that of the neutral counterpart (rLcc9, 6.5), with the acidic laccase rLacA (4.0) was used as an independent control. Treatment with the three laccases facilitated bioethanol production compared with their respective controls. The lignin contents of alkali-pretreated corncob reduced from 4.06 per cent, 5.06 per cent, and 7.80 per cent to 3.44 per cent, 3.95 per cent, and 5.03 per cent, after PIE5, rLcc9, and rLacA treatment, respectively. However, the performances of the laccases were in the order rLacA &gt; rLcc9 &gt; PIE5 in terms of decreasing total phenol concentration (0.18, 0.36, and 0.67 g/L), boosting ethanol concentration (8.02, 7.51, and 7.31 g/L), and volumetric ethanol productivity (1.34, 0.94, and 0.91 g/L·h), and shortening overall fermentation time. Our results would inform future attempts to improve laccases for ethanol production. Furthermore, based on our data and the fact that additional procedures, such as pH adjustment, are needed during neutral/alkaline fungal laccase treatment, we suggest acidic fungal laccases may be a better choice than neutral/alkaline fungal laccases in bioethanol production.

Evaluating crude whey for bioethanol production using non-Saccharomyces yeast, Kluyveromyces marxianus

Ethanol production from non-food substrate is strongly recommended to avoid competition with food production. Whey, which is rich in nutrients, is one of the non-food substrate for ethanol production by Kluyveromyces spp. The purpose of this study was to optimize ethanol from different crude (non-deproteinized, non-pH adjusted, and non-diluted) whey using K. marxianus ETP87 which was isolated from traditional yoghurt. The sterilized and non-sterilized whey were employed for K. marxianus ETP87 substrate to evaluate the yeast competition potential with lactic acid and other microflora in whey. The effect of pH and temperature on ethanol productivity from whey was also investigated. Peptone, yeast extract, ammonium sulfate ((NH4)2SO4), and urea were supplemented to whey in order to investigate the requirement of additional nutrient for ethanol optimization. The ethanol obtained from non-sterilized whey was slightly and statistically lower than sterilized whey. The whey storage at 4 °C didn’t guarantee the constant lactose presence at longer preservation time. Significantly high amount of ethanol was attained from whey without pH adjustment (3.9) even if it was lower than pH controlled (5.0) whey. The thermophilic yeast, K. marxianus ETP87, yielded high ethanol between 30 and 35 °C, and the yeast was able to produce high ethanol until 45 °C, and significantly lower ethanol was recorded at 50 °C. The ammonium sulfate and peptone enhanced ethanol productivity, whereas yeast extract and urea depressed the yeast ethanol fermentation capability. The K. marxianus ETP87, the yeast isolated from traditional yoghurt, is capable of producing ethanol from non-sterilized and non-deproteinized substrates.

Ethanol Production Potential of Sweet Sorghum in North and Central Ukraine

Background: Sweet sorghum (Sorghum saccharatum (L.) Moench) is a unique crop with great potential to serve both the food and energy industries. It is due to the possibility of (bio)ethanol production both from the juice and biomass of this crop. The sorghum stems juice contains sugar in the levels similar to that of sugarcane. Besides, low cultivation requirements for the sweet sorghum make this crop even more attractive for sugar and ethanol production. In terms of technology, sweet sorghum is seen as a transitional feedstock for the first to the second generation bioethanol production. However, effective technological development of the plant cultivation and processing in the Northern and Central Ukraine is restrained by the lack of a collection of sweet sorghum genotypes and adapted varieties for its large-scale cultivation. Additionally, no evaluations of potential (bio)ethanol productivity have been performed for this region, which is important for efficient implementation of novel biofuel-producing technologies and for successful development of a green economy. Objective: This research was aimed to create a pool of sweet sorghum genotypes with the involvement of worldwide germplasm, analyze their morphology and breed high-yielding plant lines for the efficient production of liquid biofuels for second-generation bioenergy. Based on that, we also aimed to explore the prospects regarding the efficiency of sweet sorghum cultivation for (bio)ethanol production in the Northern and Central Ukraine. Methods and Materials: A valuable gene pool of S. saccharatum (L.) Moench (41 samples) was created; in particular, high-performance genotypes were created for cultivation under the soil-climatic conditions of Ukraine. The bio-morphological features and the yield potential of the plants were determined and the biochemical composition of the phyto-raw materials was determined in different periods of vegetation, in particular, during the technical ripeness of the above-ground mass of plants. The more productive forms and varieties of sugar sorghum in terms of yield, dry matter content, sugar, and energy value of biomass during flowering and waxy ripeness are highlighted. The technological properties of plant biomass for the production of alternative liquid fuels (in particular, bioethanol) have been analyzed. Importantly, optimal cultivation conditions have been elaborated for the newly created sweet sorghum genotypes, and their productivity has also been evaluated. Moreover, for the first time, a detailed study on potential ethanol yield has been conducted. Results: Sweet sorghum has considerable potential in Ukraine as a new sugar-producing energy crop. The germplasm collection of this crop has been created (41 accessions), including introduced and acclimated genotypes and newly bred lines and varieties. The biological performance of sorghum in Ukraine and plant morphology have been analyzed. The most promising genotypes were used for breeding of new high-productive sweet sorghum varieties. The potential (bio)ethanol yield for different sugar feedstocks (juice, grain bagasse) can reach up to 11423 L/ha in total from juice, grain and bagasse. Conclusion: The estimated values of ethanol productivity are comparable to the results of other similar investigations. In conclusion, a high performance of sweet sorghum in Ukraine can be suggested.