Efficient Ethanol Production Research Articles

Assessment of the Effect of Nitrogen Concentration on Fermentation and Selection of a Highly Competitive Saccharomyces cerevisiae Strain for Efficient Ethanol Production

: The optimum nitrogen concentration for media supplementation and strain dominance are aspects of key importance to the industrial production of ethanol with a view to reducing costs and increasing yields. In this work, these two factors were investigated for four ethanologenic Saccharomyces cerevisiae strains (CLQCA-INT-001, CLQCA-INT-005, CLQCA-10-099, and UCLM 325), selected from the screening of 150 isolates, mostly from Ecuadorian yeast biodiversity. The effect of nitrogen concentration was assessed in terms of cellular growth, glucose consumption and ethanol production, and the yeast strains’ dominance was evaluated in continuous co-fermentation with cellular recycling by mitochondrial DNA analyses. Among the four selected yeast strains under study, CLQCA-INT-005 presented the highest glucose consumption at a nitrogen supplement concentration as low as 0.4 g·L−1, attaining an ethanol yield of up to 96.72% in 24 h. The same yeast strain was found to be highly competitive, showing a dominance of 80% after four cycles of fermentation in co-culture. Thus, CLQCA-INT-005 may be deemed as a very promising candidate to be used both at pilot-plant scale and at industrial scale cellulosic ethanol production.

Enhancing the efficiency of ethanol production from molasses using immobilized commercial Saccharomyces cerevisiae in two layer alginate-chitosan beads

The commercial Saccharomyces cerevisiae has been successfully inoculated and immobilized in the present study. The immobilization was conducted through a double layer encapsulated method in which 3% of alginate and 1% of chitosan become the first and second layer, respectively. The light microscopy confirmed that the inoculated-cell was dispersed and spread well in the alginate layer as the first layer. The interaction between alginate-chitosan was confirmed by Fourier Transform Infra-Red (FTIR), and the results demonstrated the physical interaction without forming any chemical interaction and binding in the immobilized cell. The immobilized cell was then used for producing ethanol from molasses through the batch fermentation process. The immobilized cell can successfully convert the glucose to ethanol and used for three cycles of fermentation before finally being saturated. The saturated was generated by breaking down of the chitosan layer generated by cell proliferation. The layer breakage forms the fouling as the effect of absorbed molasses in the surface of layer. However, the fermentation using immobilization cells provides the high-efficiency process by converting high percentages of ethanol yield production and solve the post-treatment process.

Effect of cultivar variation and Pichia stipitis NCIM 3498 on cellulosic ethanol production from rice straw

Lignocellulosic wastes such as rice straw gain importance in ensuring sustainable production of biofuels, being one of the most abundant agricultural residues worldwide. This study points the need for characterization and selection of substrate, optimizing the pretreatment method, as well as employing suitable fermenting organism, which can be of immense help in the path towards a more efficient ethanol production process. Microwave assisted alkali, acid, or water pretreated rice straw of five cultivars (PRH-10, Pusa Basmati 1121, Pusa 44, Taraori basmati and IR-36) were subjected to enzymatic hydrolysis, followed by fermentation with Pichia stipitis 3498. Saccharified hydrolysate of pretreated rice straw gave 4–5 times higher sugar recovery compared to untreated straw. Total sugar recovery from different cultivars after pretreatment and saccharification ranged between 20 and 59%. Among the cultivars, Taraori basmati gave significantly higher sugar recovery and ethanol yield of 39.65% ± 3.70 and 5.16% respectively, through microwave/alkali pretreatment which was found best. Kinetic studies done in biofermentor were able to produce the maximum yield of 6.63% (v/v) ethanol, equivalent to 92.9% of the theoretical yield, with an ethanol yield coefficient of 0.47gg-1. This study confirmed the importance of considering varietal differences of substrate, highlighted the higher efficiency of microwave/alkali pretreatment in enhancing straw digestibility and also the potential of the strain P. stipitis 3498 for ethanol production from lignocellulosic biomass.

Molecular evolutionary engineering of xylose isomerase\xa0to improve its\xa0catalytic activity and performance of micro-aerobic glucose/xylose co-fermentation\xa0in Saccharomyces cerevisiae

BackgroundExpression of d-xylose isomerase having high catalytic activity in Saccharomyces cerevisiae (S. cerevisiae) is a prerequisite for efficient and economical production of bioethanol from cellulosic biomass. Although previous studies demonstrated functional expression of several xylose isomerases (XI) in S. cerevisiae, identification of XIs having higher catalytic activity is needed. Here, we report a new strategy to improve xylose fermentation in the S. cerevisiae strain IR-2 that involves an evolutionary engineering to select top-performing XIs from eight previously reported XIs derived from various species.ResultsEight XI genes shown to have good expression in S. cerevisiae were introduced into the strain IR-2 having a deletion of GRE3 and XKS1 overexpression that allows use of d-xylose as a carbon source. Each transformant was evaluated under aerobic and micro-aerobic culture conditions. The strain expressing XI from Lachnoclostridium phytofermentans ISDg (LpXI) had the highest d-xylose consumption rate after 72 h of micro-aerobic fermentation on d-glucose and d-xylose mixed medium. To enhance LpXI catalytic activity, we performed random mutagenesis using error-prone polymerase chain reaction (PCR), which yielded two LpXI candidates, SS82 and SS92, that showed markedly improved fermentation performance. The LpXI genes in these clones carried either T63I or V162A/N303T point mutations. The SS120 strain expressing LpXI with the double mutation of T63I/V162A assimilated nearly 85 g/L d-glucose and 35 g/L d-xylose to produce 53.3 g/L ethanol in 72 h with an ethanol yield of approximately 0.44 (g/g-input sugars). An in vitro enzyme assay showed that, compared to wild-type, the LpXI double mutant in SS120 had a considerably higher Vmax (0.107 µmol/mg protein/min) and lower Km (37.1 mM).ConclusionsThis study demonstrated that LpXI has the highest d-xylose consumption rate among the XIs expressed in IR-2 under micro-aerobic co-fermentation conditions. A combination of novel mutations (T63I and V162A) significantly improved the enzymatic activity of LpXI, indicating that LpXI-T63I/V162A would be a potential construct for highly efficient production of cellulosic ethanol.

Heterozygous diploid structure of Amorphotheca resinae ZN1 contributes efficient biodetoxification on solid pretreated corn stover

BackgroundFast, complete, and ultimate removal of inhibitory compounds derived from lignocellulose pretreatment is the prerequisite for efficient production of cellulosic ethanol and biochemicals. Biodetoxification is the most promising method for inhibitor removal by its unique advantages. The biodetoxification mechanisms of a unique diploid fungus responsible for highly efficient biodetoxification in solid-state culture was extensively investigated in the aspects of cellular structure, genome sequencing, transcriptome analysis, and practical biodetoxification.ResultsThe inborn heterozygous diploid structure of A. resinae ZN1 uniquely contributed to the enhancement of inhibitor tolerance and conversion. The co-expression of gene pairs contributed to the enhancement of the degradation of lignocellulose-derived model inhibitors. The ultimate inhibitors degradation pathways and sugar conservation were elucidated by microbial degradation experimentation as well as the genomic and transcriptomic sequencing analysis.ConclusionsThe finding of the heterozygous diploid structure in A. resinae ZN1 on biodetoxification took the first insight into the global overview of biodetoxification mechanism of lignocellulose-derived inhibitors. This study provided a unique and practical biodetoxification biocatalyst of inhibitor compounds for lignocellulose biorefinery processing, as well as the synthetic biology tools on biodetoxification of biorefinery fermenting strains.

Bioethanol production from cellulose obtained from the catalytic hydro-deoxygenation (lignin-first refined to aviation fuel) of apple wood

As a renewal energy source for bioethanol production, the residuum of apple wood likely offers a major benefit by having the lignin portion transformed into biofuels firstly. In this study, we used the residuum of Raney Ni and Ru/C catalyzed and hydro-deoxygenated (for jet-fuel production) apple wood as the start substrate to produce bio-ethanol. Cellulose content could reach 83.38% for residuum of Ni catalyzed substrates (Ni-cs for short), and the enzymatic hydrolysis rate increased to 88% which is in reasonable high scale. Lignin left in residuum showed more negative effect on cellulose enzymatic hydrolysis than cellulose II. Different catalysts (Ni or Ru/C) concentration influenced the enzymatic hydrolysis and fermentation. Using quasi-simultaneous enzymatic saccharification and combined fermentation (Q-SScombF), the inhibit effect from catalysts was obvious and the ethanol production efficiency were lower than 35%. But for separate hydrolysis and fermentation process (SHF), the ethanol conversion of raw material and catalyzed residuum improved from 33% to 75% (for residuum of Ni-cs) and increased from 25% to 73% for residuum of Ru/C catalyzed substrates (Ru/C-cs). This research provided a good basis for the feasibility of bio-ethanol production from residuum of bio-refined apple wood.

A novel integrated process of high cell-density culture combined with simultaneous saccharification and fermentation for ethanol production

A novel process integration occupying high cell-density culture (HCDC) and simultaneous saccharification and fermentation (SSF) in one bioreactor achieved an efficient ethanol production in this study. The effect of inoculum sizes of S. cerevisiae for ethanol production in SSF was investigated. High ethanol production was obtained with high inoculums size of cells from 4 to 30 kg﹒m−3. The process integration including HCDC and SSF in one reactor was achieved. A production of 35.2 kg﹒m−3 cells was obtained with HCDC in aerobic conditions. Cellulase and pretreated corn stover were added into the fermentation broth in the same bioreactor, and the SSF procedure was initiated in anaerobic conditions. The highest ethanol titer of 17.08kg﹒m−3 corresponding to 101.50% theoretical yield was obtained, and the peak value time was 24 h with β-glucosidase addition in the HCDC-SSF system. The highest ethanol titer of 11.01 kg﹒m−3, corresponding 65.41% theoretical yield, was obtained at 72 h with regular SSF.

Optimized xylose isomerase uptake and expression level in Saccharomyces cerevisiae for improving ethanol production

The ability to engineer the yeast Saccharomyces cerevisiae to efficiently convert lignocellulosic biomass to ethanol remains a considerable challenge. Here, we propose a new reprogrammable strategy to optimize the expression level of the xylose isomerase (XI) gene with the induction of mutations in S . cerevisiae to improve efficient ethanol production and productivity. We sought to fine-tune the xylose uptake and catabolism abilities of S. cerevisiae during fermentation by improving efficiency of the xylose transporter, which was fused with four copies of the XI gene under the control of different promoters to obtain recombinant yeast strains. In fermentation experiments, the optimized strain CW9 cultured in yeast extract-peptone (YP) medium containing approximately 65 g/L glucose and 55 g/L xylose produced consistent ethanol yields of 0.45 g/g total sugar in about 72 h, which was close to 90% of the theoretical yield. These promising results indicate that strain CW9 is the best producer of ethanol from mixed sugar when synthetically regulating the xylose assimilation pathway. Overall, this study provides an optimal method to control XI expression levels to find better conditions for enhancing biofuel production.

Bioethanol Production by Repeated Batch Using Immobilized Yeast Cells on Sugarcane Bagasse

Delignified sugarcane bagasse from the sugar industry was used as a carrier for Saccharomyces cerevisiae SC90 immobilization and ethanol production. The proficiency of cell immobilization of S. cerevisiae SC90 on delignified sugarcane bagasse was determined by the amount of cell retention on the carrier and by scanning electron microscopy (SEM). S. cerevisiae SC90 showed the highest cell immobilization on day 1 when diluted molasses (231 g/L of total sugar) was used as a substrate. The efficiency of ethanol production by the immobilized cells was compared with cells grown in suspension in the repeated batch process. Immobilized cells exhibited a higher ethanol production than the suspended system for all five consecutive batches without any requirement for cell adaptation. The maximum ethanol yield (YP/S) of the immobilized cells was 0.42 ± 0.02 g/g (82.35% theoretical yield) in a 3 L packed bed bioreactor when the production could be prolonged up to five consecutive batches. As an additional bonus, the high protein spent yeast cells mixed with delignified sugarcane bagasse can be explored as an animal feed in the future.

Ethanol Production from Sugarcane Bagasse Using Pressurized Microwave Treatment with Inorganic Salts and Salt-Tolerant Yeast

A novel biomass pretreatment method, i.e. pressurized microwave hydrothermal treatment with inorganic salts, and the simultaneous saccharification and fermentation (SSF) using salt-tolerant cellulase, i.e. Meicelase, and salt-tolerant yeast, i.e. Saccharomyces cerevisiae BA11, were studied for the efficient ethanol production from sugarcane bagasse. The component analysis, enzymatic saccharification, and alcohol fermentation were carried out using pressurized microwave treated sugarcane bagasse with various inorganic salts. The pressurized microwave treatment using 2 wt% MgCl2 at a treatment temperature of 200 °C for a treatment time of 5 min followed by SSF without washing treatment of inorganic salts provided the maximum glucose and ethanol yields, i.e. 0.392 and 0.18 g/g-raw material, those corresponded to 99 and 90% of saccharification and ethanol conversion ratios, respectively.

A novel aeration strategy in repeated-batch fermentation for efficient ethanol production from sweet sorghum juice

Abstract To improve the efficiency of ethanol production in a batch fermentation from sweet sorghum juice under a very high gravity (VHG) condition (~ 290 g/L of total sugar) by Saccharomyces cerevisiae NP01, repeated-batch fermentation under an aerated condition (2.5 vvm for the first 4 h during every cycle) was done in a 5-L fermenter. The average ethanol concentration (P), productivity (Qp) and yield (Yp/s) for five successive cycles were 112.31 g/L, 1.55 g/L·h−1 and 0.44, respectively with 80.97% sugar consumption. To complete sugar consumption, the total sugar of the juice was reduced to a high gravity (HG) level (~ 240 g/L). The results showed that yeast extract was not necessary for ethanol production, and aeration during every other cycle i.e., alternating cycles, was sufficient to promote both yeast growth and ethanol production. The average P, Qp and Yp/s values for eight successive cycles with aeration during alternating cycles were 97.58 g/L, 1.98 g/L·h and 0.41, respectively with 91.21% sugar consumption. The total fatty acids in the yeast cells under the aerated condition were ~ 50% higher than without aeration, irrespective the initial sugar concentration, whereas the ergosterol contents under aeration condition were ~ 29% to 49% higher than those without aeration.

Reusable Floating Beads with Immobilized Xylose-Fermenting Yeast Cells for Simultaneous Saccharification and Fermentation of Lime-Pretreated Rice Straw.

Novel bioreactor beads for simultaneous saccharification and fermentation (SSF) of lime-pretreated rice straw (RS) into ethanol were prepared. Genetically modified Saccharomyces cerevisiae cells expressing genes encoding xylose reductase, xylitol dehydrogenase, and xylulokinase were immobilized in calcium alginate beads containing inorganic lightweight filler particles to reduce specific gravity. For SSF experiments, the beads were floated in slurry composed of lime-pretreated RS and enzymes and incubated under CO2 atmosphere to reduce the pH for saccharification and fermentation. Following this reaction, beads were readily picked up from the upper part of the slurry and were directly transferred to the next vessel with slurry. After 240 h of incubation, ethanol production by the beads was equivalent to that by free cells, a trend that was repeated in nine additional runs, with slightly improved ethanol yields. Slurry with pre-saccharified lime-pretreated RS was subjected to SSF with floating beads for 168 h. Although higher cell concentrations in beads resulted in more rapid initial ethanol production rates, with negligible diauxic behavior for glucose and xylose utilization, no improvement in the ethanol yield was observed. A fermentor-scale SSF experiment with floating beads was successfully performed twice, with repeated use of the beads, resulting in the production of 40.0 and 39.7 g/L ethanol. There was no decomposition of the beads during agitation at 60 rpm. Thus, this bioreactor enables reuse of yeast cells for efficient ethanol production by SSF of lignocellulosic feedstock, without the need for instruments for centrifugation or filtration of whole slurry.

Systematic optimization of gene expression of pentose phosphate pathway enhances ethanol production from a glucose/xylose mixed medium in a recombinant Saccharomyces cerevisiae

The pentose phosphate pathway (PPP) plays an important role in the synthesis of ribonucleotides and aromatic amino acids. During bioethanol production from cellulosic biomass composed mainly of d-glucose and d-xylose, the PPP is also involved in xylose metabolism by engineered Saccharomyces cerevisiae. Although the activities and thermostabilities of the four PPP enzymes (transaldolase: TAL1, transketolase: TKL1, ribose-5-phosphate ketol-isomerase: RKI1 and d-ribulose-5-phosphate 3-epimerase: RPE1) can affect the efficiency of cellulosic ethanol production at high temperatures, little is known about the suitable expression levels of these PPP genes. Here, we overexpressed PPP genes from S. cerevisiae and the thermotolerant yeast Kluyveromyces marxianus either singly or in combination in recombinant yeast strains harboring a mutant of xylose isomerase (XI) and evaluated xylose consumption and ethanol production of these yeast transformants in glucose/xylose mixed media at 36 °C. Among the PPP genes examined, we found that: (1) strains that overexpressed S. cerevisiae TKL1 exhibited the highest rate of xylose consumption relative to strains that overexpressed other PPP genes alone; (2) overexpression of RKI1 and TAL1 derived from K. marxianus with S. cerevisiae TKL1 increased the xylose consumption rate by 1.87-fold at 24 h relative to the control strain (from 0.55 to 1.03 g/L/h); (3) the strains with XI showed higher ethanol yield than strains with xylose reductase and xylitol dehydrogenase and (4) PHO13 disruption did not improve xylose assimilation under the experimental conditions. Together these results indicated that optimization of PPP activity improves xylose metabolism in genetically engineered yeast strains, which could be useful for commercial production of ethanol from cellulosic material.

Expression of a hyperthermophilic endoglucanase in hybrid poplar modifies the plant cell wall and enhances digestibility

BackgroundExpression of glycosyl hydrolases in lignocellulosic biomass has been proposed as an alternative to improve efficiency of cellulosic ethanol production. In planta production of hyperthermophilic hydrolytic enzymes could prevent the detrimental effects often seen resulting from the expression of recombinant mesophilic enzymes to plant hosts. Utilizing lignocellulosic feedstocks to produce hyperthermophilic hydrolases provides additional benefits for ethanol production in the way of transgenic feedstocks serving as both enzyme providers and cellulosic substrates.ResultsIn this study, transgenic hybrid poplar (Populus alba × grandidentata) was generated to express a hyperthermophilic endoglucanase from Thermotoga neapolitana with an optimal temperature over 100 °C. Functional hyperthermoactive endoglucanase was successfully produced in the transgenic events, and altered phenotypic growth was observed in transgenic lines. Moreover, the line with the highest TnCelB expression in both leaf and developing xylem had reduced lignin content and cellulose crystallinity, resulting in a more digestible cell wall. The activation of TnCelB by a post-harvest heat treatment resulted in enhanced saccharification efficiencies of transgenic poplar lines with moderate TnCelB expression and without alteration of cellulose and lignin when not heat-treated. In planta high-level overexpression of a hyperthermophilic endoglucanase paired with heat treatment following harvest, resulted in biomass that was comparable with wild-type lines that underwent a traditional pretreatment for saccharification.ConclusionsOverexpression of hyperthermophilic endoglucanase in feedstock had impacts on plant growth and cell wall composition, especially when the enzyme was highly expressed. Improved glucan saccharification efficiencies from transgenic lines before and after heat treatment could reduce both the economic and environmental costs associated with ethanol production from lignocellulosic biomass.

Highly efficient conversion of xylose to ethanol without glucose repression by newly isolated thermotolerant Spathaspora passalidarum CMUWF1\u20132

BackgroundEfficient bioconversion of lignocellulosic biomass to bioethanol is one of key challenges in the situation of increasing bioethanol demand. The ethanologenic microbes for such conversion are required to possess abilities of utilization of various sugars including xylose and arabinose in lignocellulosic biomass. As required additional characteristics, there are a weak or no glucose repression that allows cells to simultaneously utilize various sugars together with glucose and thermotolerance for fermentation at high temperatures, which has several advantages including reduction of cooling cost. Spathaspora passalidarum ATCC MYA-4345, a type strains, isolated previously have mainly of these abilities or characteristics but its thermotolerance is not so strong and its glucose repression on xylose utilization is revealed.ResultsNewly isolated S. passalidarum CMUWF1–2 was found to have a high ability to produce ethanol from various sugars included in lignocellulosic biomass at high temperatures. The strain achieved ethanol yields of 0.43 g, 0.40 g and 0.20 g ethanol/g xylose at 30 °C, 37 °C and 40 °C, respectively. Interestingly, no significant glucose repression was observed in experiments with mixed sugars, being consistent with the strong resistance to 2-deoxyglucose, and antimycin A showed no effect on its growth in xylose medium. Moreover, the strain was tolerant to glucose and ethanol at concentrations up to 35.0% (w/v) and 8.0% (v/v), respectively.ConclusionsS. passalidarum CMUWF1–2 was shown to achieve efficient production of ethanol from various sugars and a high ethanol yield from xylose with little accumulation of xylitol. The strain also exhibited stress-resistance including thermotolerance and no detectable glucose repression as beneficial characteristics. Therefore, S. passalidarum CMUWF1–2 has remarkable potential for conversion of lignocellulosic biomass to bioethanol.

Optimizing the coordinated transcription of central xylose-metabolism genes in Saccharomyces cerevisiae.

The efficient fermentation of xylose can improve biofuel production. We previously developed a two-stage transcriptional reprogramming (TSTR) strategy (including a glucose fermentation stage and a xylose fermentation stage) and demonstrated its application for the construction of Saccharomyces cerevisiae strains with efficient xylose utilization. In this study, we used these as initial strains to assess the effects of copy number variation (CNV) on optimal gene expression and rewiring the redox balance of the xylose utilization pathway. We obtained strains that contained several integrated copies of XYL1, XYL2, and XKS1 and showed increased ethanol yields. An examination of the individual and combined effects of CNVs of key genes and the redox balance pathway revealed that the TSTR strategy improves ethanol production efficiency. Furthermore, XYL1 or XYL2 overexpression was related to improved xylose utilization. These results showed that strains with faster growth and/or higher ethanol production produced more ethanol from xylose via the synthetic xylose-assimilation pathway. Accordingly, TSTR is an effective strategy to improve xylose metabolism in industrial yeast strains.

Improvement of inhibitors tolerance of Saccharomyces cerevisiae by overexpressing of long chain sphingoid kinases encoding gene LCB4

By-products released from pretreatment process of lignocellulose seriously hinder the development of cellulosic fuel ethanol. Therefore, the great way to increase the efficiency of cellulosic ethanol production is improvement of Saccharomyces cerevisiae tolerance to these inhibitors. In this work, the effects of LCB4 gene overexpression on cell growth and ethanol fermentation in S. cerevisiae S288C under acetic acid, furfural and vanillin stresses were studied. Compared to the control strain S288C-HO, the recombinant strain S288C-LCB4 grew better on YPD solid medium containing 10 g/L acetic acid, 1.5 g/L furfural and 1 g/L vanillin. Ethanol yields of recombinant strain S288C-LCB4 were 0.85 g/(L·h), 0.76 g/(L·h) and 1.12 g/(L·h) when 10 g/L acetic acid, 3 g/L furfural and 2 g/L vanillin were supplemented into the fermentation medium respectively, which increased by 34.9%, 85.4% and 330.8% than the control strain S288C-HO. Meanwhile, ethanol fermentation time was reduced by 30 h and 44 h under furfural and vanillin stresses respectively. Further metabolites analysis in fermentation broth showed that the recombinant strain produced more protective compounds, such as glycerol, trehalose and succinic acid, than the control strain, which could be the reason for enhancing strain tolerance to these inhibitors from pretreatment process of lignocellulose. The results indicated that overexpression of LCB4 gene could significantly improve ethanol fermentation in S. cerevisiae S288C under acetic acid, furfural and vanillin stresses.

Schizosaccharomyces japonicus has low levels of CoQ10 synthesis, respiration deficiency, and efficient ethanol production.

Coenzyme Q (CoQ) is essential for mitochondrial respiration and as a cofactor for sulfide quinone reductase. Schizosaccharomyces pombe produces a human-type CoQ10. Here, we analyzed CoQ in other fission yeast species. S. cryophilus and S. octosporus produce CoQ9. S. japonicus produces low levels of CoQ10, although all necessary genes for CoQ synthesis have been identified in its genome. We expressed three genes (dps1, dlp1, and ppt1) for CoQ synthesis from S. japonicus in the corresponding S. pombe mutants, and confirmed that they were functional. S. japonicus had very low levels of oxygen consumption and was essentially respiration defective, probably due to mitochondrial dysfunction. S. japonicus grows well on minimal medium during anaerobic culture, indicating that it acquires sufficient energy by fermentation. S. japonicus produces comparable levels of ethanol under both normal and elevated temperature (42°C) conditions, at which S. pombe is not able to grow.

Improving cellulosic ethanol fermentability of Zymomonas mobilis by overexpression of sodium ion tolerance gene ZMO0119

Inhibition of sodium ion (Na+) on Zymomonas mobilis represents an important obstacle for efficient cellulosic ethanol production. This study screened and overexpressed the genes responsible for transporting metal ions in Z. mobilis for increasing its Na+ tolerance. The ZMO0119 gene encoding Na+/H+ antiporter was identified to be highly effective for reducing intracellular Na+ concentration of Z. mobilis by improving the Na+ transport capacity. Overexpression of ZMO0119 gene in Z. mobilis significantly accelerated the cell growth, glucose consumption, and cellulosic ethanol production from the dry acid pretreated and biodetoxified corn stover feedstock. This study provided an important gene responsible for increasing the cellulosic ethanol fermentability by Z. mobilis.

Development of cell recycle technology incorporating nutrient supplementation for lignocellulosic ethanol fermentation using industrial yeast Saccharomyces cerevisiae

For the efficient production of lignocellulosic ethanol, a technology of recycling yeast cells at a high-solid load was developed. The yeast Saccharomyces cerevisiae TJ14 grew up to 5.0 × 107 cells/mL at 39 °C during the simultaneous saccharification and fermentation of hydrothermally pretreated rice straw. By exploiting the viability of the cells, a portion of the fermentation residue was recycled, yielding 52.3 g/L ethanol (90.3% yield) at 1.12 g/(L·h) in six batches. This result is attributable to the maintenance of a viable cell count (cells/mL) on the order of 107. In accordance with this principle, the effects of low-cost nutrients on yeast recycling were elucidated, where 0.125% corn steep liquor was deemed sufficient for fermenting rice straw and empty fruit bunch hydrolysates. The supplementation of sugarcane molasses into bagasse was found to be a practical approach, resulting in 63.5–67.7 g/L ethanol and 5.3 × 106–4.4 × 107 cells/mL in six batches. Here, we describe that the high cell-growth potential in industrially relevant media minimizes the retention of lignocellulosic residues in subsequent batches, thus eliminating the problematic operations in yeast recycling.