Acetic acid-and furfural-based adaptive evolution of Saccharomyces cerevisiae strains for improving stress tolerance and lignocellulosic ethanol production

Acetic acid and furfural are known as prevalent inhibitors deriving from pretreatment during lignocellulosic ethanol production.They negatively impact cell growth,glucose uptake and ethanol biosynthesis of Saccharomyces cerevisiae strains.Development of industrial S.cerevisiae strains with high tolerance towards these inhibitors is thus critical for efficient lignocellulosic ethanol production.In this study,the acetic acid or furfural tolerance of different S.cerevisiae strains could be significantly enhanced after adaptive evolution via serial cultivation for 40 generations under stress conditions.The acetic acid-based adaptive strain SPSC01-TA9 produced 30.5 g·L^(-1)ethanol with a yield of 0.46 g·g^(-1)in the presence of 9 g·L^(-1)acetic acid,while the acetic acid/furfural-based adaptive strain SPSC01-TAF94 produced more ethanol of 36.2 g·L^(-1)with increased yield up to 0.49 g·g^(-1)in the presence of both 9 g·L^(-1)acetic acid and 4 g·L^(-1)furfural.Significant improvements were also observed during non-detoxified corn stover hydrolysate culture by SPSC01-TAF94,which achieved ethanol production and yield of 29.1 g·L^(-1)and 0.49 g·g^(-1),respectively,the growth and fermentation efficiency of acetic acid/furfural-based adaptive strain in hydrolysate was 95%higher than those of wildtype strains,indicating the acetic acid-and furfural-based adaptive evolution strategy could be an effective approach for improving lignocellulosic ethanol production.The adapted strains developed in this study with enhanced tolerance against acetic acid and furfural could be potentially contribute to economically feasible and sustainable lignocellulosic biorefinery.

Trends in Starch and Sucrose Content among Sweet Sorghum Genotypes and Implications for Sucrose and Ethanol Production

Sweet sorghum has been suggested as a feedstock into the sugarcane mills for sucrose production in Zimbabwe and Swaziland. Sweet sorghum is widely grown by subsistence farmers and matures in 3 to 6 months in February, March and April, before sugarcane harvesting begins. Sweet sorghum has low sucrose content that is difficult to extract during processing. The hypothesis of the study was that sweet sorghum was a potential feedstock to sugarcane mills for the production of sugar and ethanol. The objective of this study was to investigate the trends in starch and sucrose content of four sweet sorghum genotypes namely M337, M81-E, Theis and Topper, and evaluate the potential of sweet sorghum as a feed stock for sugar and ethanol production. The sorghum juice was collected on August 10, August 24, September 8, September 18 and October 2, 2006 and starch and sucrose content were determined. There were significant （P 〈 0.001） genotypes by sampling date interaction effects. Both starch and sucrose content increased with crop sampling date. Genotypes M337 and Theis were late maturing for sucrose content compared to M81-E and Topper. All genotypes except M337 produced no significant increase in starch after 101DAP. Trends in sucrose and starch content were similar, indicating the reason sucrose was difficult to extract from sweet sorghum. The impact of this study would be boosting the incomes of small scale growers who would be subcontracted by the sugar mills to produce sweet sorghum as a feedstock to the mills before sugarcane matures.

Vintage Effect on the Strain Dependent Dynamics of Ethanol Production in Vineries of Tokaj

The dynamics of ethanol production of local strains of three yeast species and their ternary mixtures was examined in two Tokaj vineries. Although, the performance of them diverged significantly in first etaps of vinification—up to the utilization of half of the sugar content of grape juice—the variations vintages per vintages surpassed the strain-dependent alterations. The divergence in the latter aspect diminished during the last etap, and the ethanol concentration in young wines fermented by Saccharomyces cerevisiae, S. uvarum and Starmerella bacillaris (2 local strains of each) and their mixtures did not vary considerably (c.v. 4.2%). The vinification of grape juice performed more rapidly in fermentors inoculated with strains of S. cerevisiae, S. uvarum and St. bacillaris as well as with their mixtures than in spontaneously initiated ones by wild mycoflora in each vintage. The strains responded in different manners to conditions vintage per vintage, however, their ternary mixtures always fermented more intensively the grape juice than the strains alone. The strains affected the dynamics of alcohol production to different extents, but the alterations between them exceeded the variation between the average effects of the species. The circumstances of vinification significantly influenced the subsequent events of fermentation, but the maximum intensity of ethanol production was inversely proportional to the time required to start alcohol production (p > 0.05), similar to that observed in the laboratory under strictly controlled micro-vinification experiments. The maximum intensity of ethanol production (MIE) varied between 0.64 - 2.59 mM ethanol per hour. The coefficients of second-order polynomial equations describing the dynamics of alcohol production in both laboratory micro-scale and medium-scale experiments in cellars revealed similar correlations regarding the interaction of factor groups regulating the process: the constant (time-independent) and secondary (time-dependent) coefficients of these polynomes counteracted to the primary (time dependent) ones strictly in the strain-dependent manner, and the role of these three factors groups varied also in a strain dependent manner during the vinification process independently of the varying circumstances in three vintages.

Optimization on Pretreatment and Enzymatic Hydrolysis of Sugarcane Trash for Ethanol Production

The present study was conducted for the optimization of pretreatment and enzymatic hydrolysis of lignocellulosic biomass （sugarcane trash）, which is a renewable resource for the production of bioethanol. The pretreatment and enzymatic hydrolysis conditions including alkali （NaOH）/dilute acid （H2SO4）, substrate and chemical concentration for pretreatment, enzyme dosage, pH, temperature and substrate concentration for hydrolysis were varied and evaluated for sugar and ethanol production at the end. The optimum condition was accomplished using 15% w/v DS of 0-2 mm sugarcane trash in size of particle. It was pretreated with two steps of 2% w/v NaOH autoclaving followed by 2% w/v H2SO4 autoclaving with washing step after pretreatment. An enzymatic hydrolysis was then performed using 15% w/v DS pretreated substrate, hydrolyzed with cellulase 50 filter paper unit （FPU）/g DS at 50 ℃ and pH 5. After incubating at 160 r for 48 h, 117.16 g/L reducing sugar was obtained. The achieved sugar after enzymatic hydrolysis was finally fermented to ethanol by Saccharomyces cerevisiae TISTR 5596, with concentration of 48.17 g/L ethanol or yield 0.509 g/g reducing sugars which was equal to 99.81% of theoretical yield.

Optimization of wheat debranning using laboratory equipment for ethanol production

Ethanol production from starchy cereal grains is increasing rapidly due to increasing demand for alternative fuels.In Canada,wheat is the primary feedstock in ethanol plants.To improve the productivity of the ethanol plants in terms of product quality and yield,debranning of wheat grains may be employed.Debranning is advantageous in two ways.Firstly,bran removal increases the starch content of the feedstock,improving the fermentation efficiency of the ethanol plants.Secondly,bran,a valuable co-product can be used as an animal feed ingredient.In this study,experiments to optimize the debranning process were carried out using two kinds of abrasive equipment,the Satake and the TADD(tangential abrasive dehulling device)mills.Wheat samples(30 and 200 g)were debranned in the Satake mill at 1215,1412,and 1515 r/min rotational speeds,30,36,and 40 grit sizes,and 30,60,and 90 s retention times,and in the TADD mill at 900 r/min rotational speed,30,36,50,and 80 grit sizes,and 120,180,240,and 300 s retention times.In addition to debranning efficiency,the starch separation efficiencies of the two mills were calculated in different debranning conditions.In the Satake mill,the 30 g and 200 g sample size,1412 r/min and 1515 r/min rotational speeds,all grit sizes,and 60 s of retention time demonstrated the highest debranning efficiency.Correspondingly,optimal results in the TADD mill were obtained with 200 g sample size,900 r/min rotational speed,50 and 80 grit sizes,and 180 s and 240 s retention times.However,based on the experimental results,Satake mill provided better debranning values compared to the TADD mill.The starch separation efficiency values supported these results.

Direct ethanol production from rice straw by coculture with two high-performing fungi

To develop efficient and economical direct ethanol production from fine rice straw crashed mechanically, two high-performing fungi, which can secret hyperactive cellulases and/or ferment effectively various sugars, were selected from some strains belong to Mucor circinelloides preserved in our laboratory. The simultaneous saccharification and fermentation （SSF） by coculture with these fungi was investigated. The screening of high- performing fungi resulted in the selection of NBRC 4572 as an ethanol-producing fungus and NBRC 5398 as a cellulase-secreting fungus. The strain 4572 produced ethanol aerobically from glucose and xylose in high yields of 0.420 g/g at 36 h and 0.478 g/g at 60 h, respectively, but secreted fairly low cellulases. On the other hand, the strain 5398 also produced ethanol from glucose in yield of 0.340 g/g though it had a little growth in xylose culture. However, it secreted hyperactive cellulases that are essential for hydrolysis of rice straw in culture and the maximum activities of endo-β-glucanase and β-glucosi- dase were 2.11 U/L and 1.47 U/L, respectively. In SSF of rice straw by coculture with two fungi selected, the ethanol production reached 1.28 g/L after 96 h when the inoculation ratio of the strain 5398 to the strain 4572 was 9.

Hydrogen production from steam reforming of ethanol over Ni/MgO-CeO_2 catalyst at low temperature

MgO,CeO2 and MgO-CeO2 with different mole ratio of Mg:Ce were prepared by solid-phase burning method.Catalysts Ni/MgO,Ni/CeO2 and Ni/MgO-CeO2 were prepared by impregnation method.The catalytic properties were evaluated in ethanol steam reforming(ESR) reaction.Specific surface areas of the supports were measured by nitrogen adsorption-desorption at 77 K,and the catalysts were characterized with X-ray diffraction(XRD),temperature programmed reduction(TPR) and thermogravimetric(TG).The results showed that well...

Simultaneous Saccharification and Fermentation of Alkali-Acid Pretreated Sugarcane Trash to Ethanol

The Simultaneous Saccharification and Fermentation （SSF） of alkali-acid pretreated sugarcane trash to ethanol was optimized using commercial cellulase and Saccharomyces cerevisiae TISTR 5596 cells. Substrate concentration （12.5% w/v, 15% w/v, 17.5% w/v and 20% w/v）, enzyme loading （25 FPU/g Dry Substrate （DS）, 50 FPU/g DS and 75 FPU/g DS）, and temperature （30 ~C, 35 ~C and 40 ~C） were evaluated. The SSF optimal conditions for alkali-acid pretreated sugarcane trash were 20% w/v of substrate concentration, enzyme loading 50 FPU/g DS, temperature 35 ~C, initial pH 5.0 and yeast inoculum 107 cells/mL. Under the above optimal conditions, ethanol concentration was possible to reach in the range between 50.14 g/L and 55.08 g/L at 96 hrs and 144 hrs, respectively. This study could establish the effective utilization of sugarcane trash for bioethanol production using optimized fermentation parameters.

Conversion of Sugarcane Shoots and Leaves into Reducing Sugars by Pretreatment and Enzymatic Hydrolysis

Sugarcane shoots and leaves consist of 38% cellulose, 30.6% hemicellulose and 12.8% lignin on dry solid （DS） basis and have the potential to serve as low cost feedstocks for ethanol production. The pretreatment and enzymatic hydrolysis conditions include particle size, alkali （NaOH）/dilute acid （H2SO4） pretreatment, chemical and substrate concentrations, temperature, autoclaving time for pretreatment, enzyme concentration, pH and temperature for hydrolysis varied were evaluated for conversion of sugarcane shoots and leaves cellulose and hemicellulose to reducing sugar. The optimum conditions were accomplished by using 14% w/v DS of 0-10 mm sugarcane shoots and leaves in particle size, pretreated with 1.5% w/v of dilute sulfuric acid at 121℃, 15 lbs/in2 for 15 min and enzymatic saccharification using 40 FPU/g DS cellulose at 50℃ and pH 5, After incubating at 160 rpm for 12 hrs, 59 g/L or 386,38 mg/g DS of reducing sugar and 50.69% saccharification were obtained.

Nitrification characteristics of nitrobacteria immobilized in waterborne polyurethane in wastewater of corn-based ethanol fuel production

A technology to achieve stable and high ammonia nitrogen removal rates for corn distillery wastewater （ethanol fuel production） treatment has been designed.The characteristics of nitrifying bacteria entrapped in a waterborne polyurethane （WPU） gel carrier were evaluated after acclimation.In the acclimation period,nitrification rates of WPU-immobilized nitrobacteria were monitored and polymerase chain reaction （PCR） was also carried out to investigate the change in ammonium-oxidizing bacteria.The results showed that the pellet nitrification rates increased from 21 to 228 mg-N/（L-pellet·hr） and the quantity of the ammonia oxidation bacteria increased substantially during the acclimation.A continuous ammonia removal experiment with the anaerobic pond effluent of a distillery wastewater system was conducted with immobilized nitrifying bacteria for 30 days using an 80 L airlift reactor with pellets at a fill ratio of 15% （V/V）.Under the conditions of 75 mg/L influent ammonia,hydraulic retention time （HRT） of 3.7-5.6 hr,and dissolved oxygen （DO） of 4 mg/L,the effluent ammonia concentration was lower than 10 mg/L and the ammonia removal efficiency was 90%.While the highest ammonia removal rate,162 mg-N/（L-pellet·hr）,was observed when the HRT was 1.3 hr.

Potential production and spatial distribution of hybrid poplar as a biofuel crop in Connecticut,USA

The objective of this study was to assess the biomass production potential from hybrid poplars using marginal lands in the state of Connecticut,USA.A land-use suitability model was developed to identify and classify marginal lands in the state that could be used for growing hybrid poplars as a biofuel woody energy crop.The model was built on a geographic information system(GIS)platform,consisting of an exclusion area section,an ecological suitability section,and an economic/land-use suitability section.The model then was used to estimate the total biomass of the land-cover forests,annual biomass from forest and agricultural residues,and in particular the production potential of biomass from hybrid poplars over marginal lands in the state at county level.The results indicated that about 50%of the land in this state is unavailable for hybrid poplar cultivation and that less than 5%is highly suitable.The amount of usable area is highly variable on the county level.Without large-scale land use change,it appears that biofuel production in this state can only be a supplemental resource to the current energy supply.

Conversion of soy molasses,soy solubles,and dried soybean carbohydrates into ethanol

Soy molasses and soy solubles are byproducts of the conventional soy protein concentrate and soy protein isolate manufacturing processes,respectively.Conversion of the carbohydrates in these byproducts into ethanol was examined.Standardized amounts of commercial cellulase enzymes(Novozyme cellulase,β-glucosidase,and pectinase)were added to soy molasses and soy solubles solutions prepared at various solid loading rates(33%,50%,60%,75%,and 80%)to hydrolyze oligosaccharides,followed by fermentation for 96 h using Saccharomyces cerevisiae NRRL Y-2034 and Scheffersomyces stipitis NRRL Y-7124.Ethanol-extracted soybean meal(SBM)carbohydrates were also fermented for 96 h without enzymes.S.cerevisiae and S.stipitis produced about 12.5-45.0 g/L and 6.0-28.0 g/L ethanol,respectively,on molasses and solubles across these solid loading rates.The S.stipitis produced about 6.5-17 g/L ethanol and S.cerevisiae produced about 6.5-22 g/L ethanol on ethanol-extracted carbohydrates.

Enhancing short-term ethanol-type fermentation of waste activated sludge by adding saccharomycetes and the implications for bioenergy and resource recovery

Ethanol-type sludge fermentation has recently attracted much attention because it can enhance direct interspecies electron transfer and thus improve the anaerobic digestion of waste activated sludge(WAS). In this paper, the enhancement of short-term ethanol-type fermentation of WAS via adding Saccharomyces was investigated. The experimental results show that the maximum ethanol production of 1030.8 ± 20.6 mg/L was achieved, with the optimum fermentation conditions of a p H of 5.1, temperature of 26.0 ℃ and time of 8.0 hr. Although the content of volatile fatty acid(VFA) increased within 10 hr, it is one order of magnitude lower than the content of ethanol, indicating that the VFA generation did not affect the efficient production of ethanol. The analyses of changes in the microbial community during the fermentation process demonstrate that the greatest Saccharomyces activity occurred in the first 8 hr and it can play an important role in ethanol production even at a very low relative abundance. Meanwhile, most typical acid-producing bacteria were inhibited, but the hydrogenotrophic methanogens(i.e., Methanobacterium) were enriched to a certain extent. Further statistical analyses reveal that the Rhodobacter, Thermomonas, Terrimonas and Saccharomyces are responsible for ethanol production during the fermentation. However, these findings not only provide a reference for the development of enhancing ethanol-type fermentation of sludge, but also are expected to provide a new way of thinking for the efficient bioenergy and resource recovery from sludge.