Enhanced Bioethanol Production Research Articles

Enhanced bioethanol production from wheat straw hemicellulose by mutant strains of pentose fermenting organisms Pichia stipitis and Candida shehatae.

The main aim of the present study was to mutate yeast strains, Pichia stipitis NCIM 3498 and Candida shehatae NCIM 3501 and assess the mutant’s ability to utilize, ferment wheat straw hemicellulose with enhanced ethanol yield. The organisms were subjected to random mutagenesis using physical (ultraviolet radiation) and chemical (ethidium bromide) mutagens. The mutant and wild strains were used to ferment the hemicellulosic hydrolysates of wheat straw obtained by 2 % dilute sulphuric acid and enzymatic hydrolysis by crude xylanase separately. Among all the mutant strains, PSUV9 and CSEB7 showed enhanced ethanol production (12.15 ± 0.57, 9.55 ± 0.47 g/L and yield 0.450 ± 0.009, 0.440 ± 0.001 g/g) as compared to the wild strains (8.28 ± 0.54, 7.92 ± 0.89 g/L and yield 0.380 ± 0.006 and 0.370 ± 0.002 g/g) in both the hydrolysates. The mutant strains were also checked for their consistency in ethanol production and found stable for 19 cycles in hemicellulosic hydrolysates of wheat straw. A novel element in the present study was introduction of chemical mutagenesis in wild type as well as UV induced mutants. This combination of treatments i.e., UV followed by chemical mutagenesis was practically successful.

Enhanced Bioethanol Production from Blue Agave Bagasse in a Combined Extrusion–Saccharification Process

This paper describes an improved process for bioethanol production using a recently developed combined extrusion–saccharification technology. Blue agave bagasse (BAB) was pretreated via a thermo-mechano-chemical process (co-rotational twin-screw, reactive extrusion) to increase the availability of cellulose and hemicellulose for enzymatic saccharification. Then, several commercial enzyme preparations, boosted with accessory enzymes (exoglucanase, endoglucanase, hemicellulase, xylanase, and β-glucosidase), were tested with extruded BAB at 5 % consistency in a stirred vessel. The enzyme blend that produced the highest saccharification yield was evaluated at different BAB consistencies. The obtained concentration of sugars increased up to 69.5 g/L (73 % yield) when a 20 % BAB mixture was used. When the enzyme blend was fed into the extruder and with a residence time of 2 min, the yield reached 15 % of the maximum theoretical of C6 sugars along this step. This extruded and pre-saccharified BAB was further hydrolyzed and used for fermentation. The pre-saccharification step significantly enhanced cellulose degradation and ethanol production. Our results indicate that the enzymatic saccharification of BAB, coupled with reactive extrusion, produces an excellent substrate for bioethanol production.

Enhanced bioethanol production from water hyacinth (Eichhornia crassipes) by statistical optimization of fermentation process parameters using Taguchi orthogonal array design

Water hyacinth (Eichhornia crassipes), a fast growing aquatic weed and a potential lignocellulosic substrate with high hemicellulosic (44.68 ± 0.39%, w w−1) content was used for bioethanol production. The commercial fungal enzymes are expensive in nature. Taguchi orthogonal array design was employed for optimization of simultaneous saccharification and fermentation (SSF) process parameters involving recombinant Clostridium thermocellum hydrolytic enzymes and fermentative microbes for enhanced bioethanol production from mixed, microwave-assisted alkali and organosolv pretreated substrate. The factors optimized in 100 mL SSF medium were (%, v v−1): GH5 cellulase (5.6 U mg−1, 0.44 mg mL−1), 0.5; GH43 hemicellulase (3.8 U mg−1, 0.33 mg mL−1), 2.0; Saccharomyces cerevisiae (∼3.8 × 109 cells mL−1), 0.5; Candida shehatae (∼2.9 × 108 cells mL−1), 2.0; pH, 5.4 and 35 °C, temperature. GH43 hemicellulase volume, pH and temperature were the three significant factors. The optimized flask SSF with 1% (w v−1) substrate contributed ∼2-fold upturn in ethanol titre and yield (1.84 g L−1, 0.310 g of ethanol g of substrate−1) as compared with unoptimized conditions (1.0 g L−1, 0.160 gg-1). 5% (w v−1) substrate in shake flask and bioreactor contributed ethanol titre and yield of 9.78 g L−1, 0.329 gg-1 and 13.7 g L−1, 0.462 gg-1 respectively.

Enhanced Bio-Ethanol Production from Industrial Potato Waste by Statistical Medium Optimization.

Industrial wastes are of great interest as a substrate in production of value-added products to reduce cost, while managing the waste economically and environmentally. Bio-ethanol production from industrial wastes has gained attention because of its abundance, availability, and rich carbon and nitrogen content. In this study, industrial potato waste was used as a carbon source and a medium was optimized for ethanol production by using statistical designs. The effect of various medium components on ethanol production was evaluated. Yeast extract, malt extract, and MgSO4·7H2O showed significantly positive effects, whereas KH2PO4 and CaCl2·2H2O had a significantly negative effect (p-value < 0.05). Using response surface methodology, a medium consisting of 40.4 g/L (dry basis) industrial waste potato, 50 g/L malt extract, and 4.84 g/L MgSO4·7H2O was found optimal and yielded 24.6 g/L ethanol at 30 °C, 150 rpm, and 48 h of fermentation. In conclusion, this study demonstrated that industrial potato waste can be used effectively to enhance bioethanol production.

Synergism of fungal and bacterial cellulases and hemicellulases: a novel perspective for enhanced bio-ethanol production.

The complex structure of lignocellulose requires the involvement of a suite of lignocellulolytic enzymes for bringing about an effective de-polymerization. Cellulases and hemicellulases from both fungi and bacteria have been studied extensively. This review illustrates the mechanism of action of different cellulolytic and hemi-cellulolytic enzymes and their distinctive roles during hydrolysis. It also examines how different approaches can be used to improve the synergistic interaction between fungal and bacterial glycosyl hydrolases with a focus on fungal cellulases and bacterial hemicellulases. The approach entails the role of cellulosomes and their improvement through incorporation of novel enzymes and evaluates the recent break-through in the construction of designer cellulosomes and their extension towards improving fungal and bacterial synergy. The proposed approach also advocates the incorporation and cell surface display of designer cellulosomes on non-cellulolytic solventogenic strains along with the innovative application of combined cross-linked enzyme aggregates (combi-CLEAs) as an economically feasible and versatile tool for improving the synergistic interaction through one-pot cascade reactions.

Biochemical Screening of Different Types of Sweet Potato for Bioethanol Production

Today the present scenario is focused at energy production from agricultural biomass which has emerged as one of the dependable non-traditional feedstock for the production ofbioethanol. Production of this renewable fuel, especially from starchy materials such as tuber crops, holds a remarkable potential to meet the future energy. In recent years, with the increase in price of fossil fuels, the demand of biofuel production from tuber crops such as sweet potato has increased by leaps and bounds. Among different tuber crops, sweet potato (Ipomoea batatas L.) has been considered as a promising substrate for bioethanol production and has become very important to meet the future energy crisis in developing countries. In our present study, eight varieties of sweet potatoes were collected from different region of Odisha and biochemically analysed for ethanol production. Among them, the Gouri variety collected from Odisha University of Agriculture and Technology, Bhubaneswar, exhibited maximum amount of starch (293.69±2.9 gkg−1) and sugar (326.09±2.1 gkg−1) followed by Sree Krishna variety. The Gouri type also produced maximum ethanol of1 4 7.7±0.4 gkg−1 and showed ethanol yield of 90.6%. The other varieties were exhibiting low starch and sugar content (approximately 15% less) than the Gouri variety. As the Gouri variety was more potent for starch and sugar synthesis, so this variety can be utilised for enhanced bioethanol production.

Enhanced bio-ethanol production via simultaneous saccharification and fermentation through a cell free enzyme system prepared by disintegration of waste of beer fermentation broth

Current study illustrates the effect of high yeast cell density contained in the waste of beer fermentation broth (WBFB) on bio-ethanol production through simultaneous saccharification and fermentation (SSF). WBFB was disintegrated (DW) and comparatively evaluated against nondisintegrated WBFB (NDW) for bio-ethanol production at variant temperatures. Final bio-ethanol levels of 36.38 g/L and 18.65 g/L at 30 °C, 4.45 g/L and 43.23 g/L at 40 °C, and 2.32 g/L and 6.83 g/L at 50 °C were achieved with 20% NDW and DW, respectively, after 12 h. DW carried out the simultaneous saccharification and fermentation (SSF) process through cell free enzyme system and was capable of bioethanol production beyond the microbial growth temperature (>30 °C) of NDW system. The increase in sediment concentration in DW positively influenced the production capabilities of the system producing 43.23 g/L, 54.39 g/L and 62.82 g/L bio-ethanol with 20, 30 and 40% sediments at 40 °C, respectively. The retardation of bioethanol production at elevated temperature (50 °C) was expected to be caused by denaturing or digesting of certain enzymes as observed through SDS-PAGE. FTIR analysis also showed the appearance of a new band at approximately 1,590 cm−1 due to unfolding of polypeptide chains at 50 °C. The overall study reveals the positive influence of increased cell density on ethanol production and presents evidence for decreased fermentation beyond certain temperature limits.

English

Ethanol is commercially produced by fermenting agricultural products that contain high sugar or starch contents. In this study, the juices of Opilia amentacea and Curcubita pepo were used to produce bioethanol. A mixture of Urea (CON2H4), monosodium glutamate (C5H8NNaO4), potassium dihydrogen phosphate (KH2PO4), and magnesium sulfate heptahydrate (MgSO4.7H2O) is used to promote batch ethanol production with four strains of Saccharomyces cerevisiae as the fermenting organisms. The results obtained have shown that the nutrients mixture significantly affected kinetic parameters and enhanced bioethanol production. Subsequently, the highest outputs of 60.72 &plusmn; 0.68 and 50.93 &plusmn; 1.61 g ethanol/kg were obtained respectively with O. amentacea and C. pepo. In the same time, 460.97 &plusmn; 8.66 g ethanol/kg were got as maximum output from sucrose (NG). &nbsp; Key words: Fruit juices, enrichment, Saccharomyces cerevisiae, fermentation, bioethanol.

Enhanced bioethanol production from yellow poplar by deacetylation and oxalic acid pretreatment without detoxification

In order to produce ethanol from yellow poplar, deacetylation was performed using sodium hydroxide (NaOH). Optimal deacetylation conditions were determined by a response surface methodology. The highest acetic acid concentration obtained was 7.06g/L when deacetylation was performed at 60°C for 80min with 0.8% NaOH. Acetic acid was recovered by electrodialysis from the deacetylated hydrolysate. The oxalic acid pretreatment of deacetylated biomass was carried out and the hydrolysate directly used for ethanol production without detoxification. Ethanol yields ranged from 0.34 to 0.47g/g and the highest ethanol yield was obtained when pretreatment was carried out at 150°C with 50mM oxalic acid. The highest ethanol concentration obtained from pretreated biomass was 27.21g/L at 170°C, using a 50mM of oxalic acid for the simultaneous saccharification and fermentation (SSF). Overall, 20.31g of ethanol was obtained by hydrolysate and SSF from 100g of deacetylated yellow poplar.

Enhancing bioethanol production from delactosed whey permeate by upstream desalination techniques

Industrial cheese whey processing comprises generally the isolation of proteins and lactose, but the economic use for the residual molasses, the so‐called delactosed whey permeate (DWP), is still to be improved. One possibility to maximize valorization and to minimize waste water treatment is the conversion of the remaining lactose in the DWP to ethanol by the yeast Kluyveromyces marxianus. This fermentation process depends strongly on the total ash content of the DWP, as high salt concentrations inhibit yeast metabolism. Here, three different approaches were tested to lower the DWP salt content: (i) simple dilution; (ii) nanofiltration; and (iii) electrodialysis. Lactose consumption, ethanol production and time‐dependent yields were compared between the three methods. A dilution of DWP to 60% v/v led to fermentation taking less than 80 h and yield above 7% AbV (alcohol by volume). After nanofiltration, 7.5% AbV was produced in about 80 h, and after electrodialysis, 11% AbV was produced in about 52 h. On the one hand the technical treatments (nanofiltration and electrodialysis) led to enhanced productivity in the fermentations, but, on the other hand, elaborate and extensive preprocessing is needed. Overall, ethanol production from DWP could be enhanced by prior partial desalination.

Enhancement of bioethanol production in syngas fermentation with Clostridium ljungdahlii using nanoparticles

In this study, nanoparticles were used to enhance bioethanol production in syngas fermentation by Clostridium ljungdahlii. Six types of nanoparticles were tested: palladium on carbon, palladium on alumina, silica, hydroxyl-functionalized single-walled carbon nanotubes, alumina, and iron(III) oxide. Of these, silica nanoparticles at a concentration of 0.3 wt% were the best at enhancing gas-liquid mass transfer. The hydrophilic surfaces of silica nanoparticles were modified with hydrophobic functional groups such as methyl and isopropyl. Methyl-functionalized silica nanoparticles were better than unmodified and isopropyl-functionalized silica nanoparticles at enhancing mass transfer. The dissolved concentrations of CO, CO2, and H2 were enhanced by 272.9%, 200.2%, and 156.1%, respectively, by using methyl-functionalized silica nanoparticles. The use of methyl-functionalized silica nanoparticles at a concentration of 0.3 wt% during syngas fermentation by C. ljungdahlii led to significant increases in the levels of biomass, ethanol, and acetic acid production (34.5%, 166.1%, and 29.1%, respectively).

Zymomonas mobilis: a novel platform for future biorefineries.

Biosynthesis of liquid fuels and biomass-based building block chemicals from microorganisms have been regarded as a competitive alternative route to traditional. Zymomonas mobilis possesses a number of desirable characteristics for its special Entner-Doudoroff pathway, which makes it an ideal platform for both metabolic engineering and commercial-scale production of desirable bio-products as the same as Escherichia coli and Saccharomyces cerevisiae based on consideration of future biomass biorefinery. Z. mobilis has been studied extensively on both fundamental and applied level, which will provide a basis for industrial biotechnology in the future. Furthermore, metabolic engineering of Z. mobilis for enhancing bio-ethanol production from biomass resources has been significantly promoted by different methods (i.e. mutagenesis, adaptive laboratory evolution, specific gene knock-out, and metabolic engineering). In addition, the feasibility of representative metabolites, i.e. sorbitol, bionic acid, levan, succinic acid, isobutanol, and isobutanol produced by Z. mobilis and the strategies for strain improvements are also discussed or highlighted in this paper. Moreover, this review will present some guidelines for future developments in the bio-based chemical production using Z. mobilis as a novel industrial platform for future biofineries.

Ectopic expression of bacterial amylopullulanase enhances bioethanol production from maize grain

Heterologous expression of amylopullulanase in maize seeds leads to partial starch degradation into fermentable sugars, which enhances direct bioethanol production from maize grain. Utilization of maize in bioethanol industry in the United States reached ±13.3billion gallons in 2012, most of which was derived from maize grain. Starch hydrolysis for bioethanol industry requires the addition of thermostable alpha amylase and amyloglucosidase (AMG) enzymes to break down the α-1,4 and α-1,6 glucosidic bonds of starch that limits the cost effectiveness of the process on an industrial scale due to its high cost. Transgenic plants expressing a thermostable starch-degrading enzyme can overcome this problem by omitting the addition of exogenous enzymes during the starch hydrolysis process. In this study, we generated transgenic maize plants expressing an amylopullulanase (APU) enzyme from the bacterium Thermoanaerobacter thermohydrosulfuricus. A truncated version of the dual functional APU (TrAPU) that possesses both alpha amylase and pullulanase activities was produced in maize endosperm tissue using a seed-specific promoter of 27-kD gamma zein. A number of analyses were performed at 85°C, a temperature typically used for starch processing. Firstly, enzymatic assay and thin layer chromatography analysis showed direct starch hydrolysis into glucose. In addition, scanning electron microscopy illustrated porous and broken granules, suggesting starch autohydrolysis. Finally, bioethanol assay demonstrated that a 40.2±2.63% (14.7±0.90g ethanol per 100g seed) maize starch to ethanol conversion was achieved from the TrAPU seeds. Conversion efficiency was improved to reach 90.5% (33.1±0.66g ethanol per 100g seed) when commercial amyloglucosidase was added after direct hydrolysis of TrAPU maize seeds. Our results provide evidence that enzymes for starch hydrolysis can be produced in maize seeds to enhance bioethanol production.

Genetic Improvement of Plants for Enhanced Bio-Ethanol Production

The present world energy situation urgently requires exploring and developing alternate, sustainable sources for fuel. Biofuels have proven to be an effective energy source but more needs to be produced to meet energy goals. Whereas first generation biofuels derived from mainly corn and sugarcane continue to be used and produced, the contentious debate between "feedstock versus foodstock" continues. The need for sources that can be grown under different environmental conditions has led to exploring newer sources. Lignocellulosic biomass is an attractive source for production of biofuel, but pretreatment costs to remove lignin are high and the process is time consuming. Genetically modified plants that have increased sugar or starch content, modified lignin content, or produce cellulose degrading enzymes are some options that are being explored and tested. This review focuses on current research on increasing production of biofuels by genetic engineering of plants to have desirable characteristics. Recent patents that have been filed in this area are also discussed.

Optimization of dilute acid and alkaline peroxide pretreatment to enhance bioethanol production from wheat straw by co-fermentation

Optimization of dilute acid and alkaline peroxide pretreatment to enhance bioethanol production from wheat straw by co-fermentation

Enhanced Bioethanol Production in Batch Fermentation by Pervaporation Using a PDMS Membrane Bioreactor

The integration of batch fermentation and membrane-based pervaporation process in a membrane bioreactor (MBR) was studied to enhance bioethanol production compared to conventional batch fermentation operated at optimum condition. For this purpose, a laboratory-scale MBR system was designed and fabricated. Dense hydrophobic Polydimethylsiloxane (PDMS) membrane was used for pervaporation. For fermentation, pure stock culture of Saccharomyces cerevisiae as microorganism and glucose as substrate were used. Compared to conventional batch fermentation, the MBR resulted in increase of cell density, decreasing ethanol inhibition, improved productivity and yield, and resumption of clean and concentrated ethanol. These effects can be attributed to the presence of membrane as a selective separation barrier for removal of ethanol from fermentation broth. The ethanol productivity increased at least by 26.83% over conventional batch fermentation; while concentrated ethanol was obtained in the condensate of cold-trap. Furthermore, ethanol concentration in permeated side was approximately 6 to 7 times higher than that of the broth. Some biological kinetic models were investigated for determination of growth rate kinetics in conventional and MBR batch fermentation. The best results were obtained using the Monod kinetic model.

Kinetic modeling of enzymatic hydrolysis of pretreated kitchen wastes for enhancing bioethanol production

It is well known that use of low cost and abundant waste materials in microbial fermentations can reduce product costs. Kitchen wastes disposed of in large amounts from cafeterias, restaurants, dining halls, food processing plants, and household kitchens contain high amounts of carbohydrate components such as glucose, starch, and cellulose. Efficient utilization of these sugars is another opportunity to reduce ethanol costs. In this study, the effect of pretreatment methods (hot water, acid solutions, and a control) on enzymatic hydrolysis of kitchen wastes was evaluated using a kinetic modeling approach. Fermentation experiments conducted with and without traditional fermentation nutrients were assessed at constant conditions of pH 4.5 and temperature of 30°C for 48h using commercial dry baker’s yeast, Saccharomyces cerevisiae. The control, which involved no treatment, and hot water treated samples gave close glucose concentrations after 6h. The highest and lowest rates of glucose production were found as 0.644 and 0.128 (h−1) for the control (or no-pretreated (NPT)) and 1% acid solutions, respectively. The fermentation results indicated that final ethanol concentrations are not significantly improved by adding nutrients (17.2–23.3g/L). Thus, it was concluded that product cost can be lowered to a large extent if (1) kitchen wastes are used as a substrate, (2) no fermentation nutrient is used, and (3) hydrolysis time is applied for about 6h. Further optimization study is needed to increase the yield to higher levels.

Enhanced bioethanol production from pretreated corn stover via multi-positive effect of casein micelles

Casein polypeptides containing substructures of αs1-casein, β-casein, k-casein, αs2-casein were used as a lignin-blocker at above critical micelles concentration to improve the bioethanol production of dilute acid, lime, alkali, extrusion and AFEX pretreated corn stover (CS). Application of 0.5g/g glucan of casein was found to effectively increase the glucose yield of CS pretreated with dilute acid, lime, alkali, extrusion and AFEX by 31.9%, 17.0%, 22.7%, 29.5%, and 17.4%, respectively with no positive impact on Avicel. Consequently 96h simultaneous saccharification and fermentation (SSF) of these hydrolysates reduced the fermentation period by up to 48h and increased the theoretical yield of ethanol by 8.48–33.7% compared to control. Application of casein during saccharification reduced the enzyme utilization by 33.0%. Recycling of hydrolysate from casein-treated CS for a 2nd round hydrolysis resulted in average glucose yield of 36.4% compared to 29.0% control.

Improvement of multiple stress tolerance in yeast strain by sequential mutagenesis for enhanced bioethanol production

The present work deals with the improvement of multiple stress tolerance in a glucose-xylose co-fermenting hybrid yeast strain RPR39 by sequential mutagenesis using ethyl methane sulfonate, N-methyl-N'-nitro-N-nitrosoguanidine, near and far ultraviolet radiations. The mutants were evaluated for their tolerance to ethanol, temperature and fermentation inhibitors. Among these mutants, mutant RPRT90 exhibited highest tolerance to 10% initial ethanol concentration, 2 g L(-1) furfural and 8 g L(-1) acetic acid. The mutant also showed good growth at high temperature (39-40°C). A study on the combined effect of multiple stresses during fermentation of glucose-xylose mixture (3:1 ratio) was performed using mutant RPRT90. Under the combined effect of thermal (39°C) and inhibitor stress (0.25 g L(-1) vanillin, 0.5 g L(-1) furfural and 4 g L(-1) acetic acid), the mutant produced ethanol with a yield of 0.379 g g(-1), while under combined effect of ethanol (7% v/v) and inhibitor stress the ethanol yield obtained was 0.43 g g(-1). Further, under the synergistic effect of sugar (250 g L(-1)), thermal (39°C), ethanol (7% v/v) and inhibitors stress, the strain produced a maximum of 47.93 g L(-1) ethanol by utilizing 162.42 g L(-1) of glucose-xylose mixture giving an ethanol yield of 0.295 g g(-1) and productivity of 0.57 g L(-1) h(-1). Under same condition the fusant RPR39 produced a maximum of 30.0 g L(-1) ethanol giving a yield and productivity of 0.21 g g(-1) and 0.42 g L(-1) h(-1) respectively. The molecular characterization of mutant showed considerable difference in its genetic profile from hybrid RPR39. Thus, sequential mutagenesis was found to be effective to improve the stress tolerance properties in yeast.

Producing bioethanol from cellulosic hydrolyzate via co-immobilized cultivation strategy

Lignocellulose was converted into reducing sugars by using saccharification enzymes from cocultivated Trichoderma reesei and Aspergillus niger and reducing sugars as nutrients for Zymomonas mobilis to produce bioethanol in an immobilization system. After 96 h of cultivation, cocultivated T. reesei and A. niger had enzymatical synergistic effects that enabled a reducing sugar production of 1.29 g/L and a cellulose conversion rate of 23.27%. An 18% total inoculum concentration and a 1/1 inoculation ratio of T. reesei to A. niger obtained a reducing sugar production rate and a cellulose conversion rate of 2.57 g/L and 46.27%, respectively. The co-immobilization cultivation results showed that using polyurethane as a carrier optimized total saccharification enzyme activity at an inoculum ratio of 1/1 and a total inoculum concentration of 6.5×10(6)spores/mL. Based on the experimental results, the bioreactor design was further modified to enhance bioethanol production. The three strains (A. niger, T. reesei and Z. mobilis) were cocultivated with a co-immobilization cultivation system. The experimental results showed that, after 24 h cultivation, bioethanol production reached 0.56 g/L, and reducing sugar conversion rate reached 11.2% when using carboxymethylcellulose (CMC) substrates. The experimental results confirmed that the modified bioreactor enhances bioethanol production. However, further experiments are needed to determine how to prevent multi-stage failure of reducing medium volume.