Dilute oxalic acid pretreatment for biorefining giant reed (Arundo donax L.)

Biomass pretreatment is essential to overcome recalcitrance of lignocellulose for ethanol production. In the present study we pretreated giant reed (Arundo donax L.), a perennial, rhizomatous lignocellulosic grass with dilute oxalic acid. The effects of temperature (170-190 °C), acid loading (2-10% w/w) and reaction time (15-40 min) were handled as a single parameter, combined severity. We explored the change in hemicellulose, cellulose and lignin composition following pretreatment and glucan conversion after enzymatic hydrolysis of the solid residue. Two different yeast strains, Scheffersomyces (Pichia) stipitis CBS 6054, which is a native xylose and cellobiose fermenter, and Saccharomyces carlsbergensis FPL-450, which does not ferment xylose or cellobiose, were used along with commercial cellulolytic enzymes in simultaneous saccharification and fermentation (SSF). S. carlsbergensis attained a maximum ethanol concentration of 15.9 g/l after 48 h at pH 5.0, while S. stipitis, at the same condition, took 96 h to reach a similar ethanol value; increasing the pH to 6.0 reduced the S. stipitis lag phase and attained 18.0 g/l of ethanol within 72 h.     

Ethanol and co-product generation from pressurized batch hot water pretreated T85 bermudagrass and Merkeron napiergrass using recombinant Escherichia coli as biocatalyst

Pretreatment of grasses is required to maximize ethanol yield during fermentation. T85 bermudagrass and Merkeron napiergrass (Pennisetum purpureum Schumach.) were either left untreated or were pressurized batch hot water (PBHW) pretreated for 2 min at 230 °C at 5% w/v whole grass solids loading. Following a 24 h enzymatic digestion, untreated and PBHW pretreated grasses were evaluated for ethanol production and co-product generation including potential fermentation inhibitors. Fermentations of PBHW pretreated grasses with E. coli LY01 produced twice the ethanol of their untreated counterparts. PBHW pretreated Merkeron napiergrass produced 224.5 mg/g grass ethanol (73% maximum theoretical yield) and PBHW pretreated T85 bermudagrass reached 213.0 mg/g grass (70% maximum theoretical ethanol yield). Pretreatment by PBHW resulted in increased solubilization of hemicelluloses. PBHW pretreatment also produced potential fermentation inhibitors such as acetic, formic, cinnamic acids, and aldehydes. Despite some of these inhibitors remaining with the solids after PBHW pretreatment, there was more efficient hydrolysis of the cellulose and remaining hemicellulose during the enzymatic digestion of the grasses prior to fermentation when compared to the untreated grasses. This increase in digestibility observed with enzymes prior to fermentation resulted in increased ethanol yields during bioconversion using E. coli LY01 as the biocatalyst.     

Simultaneous glucose and xylose utilization for improved ethanol production from lignocellulosic biomass through SSFF with encapsulated yeast

Simultaneous glucose and xylose uptake was investigated for ethanol production using the simultaneous saccharification, filtration and fermentation (SSFF) process with pretreated wheat straw as a xylose-rich lignocellulosic biomass. A genetically engineered strain of Saccharomyces cerevisiae (T0936) with the ability to ferment xylose was used for the fermentations. SSFF was compared with a conventional method of simultaneous saccharification and fermentation (SSF) for glucose and xylose uptake, ethanol production, and cell viability on 10% and 12% suspended solids (SS) basis. With 10% SS, an ethanol yield of 90% of the theoretical level was obtained during SSFF with 80% xylose uptake while only 53% ethanol yield was observed during the SSF process. Increasing the solid load to 12% resulted in an ethanol yield of 77% of the theoretical value and 36% xylose uptake during SSFF while only 27% ethanol yield and no xylose uptake was observed during the corresponding SSF process. The SSFF process preserved the viability of the genetically engineered yeast throughout the fermentation, even when reused for 2 consecutive cultivations. The results show that the SSFF process does not only enhance effective cell performance but also facilitates simultaneous glucose and xylose utilization, which is important for broad range of biomass utilization for lignocellulosic ethanol production.     

Enzymatic hydrolysis and fermentation of crushed whole sugar beets

Pectinase and cellulase enzymes were used for hydrolysis of whole sugar beets and the hydrolyzates were fermented with Escherichia coli KO11 and Saccharomyces cerevisiae via simultaneous saccharification and fermentation (SSF). Ethanol production rate was significantly higher for S. cerevisiae than for E. coli KO11. The combined effect of pectinase and cellulase loadings on ethanol production as well as residual galacturonic acid and arabinose concentrations were modeled for fermentations with S. cerevisiae. Ethanol yields of more than 92% were reached with moderate to high cellulase and pectinase loadings at 0.51 FPU g⁻¹ and 51 U g⁻¹ of dry biomass, respectively. Ethanol yields of 85% were achieved without any enzyme addition. However, addition of cellulase and pectinase enzymes increased effluent arabinose and galacturonic acid concentrations and reduced total suspended solids. This study demonstrated the yield potential of fermentation of crushed, whole sugar beets with or without the addition of cellulase and pectinase enzymes.     

Cellulase production using biomass feed stock and its application in lignocellulose saccharification for bio-ethanol production

A major constraint in the enzymatic saccharification of biomass for ethanol production is the cost of cellulase enzymes. Production cost of cellulases may be brought down by multifaceted approaches which include the use of cheap lignocellulosic substrates for fermentation production of the enzyme, and the use of cost efficient fermentation strategies like solid state fermentation (SSF). In the present study, cellulolytic enzymes for biomass hydrolysis were produced using solid state fermentation on wheat bran as substrate. Crude cellulase and a relatively glucose tolerant BGL were produced using fungi Trichoderma reesei RUT C30 and Aspergillus niger MTCC 7956, respectively. Saccharification of three different feed stock, i.e. sugar cane bagasse, rice straw and water hyacinth biomass was studied using the enzymes. Saccharification was performed with 50 FPU of cellulase and 10 U of β-glucosidase per gram of pretreated biomass. Highest yield of reducing sugars (26.3 g/L) was obtained from rice straw followed by sugar cane bagasse (17.79 g/L). The enzymatic hydrolysate of rice straw was used as substrate for ethanol production by Saccharomyces cerevisiae. The yield of ethanol was 0.093 g per gram of pretreated rice straw.     

Identification of potential fermentation inhibitors in conversion of hybrid poplar hydrolyzate to ethanol

In the production of automobile fuel ethanol from biomass by dilute acid hydrolysis/fermentation process, degradation products from the hydrolysis substantially inhibit the bioconversion of sugar to ethanol. Majority of these inhibitors have not been previously identified due to the complexity of biomass hydrolyzate. This paper presents an analytical procedure for identification of biomass degradation products, which entails flash evaporation, anion exchange, chloroform and ethyl acetate extraction, HPLC and GC-MS analyses. More than 35 potential inhibitors to S. cerevisiae fermentation in dilute nitric acid hydrolyzates of hybrid poplar were identified by correlating the fermentability of the anion exchange treated and untreated hydrolyzate samples with their chemical compositions, and by chemical analysis of the regeneration eluate from the ion exchange resin saturated by the hydrolyzate.     

Bioethanol production from pretreated Miscanthus floridulus biomass by simultaneous saccharification and fermentation

The production of ethanol from the fast-growing perennial C4 grass Miscanthus floridulus by simultaneous saccharification and fermentation (SSF) was investigated. M. floridulus biomass was composed of 36.3% glucan, 22.8% hemicellulose, and 21.3% lignin (based on dried mass). Prior to SSF, harvested stems of M. floridulus were pretreated separately by alkali treatment at room temperature, alkali treatment at 90 °C, steam explosion, and acid-catalyzed steam explosion. The delignification rates were determined to be 73.7%, 61.5%, 42.7%, and 63.5%, respectively, by these four methods, and the hemicellulose removal rates were 51.5%, 85.1%, 70.5%, and 97.3%, respectively. SSF of residual solids after various pretreatments was performed with dried yeast (Saccharomyces cerevisiae) and cellulases (Accellerase 1000) by using 10% water-insoluble solids (WIS) of the pretreated M. floridulus as the substrate. The ethanol yields from 72-h SSF of M. floridulus biomass after these pretreatments were 48.9 ± 3.5, 78.4 ± 1.0, 46.4 ± 0.1, and 69.0 ± 0.1% (w/w), respectively, while the ethanol concentrations after 72-h SSF were determined to be 15.4 ± 1.1, 27.5 ± 0.3, 13.9 ± 0.1, and 30.8 ± 0.1 g/L, respectively. Overall, the highest amount of ethanol (0.124 g/g-dried raw material) was generated from dried raw material of M. floridulus after alkaline pretreatment at 90 °C. The acid-catalyzed steam explosion pretreatment also resulted in a high ethanol yield (0.122 g/g-dried raw material). Pretreatment resulting in high lignin and hemicellulose removal rates could make biomass more accessible to enzyme hydrolysis and lead to higher ethanol production.     

Biobutanol production from fiber-enhanced DDGS pretreated with electrolyzed water

DDGS (distiller's dried grains with solubles) is a major co-product in dry-grind ethanol production from corn. A recently developed physical process separates DDGS into two value-added components: a fiber-enriched DDGS and a portion that is rich in oil and protein. Electrolyzed water, a new pretreatment catalyst was employed to pretreat fiber-enriched DDGS. Four temperatures (130, 145, 160, and 175 °C) and three treatment times (10, 20, and 30 min) were examined in the pretreatment with a solid loading of 20% w/w. Other pretreatment methods, such as diluted sulfuric acid, alkaline solution, and hot water, were also tested for comparison purposes. Fifteen FPU cellulase/g cellulose, 40 units β-glucosidase/g cellulose, and 50 units xylanase/g dry biomass were used in the enzymatic hydrolysis at 50 °C and 10% solid loading. The hydrolyzates were fermented by Clostridium beijerinckii BA 101 at 35 °C in an auto-controlled Six-fors fermentor with continuous mixing. The highest sugar yield was achieved when using the acidic electrolyzed water treatment at 175 °C for 10 min, with 23.25 g glucose, xylose and arabinose released from 100 g fiber-enriched DDGS. The C. beijerinckii fermentation produced 5.35 g ABE (acetone, butanol, and ethanol) from 100 g dry fiber-enhanced DDGS. This study demonstrated that DDGS pretreated with electrolyzed water and hydrolyzed with commercial enzymes could be used to produce biobutanol without detoxification.     

混合菌种发酵秸秆稀酸预处理液中木糖的研究

以木糖纯种发酵菌种的筛选为基础,得到了能发酵木糖的混合菌群。将该菌群进行了稀酸预处理液发酵的筛选驯化,得到能发酵稀酸预处理液的菌群B00。与纯种发酵对比,混合菌种发酵有较广的初始pH值、温度、摇床转速的适应范围以及较广的底物应用范围和较强的发酵抑制物耐受性,因而更适合工业化的应用;在木糖含量为30 g/L、葡萄糖含量为15 g/L的稀酸预处理液中,该混合菌种的最高乙醇产量为15.8 g/L,产率为68.9%,高于纯培养的乙醇产率62.6%。     

酵母菌复合培养物对木质纤维素稀酸水解液原位脱毒乙醇发酵

为了解决木质纤维素稀酸水解产物中发酵抑制剂对微生物的抑制作用以及木糖的乙醇发酵问题,该研究用本实验室开发的能高效代谢葡萄糖产乙醇并代谢糠醛和5-羟甲基糠醛的2株酵母菌种Saccharomyces cerevisiae Y5和Ismtchenkia/orientalis Y4分别与Pichia.stipitis CBS6054组成2个复合菌种,用复合菌种对木质纤维素稀酸水解产物进行原位脱毒乙醇发酵.结果证明,复合菌种S.cerevisiae Y5,P.stipitis CBS6054显示出了很好的代谢稀酸水解液中的葡萄糖和木糖产乙醇并快速代谢糠醛和5-羟甲基糠醛的能力,乙醇产率为0.43g/g(达到理论值的85.1%).该复合培养物可作为木质纤维索稀酸水解产物不需任何脱毒处理直接进行乙醇发酵的复合菌种.     

Ethanol production from enzymatic hydrolysis of sugarcane bagasse pretreated with lime and alkaline hydrogen peroxide

In this work we evaluated ethanol production from enzymatic hydrolysis of sugarcane bagasse. Two pretreatments agents, lime and alkaline hydrogen peroxide, were compared in their performance to improve the susceptibility of bagasse to enzymatic action. Mild conditions of temperature, pressure and absence of acids were chosen to diminish costs and to avoid sugars degradation and consequent inhibitors formation. The bagasse was used as it comes from the sugar/ethanol industries, without grinding or sieving, and hydrolysis was performed with low enzymes loading (3.50 FPU g⁻¹ dry pretreated biomass of cellulase and 1.00 CBU g⁻¹ dry pretreated biomass of ??-glucosidase). The pretreatment with alkaline hydrogen peroxide led to the higher glucose yield: 691 mg g⁻¹ of glucose for pretreated bagasse after hydrolysis of bagasse pretreated for 1 h at 25 °C with 7.35% (v/v) of peroxide. Fermentation of the hydrolyzates from the two pretreatments were carried out and compared with fermentation of a glucose solution. Ethanol yields from the hydrolyzates were similar to that obtained by fermentation of the glucose solution. Although the preliminary results obtained in this work are promising for both pretreatments considered, reflecting their potential for application, further studies, considering higher biomass concentrations and economic aspects should be performed before extending the conclusions to an industrial process.     

Bioethanol production by Saccharomyces cerevisiae,Pichia stipitis and Zymomonas mobilis from delignified coconut fibre mature and lignin extraction according to biorefinery concept

In search to increase the offer of liquid, clean, renewable and sustainable energy in the world energy matrix, the use of lignocellulosic materials (LCMs) for bioethanol production arises as a valuable alternative. The objective of this work was to analyze and compare the performance of Saccharomyces cerevisiae, Pichia stipitis and Zymomonas mobilis in the production of bioethanol from coconut fibre mature (CFM) using different strategies: simultaneous saccharification and fermentation (SSF) and semi-simultaneous saccharification and fermentation (SSSF). The CFM was pretreated by hydrothermal pretreatment catalyzed with sodium hydroxide (HPCSH). The pretreated CFM was characterized by X-ray diffractometry and SEM, and the lignin recovered in the liquid phase by FTIR and TGA. After the HPCSH pretreatment (2.5% (v/v) sodium hydroxide at 180 °C for 30 min), the cellulose content was 56.44%, while the hemicellulose and lignin were reduced 69.04% and 89.13%, respectively. Following pretreatment, the obtained cellulosic fraction was submitted to SSF and SSSF. Pichia stipitis allowed for the highest ethanol yield – 90.18% – in SSSF, 91.17% and 91.03% were obtained with Saccharomyces cerevisiae and Zymomonas mobilis, respectively. It may be concluded that the selection of the most efficient microorganism for the obtention of high bioethanol production yields from cellulose pretreated by HPCSH depends on the operational strategy used and this pretreatment is an interesting alternative for add value of coconut fibre mature compounds (lignin, phenolics) being in accordance with the biorefinery concept.     

Ethanol production by Mucor indicus and Rhizopus oryzae from rice straw by separate hydrolysis and fermentation

Rice straw was successfully converted to ethanol by separate enzymatic hydrolysis and fermentation by Mucor indicus, Rhizopus oryzae, and Saccharomyces cerevisiae. The hydrolysis temperature and pH of commercial cellulase and β-glucosidase enzymes were first investigated and their best performance obtained at 45 °C and pH 5.0. The pretreatment of the straw with dilute-acid hydrolysis resulted in 0.72 g g^?1 sugar yield during 48 h enzymatic hydrolysis, which was higher than steam-pretreated (0.60 g g^?1) and untreated straw (0.46 g g^?1). Furthermore, increasing the concentration of the dilute-acid pretreated straw from 20 to 50 and 100 g L^?1 resulted in 13% and 16% lower sugar yield, respectively. Anaerobic cultivation of the hydrolyzates with M. indicus resulted in 0.36–0.43 g g^?1 ethanol, 0.11–0.17 g g^?1 biomass, and 0.04–0.06 g g^?1 glycerol, which is comparable with the corresponding yields by S. cerevisiae (0.37–0.45 g g^?1 ethanol, 0.04–0.10 g g^?1 biomass and 0.05–0.07 glycerol). These two fungi produced no other major metabolite from the straw and completed the cultivation in less than 25 h. However, R. oryzae produced lactic acid as the major by-product with yield of 0.05–0.09 g g^?1. This fungus had ethanol, biomass and glycerol yields of 0.33–0.41, 0.06–0.12, and 0.03–0.04 g g^?1, respectively.     

Effect of media sterilization and enrichment on ethanol production from carob extract in a biofilm reactor

In this study, repeated-batch fermentations in a biofilm reactor were evaluated for ethanol production from non-sterile enriched (NSE) and non-sterile non-enriched (NSNE) carob extracts by using Saccharomyces cerevisiae. For the NSE medium, the ethanol production (P) and yield (Y_P/S) were 18.46 g/L and 33.76%, respectively. However, for the NSNE medium, P and Y_P/S were 19.57 g/L and 38.14%, respectively. Results indicated that cost-effective ethanol production from non-sterile carob extract (NSCE) can be successfully applied in a biofilm reactor regarding both energy cost and recovery time.     

Comparative study on ethanol production from pretreated sugarcane bagasse using immobilized Saccharomyces cerevisiae on various matrices

Microwave alkali pretreated sugarcane bagasse was used as a substrate for production of cellulolytic enzymes, needed for biomass hydrolysis. The pretreated sugarcane bagasse was enzymatic hydrolyzed by crude unprocessed enzymes cellulase (Filter paper activity 9.4 FPU/g), endoglucanase (carboxymethylcellulase, 148 IU/g), β-glucosidase (116 IU/g) and xylanase (201 IU/g) produced by Aspergillus flavus using pretreated sugarcane bagasse as substrate under solid state fermentation. Concentrated enzymatic hydrolyzate was used for ethanol production using Saccharomyces cerevisiae immobilized on various matrices. The yield of ethanol was 0.44 g_p/g_s in case of yeast immobilized sugarcane bagasse, 0.38 g_p/g_s using Ca-alginate and 0.33 g_p/g_s using agar-agar as immobilization matrices. The immobilized yeast studied up to 10 cycles in case of immobilized sugarcane bagasse and up to 4 cycles in case of agar-agar and calcium alginate for ethanol production under repeated batch fermentation study.     

Evaluation of wet air oxidation as a pretreatment strategy for bioethanol production from rice husk and process optimization

The pretreatment of rice husk by the wet air oxidation (WAO) technique was investigated by means of a statistically designed set of experiments. Reaction temperature, air pressure, and reaction time were the process parameters considered. WAO pretreatment of rice husk increased the cellulose content of the solid fraction by virtue of lignin removal and hemicellulose solubilization. The cellulose recovery was around 92%, while lignin recovery was in the tune of 8–20%, indicating oxidation of a bulk quantity of lignin. The liquid fraction was found to be rich in hexose and pentose sugars, which could be directly utilized as substrate for ethanol fermentation. The WAO process was optimized by multi-objective numerical optimization with the help of MINITAB 14 suite of statistical software, and an optimum WAO condition of 185 °C, 0.5 MPa, and 15 min was predicted and experimentally validated to give 67% (w/w) cellulose content in the solid fraction, along with 89% lignin removal, and 70% hemicellulose solubilization; 13.1 gl^?1 glucose and 3.4 gl^?1 xylose were detected in the liquid fraction. The high cellulose content and negligible residual lignin in the solid fraction would greatly facilitate subsequent enzymatic hydrolysis, and result in improved ethanol yields from rice husk.     

Evaluation of an adapted inhibitor-tolerant yeast strain for ethanol production from combined hydrolysate of softwood

In order to evaluate the potential of an adapted inhibitor-tolerant yeast strain developed in our lab to produce ethanol from softwood, the effect of furfural and HMF presented in defined medium and pretreatment hydrolysate on cell growth was investigated. And the efficiency of ethanol production from enzymatic hydrolysate mixed with pretreatment hydrolysate of softwood by bisulfite and sulfuric acid pretreatment process was reported. The results showed that in the combined treatments of the two inhibitors, cell growth was not affected at 1 g/L each of furfural and HMF. When 3 g/L each of furfural and HMF was applied, the adapted strain responded with an extended lag phase of 24 h. Both in batch and fed-batch runs of combined hydrolysate fermentation, the final ethanol concentrations were above 20.0 g/L and the ethanol yields (Y_p/s) on the total amount of fermentable sugar presented in the pretreated materials were above 0.40 g/g. It implies the great promise of the yeast strain for improving ethanol production from softwood due to its high ability of metabolizing inhibitor compounds of furfural and HMF.     

Low temperature thermoconversion of biomass to organic chemicals in the presence of aluminum chloride

The low temperature thermoconversion of rice hulls, an agricultural residue, was accomplished at 175°C in the presence of AlCl₃ vapor. Among the volatile products were acetone, methylisobutyl ketone, and a reactive oil which formed solid carbohydrate-like products when warmed to room temperature. In the absence of AlCl₃, no products were collected at temperatures up to 225°C. The source of these compounds was found to be the hemicellulose portion of the rice hulls. Lignin and cellulose failed to produce volatile products at 175°C.     

改进的柳枝稷预处理方法及乙醇发酵研究

为了提高柳枝稷中纤维素和半纤维素糖的转化率,降低水解液中抑制剂的浓度,首先,用稀酸在温和条件下对柳枝稷进行水解,然后用碱对酸水解后的固体物进行预处理,接着用纤维素酶酶解并分别对稀酸水解液和酶解液进行乙醇发酵.结果表明:纤维素转化率达到94.26%,半纤维素转化率为60.93%,稀酸水解液乙醇发酵的乙醇产率为0.441g乙醇/g糖,达到最高理论值的86.47%.酶解液乙醇发酵的乙醇产率为0.486g乙醇/g葡萄糖,达到最高理论值的95.29%.     

Isolation of a Clostridium acetobutylicum strain and characterization of its fermentation performance on agricultural wastes

A new solvent-producing Clostridium has been isolated from soil used in intensive rice cultivation. The 16S rRNA analysis of the isolate indicates that it is closely related to Clostridium acetobutylicum, with a sequence identity of 96%. The new isolate, named C. acetobutylicum YM1, produces biobutanol from multiple carbon sources, including glucose, fructose, xylose, arabinose, glycerol, lactose, cellobiose, mannitol, maltose, galactose, sucrose and mannose. This isolate can also utilize polysaccharides such as starch and carboxylmethyl cellulose (CMC) for the production of biobutanol. The ability of isolate YM1 to produce biobutanol from agro-industrial wastes was also evaluated for rice bran, de-oiled rice bran, palm oil mill effluent and palm kernel cake. The highest concentration of biobutanol (7.27 g/L) was obtained from the fermentation medium containing 2% (w/v) fructose, with a total acetone–butanol–ethanol (ABE) concentration of 10.23 g/L. The ability of isolate YM1 to produce biobutanol from various carbon sources and agro-wastes indicates the promise of the use of this isolate for the production of biobutanol, a renewable energy resource, from readily available renewable feedstocks.