Evaluation of fuel ethanol production from aqueous ammonia-treated rice straw via simultaneous saccharification and fermentation

Rice straw (RS) has been considered a promising feedstock for ethanol production in Asia. However, the recalcitrance of biomass, particularly the presence of lignin, hinders the enzymatic saccharification of polysaccharides in RS and consequently decreases the ethanol yield. Here, we used aqueous ammonia pretreatment to remove lignin from RS (aRS). The reaction conditions were a solid:liquid ratio of 1:12, an ammonia concentration of 27% (w w⁻¹), room temperature, and a 2-week incubation. We evaluated enzymatic digestibility and the ethanol production yield. A 42% reduction in lignin content increased the glucan conversion of aRS to glucose from 20 to 71% using a combination of Cellic Ctec2 cellulases and Cellic Htec2 xylanases at enzyme loads of 15 FPU +100 XU g⁻¹ solid. Scanning electron microscopy highlighted the extensive removal of external fibres and increased porosity of aRS, which aided the accessibility of cellulose for enzymes. Using the same enzyme dosage and a solid load of 100 g L⁻¹, simultaneous saccharification and fermentation using a monoculture of Saccharomyces cerevisiae and co-culture with Candida tropicalis yielded ethanol concentrations of 22 and 25 g L⁻¹, corresponding to fermentation efficiencies of 96 and 86% fermentation, respectively. The volumetric ethanol productivities for these systems were 0.45 and 0.52 g L⁻¹ h⁻¹. However, the ethanol yield based on the theoretical glucose and xylose concentrations was lower for the co-culture (0.44 g g⁻¹) than the monoculture (0.49 g g⁻¹) due to the low xylose consumption. Further research should optimise fermentation variables or select/improve microbial strains capable of fermenting xylose to increase the overall ethanol production yield.     

Biological hydrogen production from steam-exploded straw by simultaneous saccharification and fermentation

Hydrogen was produced by simultaneous saccharification and fermentation from steam-exploded corn straw (SECS) using Clostridium butyricum AS1.209. Effect of various process parameters, such as solid to liquid ratio, enzyme loading and initial pH, etc., were examined with respect to maximum hydrogen productivity which was obtained by fitting the cumulative hydrogen production data to a modified Gompertz equation. Maximum specific hydrogen production rate and maximal hydrogen yield were 126 ml/g VSS d and 68 ml/g SECS, respectively. The yield of soluble metabolites was 197.7 mg/g SECS. Acetic acid accounted for 46% of the total was the most abundant product and this shows that hydrogen production from SECS was essentially acetate-type fermentation. Hydrogen production by simultaneous saccharification and fermentation of SECS has the predominance of short lag-stage and high maximum specific hydrogen production rate and it was a promising method for hydrogen production and straw biomass conversion.     

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.     

玉米秸秆酶解糖化液乙醇发酵的工艺优化

利用从4组混合乙醇酵母中筛选出的优势混合酵母,对玉米秸秆酶解糖化液的乙醇发酵工艺过程进行了优化试验。试验结果表明,管囊酵母和酿酒酵母组成的混合酵母具有较高的乙醇发酵能力,经60 h发酵,乙醇浓度最高可达12.55 g/L,乙醇产率为最大理论值的68.63%。根据对糖化液乙醇发酵的二次回归正交组合优化试验,当发酵温度为28.0℃,初始pH为5.2,接种量为8.1%时,实际乙醇浓度最高可达13.03g/L,乙醇产率为0.36 g/g,为最大理论值的70.59%,与所得乙醇发酵回归方程预测值基本相符。     

Acidified glycerol pretreatment for enhanced ethanol production from rice straw

Rice straw was pretreated using an industrial grade glycerol for ethanol production. The pretreatment was conducted at 130–210 °C for 1–24 h with 5% solid loading. The glucan content in the regenerated rice straw increased with increasing pretreatment temperature and time. The production of fermentable sugars initially increased as the pretreatment temperature and reaction time increased, but then decreased somewhat at the higher temperatures and with longer reaction duration. The highest amount of reducing sugar produced by the enzymatic hydrolysis was achieved at 190 °C for 10 h with 5% solid loading, optimal condition for the glycerol pretreatment of rice straw. Furthermore, it was observed that glycerol pretreatment with the addition of HCl improved the digestibility of fermentable sugars by 4–5 times that of untreated samples. Fermentation of hydrolysates resulted in an ethanol yield of 0.44 g/g sugar, corresponding to a theoretical yield of 84.3%. It was concluded that acidified glycerol is one of the good candidates of the organic solvent for the pretreatment of lignocellulosic biomass.     

Simultaneous saccharification and fermentation of sweet sorghum after acid pretreatment

The sweet sorghum bagasse pretreated with 5% (w/w) acetic acid at an accumulated solid concentration of 20% (w/v) during the 96-h fed-batch simultaneous saccharification and fermentation achieved a maximum ethanol concentration of 53.1 g/L and ethanol yield of 88.7%, compared to 25.7 g/L and 86.7% for the 96-h batch simultaneous saccharification and fermentation at a solid concentration of 10% (w/v), respectively. For comparison, the bagasse pretreated with 0.5% (w/w) sulfuric acid and water under the same fed-batch simultaneous saccharification and fermentation conditions produced maximum ethanol concentrations of 44.3 and 36.5 g/L, and ethanol yields of 77.6 and 69.7%, respectively.     

Simultaneous saccharification and co-fermentation of whole wheat in integrated ethanol production

Two of the most important ways of reducing the production cost of lignocellulosic ethanol are to increase the ethanol yield and the concentration in the fermentation broth. This can be facilitated by co-fermentation of glucose and xylose from agricultural residues such as wheat straw, due to the high amount of xylose in the hemicelluloses in these materials.Simultaneous saccharification and co-fermentation (SSCF) of steam-pretreated wheat straw (SPWS) with and without the addition of liquefied wheat meal (LWM) was performed using the pentose-fermenting yeast, TMB3400. The highest overall ethanol yield in batch operation, of around 70%, equivalent to an ethanol concentration of 43.7 g L⁻¹, was achieved using SPWS with 7.5% water-insoluble solids (WIS) and addition of LWM with 1% WIS. Using SPWS with a higher WIS (10%) resulted in a decreased yield, 60%, although the concentration of ethanol increased to 53.0 g L⁻¹. SSCF of 7.5% straw was also performed with a single (after 20 h) or fed-batch addition of 1% WIS LWM (after 20, 24 and 28 h) resulting in an increase in both ethanol yield and concentration compared to the reference, without wheat meal addition, but no significant difference compared to the batch experiments.The addition of wheat meal to SSCF did not improve xylose utilization significantly, probably due to the instant release of glucose from the liquefied meal, which hampers the uptake of xylose. The instant release of glucose was shown to be caused by the high amylase activity of the β-glucosidase enzyme preparation.     

Potential for rice straw ethanol production in the Mekong Delta,Vietnam

This study is to evaluate the potential for development of a cellulosic ethanol facility in Vietnam. Rice straw is abundant in Vietnam and highly concentrated in the Mekong Delta, where about 26 Mt year⁻¹ of rice straw has been yearly produced. To minimize the overall production cost (PC) of ethanol from rice straw, it is crucial to choose the optimal facility size. The delivered cost of rice straw varied from 20.5 to 65.4 $ dry t⁻¹ depending on transportation distance. The Mekong Delta has much lower rice straw prices compared with other regions in Vietnam because of high density and quantity of rice straw supply. Thus, this region has been considered as the most suitable location for deploying ethanol production in Vietnam. The optimal plant size of ethanol production in the region was estimated up to 200 ML year⁻¹. The improvement in solid concentration of material in the hydrothermal pre-treatment step and using residues for power generation could substantially reduce the PC in Vietnam, where energy costs account for the second largest contribution to the PC, following only enzyme costs. The potential for building larger ethanol plants with low rice straw costs can reduce ethanol production costs in Vietnam. The current estimated production cost for an optimal plant size of 200 ML year⁻¹ was 1.19 $ L⁻¹. For the future scenario, considering improvements in pre-treatment, enzyme hydrolysis steps, specific enzyme activity, and applying residues for energy generation, the ethanol production cost could reduce to 0.45 $ L⁻¹ for a plant size of 200 ML year⁻¹ in Vietnam. These data indicated that the cost-competitiveness of ethanol production could be realized in Vietnam with future improvements in production technologies.     

底物质量浓度对玉米秸秆同步糖化生物制氢的影响

利用HAU-M1光合细菌对玉米秸秆同步糖化生物制氢工艺进行实验研究,提出了同步糖化生物制氢工艺中玉米秸秆底物质量浓度与pH值、还原糖质量浓度、氢气体积分数和累积产氢量等因素之间的相关关系,探讨了底物质量浓度对玉米秸秆同步糖化生物制氢工艺的影响规律。实验结果表明:当玉米秸秆底物质量浓度为25g/L时,玉米秸秆同步糖化生物制氢工艺的累积产氢量达到最高,为186mL;当玉米秸秆底物质量浓度为15g/L时,玉米秸秆同步糖化生物制氢工艺的氢气体积分数达到最高,为48%;玉米秸秆同步糖化制氢工艺的产氢高峰期为12~48h,48h后逐渐停止产氢,可为进一步优化和完善以生物质为基质的同步糖化生物制氢工艺理论与技术提供科学参考。     

Evaluation of bio-hydrogen production using rice straw hydrolysate extracted by acid and alkali hydrolysis

This study was conducted to investigate the properties of hydrolysates obtained from acid and alkali hydrolysis and to evaluate the feasibility of employing them for bio-hydrogen production. High sugar concentrations of 16.8 g/L and 13.3 g/L were present in 0.5% and 1.0% H₂SO₄ hydrolysates, respectively. However, H₂SO₄ hydrolysis resulted in large amounts of short-chain fatty acids (SCFAs) and furan derivatives, which were removed by detoxification. In bio-hydrogen production, 1.0% H₂SO₄ hydrolysate showed a 55.6 mL of highest hydrogen production and 1.14 mol-H₂/mol-hexose equivalent_added of hydrogen yield. In control and 1.0% NaOH hydrolysate, 29.7 mL and 36.9 mL of hydrogen were produced, respectively. Interestingly, relatively high acetate and butyrate production resulted in lactate reduction. Also, NH₄OH hydrolysate produced less than 10 mL of hydrogen. Thus, these results indicate that hydrogen production and metabolite distribution can vary depending on the sugars and by-product composition in the hydrolysate.     

Hydrogen production from simultaneous saccharification and fermentation of lignocellulosic materials in a dual-chamber microbial electrolysis cell

In this work, a dual-chamber microbial electrolysis cell (MEC) with concentric cylinders was fabricated to investigate hydrogen production of three different lignocellulosic materials via simultaneous saccharification and fermentation (SSF). The maximal hydrogen production rate (HPR) was 2.46 mmol/L/D with an energy recovery efficiency of 215.33 % and a total energy conversion efficiency of 11.29 %, and the maximal hydrogen volumetric yield was 28.67 L/kg from the mixed substrate. The concentrations of reducing sugar and organic acids, the pH, and the current in the MEC system during hydrogen production were monitored. The concentrations of reducing sugar, butyrate, lactate, formate, and acetate initially increased during SSF and then decreased due to hydrogen production. Moreover, the highest current was obtained from the mixed substrate, which means that the mixed substrates are beneficial to microbial growth and metabolism. These results suggest that lignocellulosic materials can be used as substrate in a low-energy-input dual-chamber MEC system for hydrogen production.     

Influence of initial anolyte pH and temperature on hydrogen production through simultaneous saccharification and fermentation of lignocellulose in microbial electrolysis cell

An investigation on the performance of hydrogen production by simultaneous saccharification and fermentation (SSF) in a dual-chamber microbial electrolysis cell (MEC) was carried out to consider different anolyte pH levels and culture temperatures, and the influences of anolyte pH value and culture temperature on changes of current, organic acid and pH value were also evaluated. The maximal hydrogen production rate (HPR) of 2.46 mmol/L/D (hydrogen energy recovery 219.02%) was obtained at the initial anolyte pH of 6.5. Within the range of the tested operation temperatures (30–50 °C), the optimal temperature for hydrogen production by SSF in the MEC systems was 35 °C. Moreover, the contents of organic acids and reducing sugar significantly changed with varying in initial anolyte pH and temperature levels. The result indicates that a low initial anolyte pH value and high culture temperature was beneficial to hydrolysis of cellulose, and a high initial anolyte pH value and a moderate culture temperature to hydrogen production.     

The addition of hydrolyzed rice straw in xylose fermentation by Pichia stipitis to increase bioethanol production at the pilot-scale

The pretreatment of agricultural biomass by diluted acid is often employed to facilitate the release of monosaccharide for the subsequent enzyme hydrolysis for lignocellulosic ethanol production. However, furfural and hydroxymethylfurfural are usually generated and markedly decrease the yield of pentose fermentation during this pretreatment. In the present study, the enhancement of lignocellulosic ethanol production was successfully demonstrated at pilot scale with extra addition of hydrolyzed rice straw into pentose fermentation by Pichia stiptis. This way has resulted into the increase of P. stiptis cell mass was shown to play a positive role. The ethanol yield, 0.45 g_p/g_s, with the addition of hydrolyzed rice straw in hemicellulosic hydrolysate from plywood, bagasse and bamboo were increase 20–51% to demonstrate the applicability of this technology in a variety of lignocellulosic ethanol processes due to the efficient conversion of xylose.     

Contents of various sources of glucose and fructose in rice straw, a potential feedstock for ethanol production in Japan

The major carbohydrates of rice straw samples were determined in order to evaluate the potential of using rice straw as a feedstock for ethanol production in Japan. Straw samples were harvested by cutting the plants at ground level when the grain was mature and immediately heating or chilling the samples. In all cases, significant amounts (62-303 g kg⁻¹) of soft carbohydrates defined as consisting of glucose, fructose, sucrose, starch and ??-1,3-1,4-glucan were detected, in addition to structural carbohydrates (cellulose and xylan). These results indicate that rice straw is a rich source of fermentable sugars from both soft carbohydrates and lignocellulosic portions of the cell wall.     

Organosolv pretreatment of Liriodendron tulipifera and simultaneous saccharification and fermentation for bioethanol production

An acid-free organosolv process was proposed to overcome the problems caused by acid catalyst in organosolv process, thereby producing ethanol from Liriodendron tulipifera effectively. Although relative lignin contents were above 20%, enzymatic conversion increased significantly to 65% at all conditions, and thus correlation between lignin and enzymatic conversion could not be explained using relative lignin content. Enzymatic conversion increased significantly above 65% regardless of temperature, which suggests the organosolv pretreatment with sodium hydroxide can be performed at lower temperature. FE-SEM showed that the process made the structure loose and broke down biomass through lignin dissolution. Wrinkle formation by alkaline swelling was also observed and it might increase surface area. Although pore-volume increased slightly, it was not the sole key factor for the organosolv pretreatment with sodium hydroxide. Increase in surface area and enzyme adsorption enhanced the enzymatic hydrolysis. Ethanol of 96% could be produced theoretically and it suggested that the acid-free organosolv process was an effective pretreatment method for bioethanol production from L. tulipifera.     

Enhanced ethanol production by removal of cutin and epicuticular waxes of wheat straw by plasma assisted pretreatment

The removal of cutin and epicuticular waxes of wheat straw by PAP (plasma assisted pretreatment) was investigated. Wax removal was observed by Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) as chemical change on the surface of most intensively pretreated samples as well as with Scanning Electron Microscopy (SEM) imaging. Compounds resulting from wax degradation were analyzed in the washing water of PAP wheat straw. The wax removal enhanced enzymatic hydrolysis yield and, consequently, the efficiency of wheat straw conversion into ethanol. In total, PAP increased the conversion rate of the raw material carbohydrate content up to 67%, compared to untreated raw material.     

Hydrolysis of pretreated rice straw by an enzyme cocktail comprising acidic xylanase from Aspergillus sp. for bioethanol production

The most crucial enzyme involved in xylan hydrolysis is endoxylanase which cleaves the internal glycosidic bonds of xylan. The aim of this work was to study the production of extracellular xylanase by a locally isolated strain of Aspergillus sp. under solid-state fermentation (SSF) and to evaluate the potential of the enzyme in enzymatic hydrolysis of pretreated rice straw. Xylanase production reached maximum with incubation period (96 h), moisture level (80%), inoculum size (3 × 10⁶ spores/mL), pH (4.8), temperature (25 °C), carbon source (wheat bran) and nitrogen source (yeast extract). Under optimized conditions, xylanase production reached to 5059 IU/gds. Crude xylanase was used for supplementing the enzyme cocktail comprising cellulases (Zytex, India), β-glucosidase (In-house) and xylanase (In-house) for the saccharification of alkali-pretreated rice straw to get the maximum reducing sugar production. The cocktail containing the three enzymes resulted a maximum of 574.8 mg/g of total reducing sugars in comparison to 430.2 mg/g sugars by the cocktail without xylanase. These results proved that the crude xylanase preparation from Aspergillus sp. could be a potent candidate for the enzyme cocktail preparation for biomass hydrolysis in lignocellulosic bioethanol program.     

Cellulolytic enzymes produced by a newly isolated soil fungus Penicillium sp. TG2 with potential for use in cellulosic ethanol production

A newly isolated soil fungus, Penicillium sp. TG2, had cellulase activities that were comparable to those of Trichoderma reesei RUT-C30, a common commercial strain used for cellulase production. The maximal and specific activities were 1.27 U/mL and 2.28 U/mg for endoglucanase, 0.31 U/mL and 0.56 U/mg for exoglucanase, 0.54 U/mL and 1.03 U/mg for β-glucosidase, and 0.45 U/mL and 0.81 U/mg for filter paper cellulase (FPase), respectively. Optimal FPase activity was at pH 5.0 and 50 °C. We used a simultaneous saccharification and fermentation (SSF) process, which employed the yeast Kluyveromyces marxianus and Penicillium sp. TG2 cellulolytic enzymes, to produce ethanol from empty palm fruit bunches (EFBs), a waste product from the palm oil industry. The present findings indicate that Penicillium sp. TG2 has great potential as an alternative source of enzymes for saccharification of lignocellulosic biomass.     

双菌比较优化稻草“液化”发酵产乙醇的研究

利用稻草液化产物为底物,分别采用酿酒酵母和休哈塔假丝酵母发酵生产乙醇,对影响发酵阶段的各因素进行优化,选取最佳菌种完成秸秆到乙醇的转化。结果表明,液化产物经酶解后葡萄糖浓度可达69.5mg/mL,是发酵制备乙醇的良好底物。优化发酵后,酿酒酵母更适合做液化产物的发酵菌种。适宜的发酵条件:初始葡萄糖浓度60~65 mg/mL,温度30℃,pH=6.0,装液量80 mL,接种量10%,发酵时间36 h,在此条件下乙醇得率可达49.3%,能达到理论得率的96.1%,转化率最高为0.27 g/g(乙醇/液化产物)。     

Covalent immobilization of enzymes and yeast: Towards a continuous simultaneous saccharification and fermentation process for cellulosic ethanol

The immobilization of enzymes and yeast cells is a key factor for establishing a continuous process of cellulosic ethanol production, which can combine the benefits of a separated hydrolysis and fermentation process and a simultaneous saccharification and fermentation process. This paper investigates the use of cellulase enzyme and yeast cell immobilization under a flow regime of ethanol production from soluble substrates such as cellobiose and carboxymethyl cellulose. The immobilization was achieved by incubating enzymes and yeast cells on polystyrene surfaces which had been treated by nitrogen ion implantation. The saccharification by immobilized enzymes and the fermentation by immobilized yeast cells were conducted in two separate vessels connected by a pump. During the experiments, glucose concentrations were always maintained at low levels which potentially reduce product inhibition effects on the enzymes. Covalent immobilization of enzymes and yeast cells on the plasma treated polymer reduces loss by shear flow induced detachment. The potential for continuous flow production of ethanol and the influence of daughter yeast cells in the circulating flow on the immobilized enzyme activity are discussed.