Simultaneous saccharification and fermentation (SSF) of non-starch polysaccharides and starch from fresh tuber of Canna edulis ker at a high solid content for ethanol production

Canna edulis ker is a potential feedstock for ethanol production because of its low nutrition requirements and the high starch content of its tubers. The processing of C. edulis is limited by the high viscosity of the biomass. In this study, cell wall degrading enzymes (CWDEs) containing acid xylanase and β-glucanase were successful in reducing the viscosity (from 167.30 Pa s to 8.66 Pa s) at 50 °C for 120 min. The effect of CWDEs on simultaneous saccharification and fermentation (SSF) was investigated. Addition of CWDEs before SSF, resulted in an increase in total sugar and fermentable sugar. Meanwhile, the viscosity decreased sharply from approximately 200.00 Pa s to 2.98 Pa s, thereby improving the fermentation parameters and the mass fraction of the theoretical ethanol yield was 94.5%. Only special demand of nutritional ingredients was nitrogen, urea at 750 mg kg⁻¹ was found to be suitable for this purpose. In the verification experiments, the mass fraction of the theoretical ethanol yield in a 5 L fermentor was 98.3%. In conclusion, the pretreatment with CWDEs has significant effect on high level ethanol production using roots and tubers on an industrial scale from the biomass utilization efficiency and economic standpoint.     

Optimization of very high gravity fermentation process for ethanol production from industrial sugar beet syrup

In order to reduce production costs and environmental impact of bioethanol from sugar beet low purity syrup 2, an intensification of the industrial alcoholic fermentation carried out by Saccharomyces cerevisiae is necessary. Two fermentation processes were tested: multi-stage batch and fed-batch fermentations with different operating conditions. It was established that the fed-batch process was the most efficient to reach the highest ethanol concentration. This process allowed to minimize both growth and ethanol production inhibitions by high sugar concentrations or ethanol. Thus, a good management of the operating conditions (initial volume and feeding rate) could produce 15.2% (v/v) ethanol in 53 h without residual sucrose and with an ethanol productivity of 2.3 g L h⁻¹.     

Modeling semi-simultaneous saccharification and fermentation of ethanol production from cellulose

Using a highly refined standard cellulose (microcrystalline cellulose, (Avicel PH101)), the kinetics of the simultaneous saccharification and fermentation (SSF) of cellulose to ethanol was studied with a prior hydrolysis phase (semi-simultaneous saccharification and fermentation (SSSF)) conducted under optimal conditions for enzymatic hydrolysis. Four cases have been studied: 24-h pre-hydrolysis + 48-h SSF (SSSF 24), 12-h pre-hydrolysis + 60-h SSF (SSSF 12), 72-h SSF, and 48-h hydrolysis + 24-h fermentation. SSSF 24 produced higher yield and higher productivity of ethanol than the other operating modes. A coupled set of differential equations were developed to describe the change rates of cellobiose, glucose, microorganism, ethanol, glycerol, acetic acid, and lactic acid concentrations in the batch operation of separate hydrolysis and SSF of ethanol production from Avicel PH101. The model parameters were determined by a MATLAB program based on the batch experimental data of the SSSF. The analysis of the reaction rates of cellobiose, glucose, cell, and ethanol using the model showed that the conversion of cellulose to cellobiose was the rate-controlling step in the SSSF process of ethanol production from cellulose. The batch SSSF model was extended to the continuous and fed-batch operating modes. For the continuous operation in the SSSF, the productivity of SSSF 24 was much higher than that of SSSF 12 though the ethanol concentrations of both cases have not a great difference.     

餐厨废弃物糖化发酵炼制燃料乙醇工艺优化

利用高效液化酶(Liquozyme)和糖化酶(AMG)对餐厨废弃物进行液化糖化,优化了酶组成、加酶量、pH、操作温度、操作时间、酵母接种量等参数,建立了由餐厨废弃物炼制燃料乙醇的最佳工艺。结果表明:通过液化酶和糖化酶复配,可降低原料粘度、提高传质效率,使淀粉类多糖快速转化为可发酵性单糖;液化酶最优作用条件为85℃,pH值5.1~5.2,加酶量为0.75 U/g(干基),液化时间为40 min;糖化酶最优作用条件为45℃,pH值5.0,加酶量0.5 U/g(干基),糖化时间为30 min;最佳的发酵条件是酵母接种量3 g/L,发酵时间20 h,所得乙醇浓度为54 g/L,相当于0.438 g/g(乙醇/葡萄糖),达到理论产量的86%。     

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.     

Arrowroot as a novel substrate for ethanol production by solid state simultaneous saccharification and fermentation

Ethanol production from Canna edulis Ker was successfully carried out by solid state simultaneous saccharification and fermentation. The enzymatic hydrolysis conditions of C. edulis were optimized by Plackett–Burman design. The effect of inert carrier (corncob and rice bran) on ethanol fermentation and the kinetics of solid state simultaneous saccharification and fermentation was investigated. It was found that C. edulis was an alternative substrate for ethanol production, 10.1% (v/v) of ethanol concentration can attained when 40 g corncob and 10 g rice bran per 100 g C. edulis powder were added for ethanol fermentation. No shortage of fermentable sugars was observed during solid state simultaneous saccharification and fermentation. There was no wastewater produced in the process of ethanol production from C. edulis with solid state simultaneous saccharification and fermentation and the ethanol yield of more than 0.28 tonne per one tonne feedstock was achieved. This is first report for ethanol production from C. edulis powder.     

Simultaneous saccharification and fermentation of cellulose for bio-hydrogen production by anaerobic mixed cultures in elephant dung

The objective of this study was to optimize the culture conditions for simultaneous saccharification and fermentation (SSF) of cellulose for bio-hydrogen production by anaerobic mixed cultures in elephant dung under thermophilic temperature. Carboxymethyl cellulose (CMC) was used as the model substrate. The investigated parameters included initial pH, temperature and substrate concentration. The experimental results showed that maximum hydrogen yield (HY) and hydrogen production rate (HPR) of 7.22 ± 0.62 mmol H₂/g CMC_added and 73.4 ± 3.8 mL H₂/L h, respectively, were achieved at an initial pH of 7.0, temperature of 55 °C and CMC concentration of 0.25 g/L. The optimum conditions were then used to produce hydrogen from the cellulose fraction of sugarcane bagasse (SCB) at a concentration of 0.40 g/L (equivalent to 0.25 g/L cellulose) in which an HY of 7.10 ± 3.22 mmol H₂/g cellulose_added. The pre-dominant hydrogen producers analyzed by polymerase chain reaction-denaturing gel gradient electrophoresis (PCR-DGGE) were Thermoanaerobacterium thermosaccharolyticum and Clostridium sp. The lower HY obtained when the cellulose fraction of SCB was used as the substrate might be due to the presence of lignin in the SCB as well as the presence of Lactobacillus parabuchneri and Lactobacillus rhamnosus in the hydrogen fermentation broth.     

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.     

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.     

Biological hydrogen production: Simultaneous saccharification and fermentation with nitrogen and phosphorus removal from wastewater effluent

A simple anaerobic biodegradation process using wastewater treatment plant (WWTP) effluent, shredded paper, and a purge of nitrogen gas was used to produce hydrogen and simultaneously capture nitrogen and phosphorus. Two reactor configurations, a sequencing batch reactor (SBR) and a classic batch reactor (CBR) were tested as simultaneous saccharification and fermentation reactors (enzymatic hydrolysis and fermentation in one tank). The CBR demonstrated greater stability of hydrogen production and simplicity of operation, while the SBR provided better nitrogen and phosphorus removal efficiencies. Nuclear magnetic resonance analyses showed acetic acid to be the main product from both reactors. Optimal CBR conditions were found to be pH 5, 4 g/L loading, 0.45 ml/g Accellerase 1500, and 38 °C. Experiments with an argon purge in place of nitrogen and with ammonium chloride spiking suggested that hydrogenase and nitrogenase enzymes contributed similarly to hydrogen production in the cultures. Analysis of a single fermentation showed that hydrogen production occurred relatively early in the course of TOC removal, and that follow-on treatments might extract more energy from the products.     

Enzymatic saccharification and fermentation of paper and pulp industry effluent for biohydrogen production

Paper and pulp industry effluent was enzymatically hydrolysed using crude cellulase enzyme (0.8–2.2FPU/ml) obtained from Trichoderma reesei and from the hydrolysate biohydrogen was produced using Enterobacter aerogenes. The influence of temperature and incubation time on enzyme production was studied. The optimum temperature for the growth of T. reesei was found to be around 29 °C. The enzyme activity of 2.5 FPU/ml was found to produce about 22 g/l of total sugars consisting mainly of glucose, xylose and arabinose. Relevant kinetic parameters with respect to sugars production were estimated using two fraction model. The enzymatic hydrolysate was used for the biohydrogen production using E. aerogenes. The growth data obtained for E. aerogenes were fitted well with Monod and Logistic equations. The maximum hydrogen yield of 2.03 mol H₂/mol sugar and specific hydrogen production rate of 225 mmol of H₂/g cell/h were obtained with an initial concentration of 22 g/l of total sugars. The colour and COD of effluent was also decreased significantly during the production of hydrogen. The results showed that the paper and pulp industry effluent can be used as a substrate for biohydrogen production.     

Simultaneous cold hydrolysis and fermentation of fresh sweet potato

In recent decades, environmental and economic issues have pushed the production of biofuels worldwide. In this scenario, ethanol is the most produced biofuel. Starch is a potential substrate for this purpose, but the extra cost needed to hydrolyze it into glucose is still a drawback. As an alternative for the expensive and energy demanding conventional hydrolysis process, the cold hydrolysis is being studied. In this process, granular starch degrading enzymes act directly on raw starch granules; therefore, this hydrolysis is carried out below gelatinization temperature. As a consequence, the energy requirement can be significantly reduced. In this work, the cold hydrolysis and fermentation of fresh sweet potato were experimentally studied. For that, it was employed the sweet potato strain BRS Cuia, whose carbohydrate level reaches 28.7%. It can be translated into a potential to produce 185 L t⁻¹ ethanol, or equivalently 7400 L ha⁻¹. The enzymes blend adopted for the hydrolysis stage was Stargen™ 002. The surface response method indicated 200 g L⁻¹ of sweet potato and 45 GAU g⁻¹ of sweet potato as the best balance between high glucose formation rate and low enzyme consume. The 1 h pretreatment that achieved the highest glucose concentration was at 52 °C in the presence of the enzymes blend. Finally, the study of the simultaneous hydrolysis and fermentation showed that the medium supplementation has no significant effect over the fermentation performance, while the pH control is beneficial, improving the ethanol production in 54%.     

甜龙竹蛀粉纤维素燃料乙醇发酵的糖化研究

纤维素糖化是甜龙竹蛀粉燃料乙醇发酵研究中的重要内容.正交试验和对比试验结果表明,在pH值为4.4、温度为55℃,酶用量为200 u/g,酶解时间小于20 h条件下,进行纤维素酶解较合适,但酶解率较低.通过酸或碱并结合有机溶剂进行预处理能显著提高蛀粉纤维素糖化率.同时添加半纤维素复合酶进行酶解,能使蛀粉中大部分半纤维素水解,并能促进纤维素的酶解.     

Effect of nutrient supplementation on ethanol production in different strategies of saccharification and fermentation from acid pretreated rice straw

The effect of nutrient supplementation on ethanol production by recently selected thermotolerant yeast (Kluyveromyces marxianus NRRL Y-6860) was investigated in different strategies of saccharification and fermentation employing rice straw pretreated by dilute acid. Among the evaluated strategies, similar ethanol yields (Y_P/S ∼ 0.23 g g⁻¹) were obtained with or without nutrient addition. However, considering the whole process time, the strategy based on simultaneous saccharification and fermentation (SSF), without pre-hydrolysis, was assigned as the most suitable configuration due to the highest ethanol volumetric productivity (1.4 g L⁻¹ h⁻¹), about 2-fold higher in relation to the others. The impact of enzymatic preparation employed in this study was also evaluated on glucose fermentation in semi-synthetic medium. The enzymatic preparation affected both glucose consumption and ethanol production by K. marxianus NRRL Y-6860, but just in the absence of nutrients. Therefore, the enzyme type and loading should be carefully defined, not only by the capital costs involved, but also by the possibility of increasing the fermentation inhibitors.     

Influence of phytase and supportive enzymes applied during high gravity mash preparation on the improvement of technological indicators of the alcoholic fermentation process

Phytic acid is one of the compounds that can limit the availability of biogenic substances responsible for the environmental stress response of the yeast. The aim of the study was determining the influence of hydrolysis of the phytic acid complexes in the HG maize mashes on the availability of free amino nitrogen (FAN) released from hydrolyzed proteins and the resulting change in the fermentation activity of the yeast. Prospects for the optimization of the alcoholic fermentation process by combined application of phytase, protease and augmented set of amylolytic enzymes were also assessed. The application of protease phytase resulted in an increase of FAN concentration during subsequent hours of the alcoholic fermentation process which indicated an increased availability of proteins susceptible to hydrolysis. A positive effect of combined application of phytase, protease and amylolytic enzymes (with pullulanase) on technological indicators of the fermentation process was observed. The final ethanol concentration increased by 1% v v⁻¹ and the yield increased by 6 L EtOH 100 kg⁻¹ of starch. The optimization of the mashing process, involving the application of additional enzymes (phytase, pullulanase, protease), improved the yield of fermentation in relation to the theoretical yield to the level of 93%. A combined application of α-amylase, pullulanase, glucoamylase, protease and phytase during HG mashes preparation lowered the concentration of higher alcohols by 450 mg L⁻¹ EtOH, on average, as compared to the control variant. At the same time, the concentration of acetaldehyde in the spirits increased above 480 mg L⁻¹ EtOH.     

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.     

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.     

狗牙根糖化液发酵制备乙醇的工艺研究

禾本科狗尾草属植物——狗牙根,纤维素含量非常丰富,纤维素经过稀酸、纤维素酶和木聚糖酶处理后,接种嗜鞣管囊酵母发酵可得到生物乙醇。试验以乙醇得率为指标,研究了葡萄糖、酵母膏、蛋白胨、接种量、温度、时间、pH等因素对乙醇得率的影响,通过正交试验,得到最佳的试验组合,最后用狗牙根糖化液代替葡萄糖进行发酵,生产制备生物乙醇。结果表明,在以葡萄糖为原料的乙醇生产过程中的最佳条件是:100 mL的培养基中,葡萄糖20 g、酵母膏1.5 g、蛋白胨0.5 g、接种量13 mL、温度30℃、时间60 h、pH5.5、硫酸铵1 g、磷酸二氢钾0.2 g、硫酸镁0.1 g,乙醇的最高产率为43.35%;狗牙根糖化液最终乙醇得率为7.04%。     

Fed-batch Saccharomyces cerevisiae fermentation of hydrolysate sugars: A dynamic model-based approach for high yield ethanol production

The efficient fermentation of hydrolyzed sugars from lignocellulosic biomass feedstock to ethanol remains a complex multi-parametric problem. Thus, in the present study, an advanced structured dynamic model for the simulation of the fermentative ethanol production from hydrolysate sugars is developed. The model is combined with a statistical experimental design to determine an optimal operating strategy that maximizes ethanol production and serves for the systematic evaluation of critical process variables. In particular, the effects of various operating conditions and feeding strategies on the dynamic behavior of batch and fed-batch fermentation processes are explored. The deviation from the desired product or the metabolic inhibition of ethanol production are related with the applied environmental conditions and substrate and product inhibition phenomena. The operating strategy, designed with the assistance of the mathematical tools proposed in this study, includes an exponential addition policy of substrate. This strategy is experimentally proved to enhance the final product concentration, raising the ethanol productivity to 2.27 g L⁻¹ h⁻¹ and the ethanol yield to 53.5% of the maximum theoretical value. Moreover, the simulated strategies were in excellent agreement with the experimental results obtained from the real process using low and high glucose initial concentration, under batch and fed-batch conditions, in both flask- and bioreactor-scale cultivations, proving the model's predictive and optimization capabilities. Further improvement of process performance is expected when combining the proposed dynamic model with advanced optimization algorithms to derive the optimal bioprocess operating strategy.