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Abstract Aim of the study was to assess the suitability of the combined use of microwave radiation and sodium cumene sulfonate under optimized process conditions for the preparation of maize stillage biomass as a raw material for the production of cellulosic ethanol. The key parameter guaranteeing a high level of lignin removal from biomass (ca. 44%) was concentration of hydrotrope. Even at high biomass concentration (16% w/v) and a cellulase enzyme dose of about 4 filter-paper units/g, maize stillage biomass subjected to microwave-assisted hydrotropic pretreatment was highly susceptible to enzymatic degradation, which resulted in 80% hydrolysis yield. It is possible to obtain a fermentation medium with a very high glucose concentration (up to 80 g/L), without fermentation inhibitors and, as a consequence, to reach a very high level of sugar conversion to ethanol (concentration above 40 g/L), even as much as 95% of theoretical yield. Microwave hydrotropic treatment with sodium cumene sulfonate is a very effective way to prepare waste maize stillage biomass for the production of cellulosic ethanol. The degradation of the lignocellulose structure by the simultaneous use of microwaves and hydrotropes ensured a high degree of conversion of structural polysaccharides to bioethanol. The method provides a high level of enzymatic degradation of cellulose, leading to a medium with high content of released sugars suitable for bioconversion, which is in line with assumptions of the second-generation ethanol production technology. Key points • Microwave-assisted hydrotropic pretreatment is a new way to cellulosic ethanol production. • Microwave-assisted hydrotropic delignification removes 44% of lignin from biomass. • No fermentation inhibitors are obtained after microwave-assisted hydrotropic pretreatment. • High ethanol concentration (above 40 g/L) and fermentation yield (95% of theoretical yield) from biomass after microwave-assisted hydrotropic pretreatment.

The utilized biomass is an important consideration for sustainable biofuel production. To avoid competing with food needs, researchers have turned their attention to non-food lignocellulosic biomasses as potential feedstocks for biofuel production. However, the saccharification of a lignocellulosic biomass produces a large amount of lignin as waste. To overcome this hurdle, biomass gasification has been suggested as an alternative to saccharification. During biomass gasification, oxides of carbon (CO, CO2) and hydrogen are produced as a major product. Accordingly, microorganisms capable of utilizing these oxides of carbon have gained attention as hosts for the production of biofuels, such as ethanol and butanol. In this work, we reviewed the Calvin cycle and Wood-Ljungdahl pathway for utilizing oxides of carbon in cyanobacteria and acetogens, respectively, and discussed the metabolic engineering strategies that may be used to produce ethanol and butanol from oxides of carbon through these routes.

Straw is an agricultural residue of the production of e.g. cereals, rapeseed or sunflowers. It includes dried stalks, leaves, and empty ears and corncobs, which are separated from the grains during harvest. Straw is a promising lignocellulosic feedstock with a beneficial greenhouse gas balance for the production of biofuels and chemicals. Like all lignocellulosic materials, straw is recalcitrant and requires thermochemical and enzymatic pretreatment to enable access to the three major biopolymers of straw-the polysaccharides cellulose and hemicellulose and the polyaromatic compound lignin. Straw is used for commercial ethanol and biogas production. Considerable research has also been conducted to produce biobutanol, biodiesel and biochemicals from this raw material, but more research is required to establish them on a commercial scale. The major hindrance for launching industrial biofuel and chemicals' production from straw is the high cost necessitated by pretreatment of the material. Improvements of microbial strains, production and extraction technologies, as well as co-production of high-value compounds represent ways of establishing straw as feedstock for the production of biofuels, chemicals and food.

Butanol is a platform chemical that is utilized in a wide range of industrial products and is considered a suitable replacement or additive to liquid fuels. So far, it is mainly produced through petrochemical routes. Alternative production routes, for example through biorefinery, are under investigation but are currently not at a market competitive level. Possible alternatives, such as acetone-butanol-ethanol (ABE) fermentation by solventogenic clostridia are not market-ready to this day either, because of their low butanol titer and the high costs of feedstocks. Here, we analyzed wheat middlings and wheat red dog, two wheat milling byproducts available in large quantities, as substrates for clostridial ABE fermentation. We could identify ten strains that exhibited good butanol yields on wheat red dog. Two of the best ABE producing strains, Clostridium beijerinckii NCIMB 8052 and Clostridium diolis DSM 15410, were used to optimize a laboratory-scale fermentation process. In addition, enzymatic pretreatment of both milling byproducts significantly enhanced ABE production rates of the strains C. beijerinckii NCIMB 8052 and C. diolis DSM 15410. Finally, a profitability analysis was performed for small- to mid-scale ABE fermentation plants that utilize enzymatically pretreated wheat red dog as substrate. The estimations show that such a plant could be commercially successful.Key points• Wheat milling byproducts are suitable substrates for clostridial ABE fermentation.• Enzymatic pretreatment of wheat red dog and middlings increases ABE yield.• ABE fermentation plants using wheat red dog as substrate are economically viable.

Distillers' dried grain with solubles (DDGS) is a byproduct of bioethanol fermentation, which uses the dry milling technology for starch-rich grains such as corn, wheat, and barley. The current interest in bioethanol is increasing due to the need for renewable liquid fuels specifically in the transportation sector. Since DDGS is rich in crude protein, fat, fiber, vitamins, and minerals, it is currently used as aquaculture, livestock, and poultry feeds. In recent years, DDGS has been used as feedstock in the production of value-added products via microbial fermentation. Numerous studies reported the production organic acids, methane, biohydrogen, and hydrolytic enzymes using DDGS. While DDGS contains remarkable amounts of macronutrients, pre-treatment of DDGS is required for release of the fermentable sugars. The pre-treatment methods such as chemical, physical, and biological origin are either solely used or combined to obtain maximal yields for different applications. Therefore, this review summarizes some of the most prominent pre-treatment processes generating high fermentable sugar yields for the productions of value-added products in the last 5 years. A special focus has been given to the effect of the variability of DDGS on the final product. Integration of hydrolytic enzyme production with the traditional bioethanol production facilities has been discussed for further improvement of bioethanol, methane, and biohydrogen using DDGS as fermentation feedstock.Key points• Distillers' dried grain with solubles (DDGS) has high nutritional value, but the nutritional profile is variable.• DDGS can be used for microbial fermentation feedstock to produce value-added products.• A review of the microbial products using DDGS is given for the last 5 years.• DDGS has the potential to replace expensive feedstocks of value-added products.

With growing interest in alternative fuels to minimize carbon and particle emissions, research continues on the production of lignocellulosic ethanol and on the development of suitable yeast strains. However, great diversities and continued technical advances in pretreatment methods for lignocellulosic biomass complicate the evaluation of developed yeast strains, and strain development often lags industrial applicability. In this review, recent studies demonstrating developed yeast strains with lignocellulosic biomass hydrolysates are compared. For the pretreatment methods, we highlight hydrothermal pretreatments (dilute acid treatment and autohydrolysis), which are the most commonly used and effective methods for lignocellulosic biomass pretreatment. Rather than pretreatment conditions, the type of biomass most strongly influences the composition of the hydrolysates. Metabolic engineering strategies for yeast strain development, the choice of xylose-metabolic pathway, adaptive evolution, and strain background are highlighted as important factors affecting ethanol yield and productivity from lignocellulosic biomass hydrolysates. A comparison of the parameters from recent studies demonstrating lignocellulosic ethanol production provides useful information for future strain development.

The microbes present in bioethanol production processes have been previously studied in laboratory-scale experiments, but there is a lack of information on full-scale industrial processes. In this study, the microbial communities of three industrial bioethanol production processes were characterized using several methods. The samples originated from second-generation bioethanol plants that produce fuel ethanol from biowaste, food industry side streams, or sawdust. Amplicon sequencing targeting bacteria, archaea, and fungi was used to explore the microbes present in biofuel production and anaerobic digestion of wastewater and sludge. Biofilm-forming lactic acid bacteria and wild yeasts were identified in fermentation samples of a full-scale plant that uses biowaste as feedstock. During the 20-month monitoring period, the anaerobic digester adapted to the bioethanol process waste with a shift in methanogen profile indicating acclimatization to high concentrations of ammonia. Amplicon sequencing does not specifically target living microbes. The same is true for indirect parameters, such as low pH, metabolites, or genes of lactic acid bacteria. Since rapid identification of living microbes would be indispensable for process management, a commercial method was tested that detects them by measuring the rRNA of selected microbial groups. Small-scale testing indicated that the method gives results comparable with plate counts and microscopic counting, especially for bacterial quantification. The applicability of the method was verified in an industrial bioethanol plant, inspecting the clean-in-place process quality and detecting viability during yeast separation. The results supported it as a fast and promising tool for monitoring microbes throughout industrial bioethanol processes.

Besides free sugars, sweet sorghum stalks contain cellulose and hemicellulose that can be used for biofuel production. The pretreatment of stalks without the extraction of free sugars is more complicated than typical lignocelluloses, because of the degradation of free sugars during most pretreatment processes. In this study, the bioconversion of sweet sorghum stalks into biogas and bioethanol was studied using an improved organosolv pretreatment within a biorefinery framework. The organosolv pretreatment was developed using an aqueous solution of ethanol (EtOH) and isopropanol (IPOH). The process was optimized to obtain a liquor containing free sugars with the least sugar degradations together with a highly degradable solid fraction. The liquor was subjected to anaerobic digestion for biomethane production, while the solid was used for ethanol production via simultaneous saccharification and fermentation (SSF). The most influencing pretreatment parameters, i.e., temperature, time, alcohol to water ratio, EtOH to IPOH ratio, and the presence or absence of sulfuric acid (as a catalyst), were adjusted to achieve the highest yields of bioconversion. The maximum methane and ethanol production yields of 271.2 mL CH4/g VS and 87.8% (equal to the gasoline equivalent of 0.170 and 0.241 L/kg, respectively) were achieved from the liquor and pretreated solid, respectively; however, they were obtained at different optimum conditions. Considering the biorefinery perspective, the highest gasoline equivalent of 0.249 L/kg was efficiently obtained from the whole process after pretreatment at 140 °C for 30 min using 60:20 EtOH/IPOH ratio in the presence of 1% sulfuric acid. Further analyses, including enzymatic adsorption/desorption, compositional analysis, FTIR, and SEM, were conducted to investigate the effects of this newly developed pretreatment on the substrate.

The mixed culture fermentation is an important environmental biotechnology that converts biodegradable organic wastes to valuable chemicals such as hydrogen, methane, acetate, ethanol, propionate, and so on. For the multistep process of hydrolysis, acidogenesis, acetogenesis/homoacetogensis, and methanogenesis, the typical metabolic reactions are firstly summarized. And then, since the final metabolites are always a mixture, the separation and purification processes are necessary to couple with anaerobic fermentation. Therefore, several typical coupling technologies including biogas upgrading, two-stage fermentation, gas stripping, membrane technology of pervaporation, membrane distillation, electrodialysis, bipolar membrane electrodialysis, and microbial fuel cells are summarized to separate the metabolites and recover energy. At last, the novel technologies such as the controlled metabolite production, medium chain carboxylic acid production, and high temperature ethanol recovery in thermophilic mixed culture fermentation are also reviewed. However, the novel concepts are still needed to meet the demands of better overall performances and lower total costs.

Stillage, which is generated during bioethanol production, constitutes a promising substrate for biogas production within the scope of an integrated biorefinery concept. In this study, a microbial community was grown on thin stillage as mono-substrate in a continuous stirred tank reactor (CSTR) at a constant temperature of 55 °C, at an organic loading rate of 1.5 goTS/L*d and a retention time of 25 days. Using an amplicon-based dataset of 17,400 high-quality sequences of 16S rRNA gene fragments (V2-V3 regions), predominance of Bacteria assigned to the families Thermotogaceae and Elusimicrobiaceae was detected. Dominant members of methane-producing Euryarchaeota within the CSTR belonged to obligate acetoclastic Methanosaetaceae and hydrogenotrophic Methanobacteriaceae. In order to investigate population dynamics during reactor acidification, the organic loading rate was increased abruptly, which resulted in an elevated concentration of volatile fatty acids. Acidification led to a decrease in relative abundance of Bacteria accompanied with stable numbers of Archaea. Nevertheless, the abundance of Methanosaetaceae increased while that of Methanobacteriales decreased successively. These findings demonstrate that a profound intervention to the biogas process may result in persistent community changes and reveals uncommon bacterial families as process-relevant microorganisms.

Biochemical processes in biogas plants are still not fully understood. Especially, the identification of possible bottlenecks in the complex fermentation processes during biogas production might provide potential to increase the performance of biogas plants. To shed light on the question which group of organism constitutes the limiting factor in the anaerobic breakdown of organic material, biogas sludge from different mesophilic biogas plants was examined under various conditions. Therefore, biogas sludge was incubated and analyzed in anaerobic serum flasks under an atmosphere of N2/CO2. The batch reactors mirrored the conditions and the performance of the full-scale biogas plants and were suitable test systems for a period of 24 h. Methane production rates were compared after supplementation with substrates for syntrophic bacteria, such as butyrate, propionate, or ethanol, as well as with acetate and H2+CO2 as substrates for methanogenic archaea. Methane formation rates increased significantly by 35 to 126 % when sludge from different biogas plants was supplemented with acetate or ethanol. The stability of important process parameters such as concentration of volatile fatty acids and pH indicate that ethanol and acetate increase biogas formation without affecting normally occurring fermentation processes. In contrast to ethanol or acetate, other fermentation products such as propionate, butyrate, or H2 did not result in increased methane formation rates. These results provide evidence that aceticlastic methanogenesis and ethanol-oxidizing syntrophic bacteria are not the limiting factor during biogas formation, respectively, and that biogas plant optimization is possible with special focus on methanogenesis from acetate.

Microbial production of ethanol might be a potential route to replace oil and chemical feedstocks. Bioethanol is by far the most common biofuel in use worldwide. Lignocellulosic biomass is the most promising renewable resource for fuel bioethanol production. Bioconversion of lignocellulosics to ethanol consists of four major unit operations: pretreatment, hydrolysis, fermentation, and product separation/distillation. Conventional bioethanol processes for lignocellulosics apply commercial fungal cellulase enzymes for biomass hydrolysis, followed by yeast fermentation of resulting glucose to ethanol. The fungus Neurospora crassa has been used extensively for genetic, biochemical, and molecular studies as a model organism. However, the strain's potential in biotechnological applications has not been widely investigated and discussed. The fungus N. crassa has the ability to synthesize and secrete all three enzyme types involved in cellulose hydrolysis as well as various enzymes for hemicellulose degradation. In addition, N. crassa has been reported to convert to ethanol hexose and pentose sugars, cellulose polymers, and agro-industrial residues. The combination of these characteristics makes N. crassa a promising alternative candidate for biotechnological production of ethanol from renewable resources. This review consists of an overview of the ethanol process from lignocellulosic biomass, followed by cellulases and hemicellulases production, ethanol fermentations of sugars and lignocellulosics, and industrial application potential of N. crassa.

Ethanol produced from various lignocellulosic materials such as wood, agricultural and forest residues has the potential to be a valuable substitute for, or complement to, gasoline. One of the major resources in the Northern hemisphere is softwood. This paper reviews the current status of the technology for ethanol production from softwood, with focus on hemicellulose and cellulose hydrolysis, which is the major problem in the overall process. Other issues of importance, e.g. overall process configurations and process economics are also considered.

Continuous production of acetone, n-butanol, and ethanol (ABE) was carried out using immobilized cells of Clostridium acetobutylicum DSM 792 using glucose and sugar mixture as a substrate. Among various lignocellulosic materials screened as a support matrix, coconut fibers and wood pulp fibers were found to be promising in batch experiments. With a motive of promoting wood-based bio-refinery concept, wood pulp was used as a cell holding material. Glucose and sugar mixture (glucose, mannose, galactose, arabinose, and xylose) comparable to lignocellulose hydrolysate was used as a substrate for continuous production of ABE. We report the best solvent productivity among wild-type strains using column reactor. The maximum total solvent concentration of 14.32 g L(-1) was obtained at a dilution rate of 0.22 h(-1) with glucose as a substrate compared to 12.64 g L(-1) at 0.5 h(-1) dilution rate with sugar mixture. The maximum solvent productivity (13.66 g L(-1) h(-1)) was obtained at a dilution rate of 1.9 h(-1) with glucose as a substrate whereas solvent productivity (12.14 g L(-1) h(-1)) was obtained at a dilution rate of 1.5 h(-1) with sugar mixture. The immobilized column reactor with wood pulp can become an efficient technology to be integrated with existing pulp mills to convert them into wood-based bio-refineries.

To save cost and input energy for bioethanol production, a consolidated continuous solid-state fermentation system composed of a rotating drum reactor, a humidifier, and a condenser was developed. Biomass, saccharifying enzymes, yeast, and a minimum amount of water are introduced into the system. Ethanol produced by simultaneous saccharification and fermentation is continuously recovered as vapor from the headspace of the reactor, while the humidifier compensates for the water loss. From raw corn starch as a biomass model, 95 +/- 3, 226 +/- 9, 458 +/- 26, and 509 +/- 64 g l(-1) of ethanol solutions were recovered continuously when the ethanol content in reactor was controlled at 10-20, 30-50, 50-70 and 75-85 g kg-mixture(-1), respectively. The residue showed a lesser volume and higher solid content than that obtained by conventional liquid fermentation. The cost and energy for intensive waste water treatment are decreased, and the continuous fermentation enabled the sustainability of enzyme activity and yeast in the system.

Xylitol production from corncob hemicellulose is a popular process in China. Microbial conversion of xylose to xylitol, as a biological process with many advantages, has drawn increasing attention. As a by-product from the manufacturing of xylitol, corncob cellulosic residues are produced in very large amounts and represent an environmental problem. As a result, considering the large amount of xylitol production in China, the conversion of corncob cellulosic residues has become a widespread issue having to be tackled. After the hemicellulose in corncob has been hydrolyzed for xylitol production, the corncob cellulosic residue is porous and can easily be hydrolyzed by cellulases into glucose and further converted to ethanol, another high-added-value chemical. Based on the latest technology advancements in xylitol, cellulase, and ethanol production, the integrated production of ethanol from corncob cellulosic residues appears as a promising way to improve the profit of the whole xylitol production process.

The process of ethanol fermentation has a long history in the production of alcoholic drinks, but much larger scale production of ethanol is now required to enable its use as a substituent of gasoline fuels at 3%, 10%, or 85% (referred to as E3, E10, and E85, respectively). Compared with fossil fuels, the production costs are a major issue for the production of fuel ethanol. There are a number of possible approaches to delivering cost-effective fuel ethanol production from different biomass sources, but we focus in our current report on high-temperature fermentation using a newly isolated thermotolerant strain of the yeast Kluyveromyces marxianus. We demonstrate that a 5 degrees C increase only in the fermentation temperature can greatly affect the fuel ethanol production costs. We contend that this approach may also be applicable to the other microbial fermentations systems and propose that thermotolerant mesophilic microorganisms have considerable potential for the development of future fermentation technologies.

China is one of the few countries, which maintained the fermentative acetone-butanol-ethanol (ABE) production for several decades. Until the end of the last century, the ABE fermentation from grain was operated in a few industrial scale plants. Due to the strong competition from the petrochemical industries, the fermentative ABE production lost its position in the 1990s, when all the solvent fermentation plants in China were closed. Under the current circumstances of concern about energy limitations and environmental pollution, new opportunities have emerged for the traditional ABE fermentation industry since it could again be potentially competitive with chemical synthesis. From 2006, several ABE fermentation plants in China have resumed production. The total solvent (acetone, butanol, and ethanol) production capacity from ten plants reached 210,000 tons, and the total solvent production is expected to be extended to 1,000,000 tons (based on the available data as of Sept. 2008). This article reviews current work in strain development, the continuous fermentation process, solvent recovery, and economic evaluation of ABE process in China. Challenges for an economically competitive ABE process in the future are also discussed.

Production of ethanol by bioconversion of lignocellulosic biomass has attracted much interest in recent years. However, the pretreatment process for increasing the enzymatic digestibility of cellulose has become a key step in commercialized production of cellulosic ethanol. During the last decades, many pretreatment processes have been developed for decreasing the biomass recalcitrance, but only a few of them seem to be promising. From the point of view for integrated utilization of lignocellulosic biomass, organosolv pretreatment provides a pathway for biorefining of biomass. This review presents the progress of organosolv pretreatment of lignocellulosic biomass in recent decades, especially on alcohol, organic acid, organic peracid and acetone pretreatments, and corresponding action mechanisms. Evaluation and prospect of organosolv pretreatment were performed. Finally, some recommendations for future investigation of this pretreatment method were given.

In this study, the repeated-batch fermentation of liquefied cassava medium using the flocculent hybrid Saccharomyces cerevisiae CHFY0321 was investigated for semicontinuous, high-throughput production of bioethanol. Cassava medium was selected due to the industrial requirement for a cheap starchy substrate. Fermentations were performed by simultaneous saccharification and fermentation (SSF) with a set of ten batches successfully completing a series within the repeated fermentation process. In addition, pH of the culture medium was not controlled to simplify ethanol production for future use in industry. Optimal recycling volume was found to be 5%. Volumetric productivity, final ethanol concentration, and ethanol yield were measured at 3.34 g l(-1) h(-1), 84.5 g l(-1), and 90.7%, respectively. Cell recycling (24.5 g DCW l(-1)) resulted in 1.8-fold decrease in fermentation time (24 h) and 1.8-fold increase in volumetric productivity compared with the ordinary batch fermentation. Therefore, repeated-batch SSF using flocculent CHFY0321 demonstrates the possibility of cost-effective bioethanol production by eliminating additional saccharification and inoculation steps.

An integrative simultaneous saccharification and fermentation (SSF) modeling is a useful guiding tool for rapid process optimization to meet the techno-economic requirement of industrial-scale lignocellulosic ethanol production. In this work, we have developed the SSF model composing of a metabolic network of a Saccharomyces cerevisiae cell associated with fermentation kinetics and enzyme hydrolysis model to quantitatively capture dynamic responses of yeast cell growth and fermentation during SSF. By using model-based design of feeding profiles for substrate and yeast cell in the fed-batch SSF process, an efficient ethanol production with high titer of up to 65 g/L and high yield of 85 % of theoretical yield was accomplished. The ethanol titer and productivity was increased by 47 and 41 %, correspondingly, in optimized fed-batch SSF as compared to batch process. The developed integrative SSF model is, therefore, considered as a promising approach for systematic design of economical and sustainable SSF bioprocessing of lignocellulose.

In recent years, growing attention has been devoted to the conversion of biomass into fuel ethanol, considered the cleanest liquid fuel alternative to fossil fuels. Significant advances have been made towards the technology of ethanol fermentation. This review provides practical examples and gives a broad overview of the current status of ethanol fermentation including biomass resources, microorganisms, and technology. Also, the promising prospects of ethanol fermentation are especially introduced. The prospects included are fermentation technology converting xylose to ethanol, cellulase enzyme utilized in the hydrolysis of lignocellulosic materials, immobilization of the microorganism in large systems, simultaneous saccharification and fermentation, and sugar conversion into ethanol.

Poly 3-hydroxybutyric acid (PHB) and related copolymers can be advantageously produced when integrated into a sugarcane mill. In this favorable scenario, the energy necessary for the production process is provided by biomass. Carbon dioxide emissions to the environment are photosynthetically assimilated by the sugarcane crop and wastes are recycled to the cane fields. The polymer can be produced at low cost considering the availability of a low-price carbon source and energy.

Nutrient cost is an important aspect in the fermentation of biomass to ethanol. With a goal of developing a cost-effective fermentation medium, several industrially available nutrient sources were evaluated for their effectiveness in the simultaneous saccharification and fermentation of pretreated poplar with Saccharomyces cerevisiae D5A. These studies showed that a low-cost medium containing 0.3% corn steep liquor and 2.5 mM MgSO4 · 7H2O was similar in performance to a nutrient-rich medium. Besides its low cost, this alternative medium consists of components that are available on a commercial scale, thereby making it industrially relevant.

The production of ethanol from carob pods by Saccharomyces cerevisiae in solid-state fermentation was investigated. The maximal ethanol concentration (160±3 g/kg dry pods), ethanol productivity (6.7 ± 0.2 g/kg per hour), ethanol yield (40 ± 1.8%), biomass concentration (7.5 ± 0.4 x 108 cells/g carob pulp) and fermentation efficiency (80 ± 2%) were obtained at an inoculum amount of 3%, a particle size of 0.5 mm, a moisture level of 70%, a pH of 4.5 and a temperature of 30°C. Under the same fermentation conditions both sterilized and non-sterilized carob pods pulp gave the same maximum ethanol concentration.

Ethanol is a potential energy source and its production from renewable biomass has gained lot of popularity. There has been worldwide research to produce ethanol from regional inexpensive substrates. The present study deals with the optimization of process parameters (viz. temperature, pH, initial total reducing sugar (TRS) concentration in sugar cane molasses and fermentation time) for ethanol production from sugar cane molasses by Zymomonas mobilis using Box–Behnken experimental design and genetic algorithm (GA). An empirical model was developed through response surface methodology to analyze the effects of the process parameters on ethanol production. The data obtained after performing the experiments based on statistical design was utilized for regression analysis and analysis of variance studies. The regression equation obtained after regression analysis was used as a fitness function for the genetic algorithm. The GA optimization technique predicted a maximum ethanol yield of 59.59 g/L at temperature 31 °C, pH 5.13, initial TRS concentration 216 g/L and fermentation time 44 h. The maximum experimental ethanol yield obtained after applying GA was 58.4 g/L, which was in close agreement with the predicted value.

Lignocellulosic materials are the most abundant renewable organic resources (~200 billion tons annually) on earth that are readily available for conversion to ethanol and other value-added products, but they have not yet been tapped for the commercial production of fuel ethanol. The lignocellulosic substrates include woody substrates such as hardwood (birch and aspen, etc.) and softwood (spruce and pine, etc.), agro residues (wheat straw, sugarcane bagasse, corn stover, etc.), dedicated energy crops (switch grass, and Miscanthus etc.), weedy materials (Eicchornia crassipes, Lantana camara etc.), and municipal solid waste (food and kitchen waste, etc.). Despite the success achieved in the laboratory, there are limitations to success with lignocellulosic substrates on a commercial scale. The future of lignocellulosics is expected to lie in improvements of plant biomass, metabolic engineering of ethanol, and cellulolytic enzyme-producing microorganisms, fullest exploitation of weed materials, and process integration of the individual steps involved in bioethanol production. Issues related to the chemical composition of various weedy raw substrates for bioethanol formation, including chemical composition-based structural hydrolysis of the substrate, need special attention. This area could be opened up further by exploring genetically modified metabolic engineering routes in weedy materials and in biocatalysts that would make the production of bioethanol more efficient.

In order to develop a method for producing fuel ethanol from cassava pulp using cell surface engineering (arming) technology, an arming yeast co-displaying α-amylase (α-AM), glucoamylase, endoglucanase, cellobiohydrase, and β-glucosidase on the surface of the yeast cells was constructed. The novel yeast strain, possessing the activities of all enzymes, was able to produce ethanol directly from soluble starch, barley β-glucan, and acid-treated Avicel. Cassava is a major crop in Southeast Asia and used mainly for starch production. In the starch manufacturing process, large amounts of solid wastes, called cassava pulp, are produced. The major components of cassava pulp are starch (approximately 60%) and cellulose fiber (approximately 30%). We attempted simultaneous saccharification and ethanol fermentation of cassava pulp with this arming yeast. During fermentation, ethanol concentration increased as the starch and cellulose fiber substrates contained in the cassava pulp decreased. The results clearly showed that the arming yeast was able to produce ethanol directly from cassava pulp without addition of any hydrolytic enzymes.

Lodgepole pine from forest thinnings is a potential feedstock for ethanol production. In this study, lodgepole pine was converted to ethanol with a yield of 276 L per metric ton of wood or 72% of theoretical yield. The lodgepole pine chips were directly subjected to sulfite pretreatment to overcome recalcitrance of lignocellulose (SPORL) pretreatment and then disk-milled; the recovered cellulose substrate was quais-simultaneously saccharified enzymatically and fermented to ethanol using commercial cellulases and Saccharomyces cerevisiae D5A. The liquor stream from the pretreatment containing hydrolyzed sugars mainly from hemicelluloses was fermented by the same yeast strain after detoxification using an XAD resin column. The SPORL pretreatment was conducted at 180 degrees C for a period of 25 min with a liquor-to-wood ratio of 3:1 (v/w) in a laboratory digester. Three levels of sulfuric acid charge (0.0%, 1.4%, and 2.2% on an oven dried wood basis in w/w) and three levels of sodium bisulfite charge (0.0%, 4.0%, and 8.0% in w/w) were applied. Mechanical and thermal energy consumption for milling and pretreatment were determined. These data were used to determine the efficiency of sugar recoveries and net ethanol energy production values and to formulate a preliminary mass and energy balance.

The biorefinery produces fuels, solvents, plastics and food for human beings. In some countries, these biorefinery products are made from waste biomass. The main processes in the biorefinery involve ethanol fermentation and lactic acid fermentation. For the biorefinery, many hybrid technologies were developed from different fields, such as bioengineering, polymer chemistry, food science and agriculture.

A circulating loop bioreactor (CLB) with cells immobilized in loofa sponge was constructed for simultaneous aerobic and anaerobic processes. The CLB consists of an aerated riser and a non-aerated downcomer column connected at the top and bottom by cylindrical pipes. Ethanol production from raw cassava starch was investigated in the CLB. Aspergillus awamori IAM 2389 and Saccharomyces cerevisiae IR2 immobilized on loofa sponge were placed, respectively, in the aerated riser column and non-aerated downcomer column. Both alpha-amylase and glucoamylase activities increased as the aeration rate was increased. Ethanol yield and productivity increased with an increase in the aeration rate up to 0.5 vvm, but decreased at higher aeration rates. The CLB was operated at an aeration rate of 0.5 vvm for more than 600 h, resulting in an average ethanol productivity and yield from raw cassava starch of 0.5 g-ethanol l(-1) x h(-1) and 0.45 g ethanol/g starch, respectively. In order to increase ethanol productivity, it was necessary to increase the dissolved oxygen (DO) concentration in the riser column and decrease the DO concentration in the downcomer column. However, increasing the aeration rate resulted in increases in the DO concentration in both the riser and the downcomer columns. At high aeration rate, there was no significant difference in the DO concentration in the riser and downcomer columns. The aeration rate was therefore uncoupled from the liquid circulation by attaching a time-controlled valve in the upper connecting pipe. By optimizing the time and frequency of valve opening, and operation at high aeration rate, it was possible to maintain a very high DO concentration in the riser column and a low DO concentration in the downcomer column. Under these conditions, ethanol productivity increased by more than 100%, to 1.17 g l(-1) x h(-1).

A new control strategy was experimentally assessed on a laboratory scale by means of a two-stage fermentation for yeast production. The two-stage fermentation includes a batch culture to grow inoculum and a subsequent fed-batch culture. The strategy was based on controlling biomass concentration throughout the two-stage fermentation by manipulating the glucose feed rate. An L/A algorithm continuously tuned by ethanol concentration and respiratory quotient was used with constant external settings. The L/A controller performed satisfactorily throughout the two-stage fermentation and was able to move smoothly from one phase of operation to another. An average specific growth rate of 0.25 h−1 and a cellular yield of 0.5 g·g−1 were achieved during the fed-batch phase while over 40 g·l−1 biomass was obtained.

Saccharomyces cerevisiae was immobilised by adsorption to untreated sugar cane bagasse in a packed bed reactor. Complete conversion of glucose to ethanol was obtained at a dilution rate of 0.19 h−1. Continuous ethanol production was maintained for up to 57 days. Reactor productivity increased with increasing packing density of the bagasse. Plugging of void spaces due to cell overgrowth led to channelling of the feed and decreased reactor productivity. Increasing the average column temperature alleviated plugging and restored column performance over a short period; however prolonged exposure to the high temperature resulted in decreased ethanol production rates. Bagasse has advantages as a support material for ethanol production from sugar cane or beet, including negligible cost, ready availability and the capacity to support a high yeast population.

Pectin-rich agro-industrial residues are feedstocks with potential for sustainable biorefineries. They are generated in high amounts worldwide from the industrial processing of fruits and vegetables. The challenges posed to the industrial implementation of efficient bioprocesses are however manyfold and thoroughly discussed in this review paper, mainly at the biological level. The most important yeast cell factory platform for advanced biorefineries is currently Saccharomyces cerevisiae, but this yeast species cannot naturally catabolise the main sugars present in pectin-rich agro-industrial residues hydrolysates, in particular d-galacturonic acid and l-arabinose. However, there are non-Saccharomyces species (non-conventional yeasts) considered advantageous alternatives whenever they can express highly interesting metabolic pathways, natively assimilate a wider range of carbon sources or exhibit higher tolerance to relevant bioprocess-related stresses. For this reason, the interest in non-conventional yeasts for biomass-based biorefineries is gaining momentum. This review paper focuses on the valorisation of pectin-rich residues by exploring the potential of yeasts that exhibit vast metabolic versatility for the efficient use of the carbon substrates present in their hydrolysates and high robustness to cope with the multiple stresses encountered. The major challenges and the progresses made related with the isolation, selection, sugar catabolism, metabolic engineering and use of non-conventional yeasts and S. cerevisiae-derived strains for the bioconversion of pectin-rich residue hydrolysates are discussed. The reported examples of value-added products synthesised by different yeasts using pectin-rich residues are reviewed. Key Points• Review of the challenges and progresses made on the bioconversion of pectin-rich residues by yeasts.• Catabolic pathways for the main carbon sources present in pectin-rich residues hydrolysates.• Multiple stresses with potential to affect bioconversion productivity.• Yeast metabolic engineering to improve pectin-rich residues bioconversion.

Applications of microbiome research through metagenomics promise to generate microbiome manipulation strategies for improved larval survival in aquaculture. However, existing lacunae on the effects of sample preservation methods in metagenome profiles hinder the successful application of this technique. In this context, four preservation methods were scrutinized to identify reliable methods for fish larval microbiome research. The results showed that a total of ten metagenomics metrics, including DNA yield, taxonomic and functional microbiome profiles, and diversity measures, were significantly (P 0.05) influenced by the preservation method. Activity ranking based on the performance and reproducibility showed that three methods, namely immediate direct freezing, room temperature preservation in absolute ethanol, and preservation at - 20 °C in lysis, storage, and transportation buffer, could be recommended for larval microbiome research. Furthermore, as there was an apparent deviation of the microbiome profiles of ethanol preserved samples at room temperature, the other methods are preferred. Detailed analysis showed that this deviation was due to the bias towards Vibrionales and Rhodobacterales. The microbial taxa responsible for the dissimilarity across different methods were identified. Altogether, the paper sheds light on the preservation protocols of fish larval microbiome research for the first time. The results can help in cross-comparison of future and past larval microbiome studies. Furthermore, this is the first report on the activity ranking of preservation methods based on metagenomics metrics. Apart from methodological perspectives, the paper provides for the first time certain insights into larval microbial profiles of Rachycentron canadum, a potential marine aquaculture species. KEY POINTS: • First report on effects of preservation methods on fish larval microbiome profiles. • First report on activity ranking of preservation methods based on metagenomics metrics. • Storage methods influenced DNA yield, taxonomic and functional microbiome profiles.

With the aim of improving current ethanologenic Escherichia coli strains, we screened a metagenomic library from bovine ruminal fluid for cellulolytic enzymes. We isolated one fosmid, termed Csd4, which was able to confer to E. coli the ability to grow on complex cellulosic material as the sole carbon source such as avicel, carboxymethyl cellulose, filter paper, pretreated sugarcane bagasse, and xylan. Glucanolytic activity obtained from E. coli transformed with Csd4 was maximal at 24 h of incubation and was inhibited when glucose or xylose were present in the media. The 34,406-bp DNA fragment of Csd4 was completely sequenced, and a putative endoglucanase, a xylosidase/arabinosidase, and a laccase gene were identified. Comparison analysis revealed that Csd4 derived from an organism closely related to Prevotella ruminicola, but no homologies were found with any of the genomes already sequenced. Csd4 was introduced into the ethanologenic E. coli MS04 strain and ethanol production from CMC, avicel, sugarcane bagasse, or filter paper was observed. Exogenously expressed β-glucosidase had a positie effect on cell growth in agreement with the fact that no putative β-glucosidase was found in Csd4. Ethanol production from sugarcane bagasse was improved threefold by Csd4 after saccharification by commercial Trichoderma reesei cellulases underlining the ability of Csd4 to act as a saccharification enhancer to reduce the enzymatic load and time required for cellulose deconstruction.

Lignocellulosic biomass (LCB) is globally available and sustainable feedstock containing sugar-rich platform that can be converted to biofuels and specialty products through appropriate processing. This review focuses on the efforts required for the development of sustainable and economically viable lignocellulosic biorefinery to produce carbon neutral biofuels along with the specialty chemicals. Sustainable biomass processing is a global challenge that requires the fulfillment of fundamental demands concerning economic efficiency, environmental compatibility, and social responsibility. The key technical challenges in continuous biomass supply and the biological routes for its saccharification with high yields of sugar sources have not been addressed in research programs dealing with biomass processing. Though many R&D endeavors have directed towards biomass valorization over several decades, the integrated production of biofuels and chemicals still needs optimization from both technical and economical perspectives. None of the current pretreatment methods has advantages over others since their outcomes depend on the type of feedstock, downstream process configuration, and many other factors. Consolidated bio-processing (CBP) involves the use of single or consortium of microbes to deconstruct biomass without pretreatment. The use of new genetic engineering tools for natively cellulolytic microbes would make the CBP process low cost and ecologically friendly. Issues arising with chemical characteristics and rigidity of the biomass structure can be a setback for its viability for biofuel conversion. Integration of functional genomics and system biology with synthetic biology and metabolic engineering undoubtedly led to generation of efficient microbial systems, albeit with limited commercial potential. These efficient microbial systems with new metabolic routes can be exploited for production of commodity chemicals from all the three components of biomass. This paper provides an overview of the challenges that are faced by the processes converting LCB to commodity chemicals with special reference to biofuels.

Arthrobacter simplex has received considerable interests due to its superior Δ1-dehydrogenation ability. Ethanol used as co-solvent is a stress commonly encountered during biotransformation. Therefore, studies of ethanol tolerance of A. simplex are of great importance to improve the biotransformation efficiency. In this paper, the combined analysis of physiological properties, cell compositions, stress-responsive metabolites, and proteome profiles was carried out to achieve a global view of ethanol tolerance of A. simplex. Under sublethal conditions, cell permeability and membrane fluidity exhibited concentration-dependent increase by affecting the contents or compositions of cell peptidoglycan, lipids, phospholipids, and fatty acids. Among them, cis-trans isomerization of unsaturated fatty acids was a short-term and reversible process, while the changes in phospholipid headgroups and increase in saturation degree of fatty acids were long-term and irreversible processes, which collectively counteracted the elevated membrane fluidity caused by ethanol and maintained the membrane stability. The decreased intracellular ATP content was observed at high ethanol concentration since proton motive force responsible for driving ATP synthesis was dissipated. The involvement of trehalose and glycerol, oxidative response, and DNA damage were implicated due to their changes in positive proportion to ethanol concentration. Proteomic data supported that ethanol invoked a global alteration, among which, the change patterns of proteins participated in the biosynthesis of cell wall and membrane, energy metabolism, compatible solute metabolism, and general stress response were consistent with observations from cell compositions and stress-responsive metabolites. The protective role of proteins participated in DNA repair and antioxidant system under ethanol stress was validated by overexpression of the related genes. This is the first demonstration on ethanol tolerance mechanism of A. simplex, and the current studies also provide targets to engineer ethanol tolerance of A. simplex.

An abundant agricultural residue, rice straw (RS) was pretreated using ammonia fiber expansion (AFEX) process with less than 3% sugar loss. Along with commercial cellulase (Spezyme® CP) at 15 filter paper unit/g of glucan, the addition of Multifect® Xylanase at 2.67 mg protein/g glucan and Multifect® Pectinase at 3.65 mg protein/g glucan was optimized to greatly increase sugar conversion of AFEX-treated RS. During enzymatic hydrolysis even at 6% glucan loading (equivalent to 17.8% solid loading), about 80.6% of glucan and 89.6% of xylan conversions (including monomeric and oligomeric sugars) were achieved. However, oligomeric glucose and xylose accounted for 12.3% of the total glucose and 37.0% of the total xylose, respectively. Comparison among the three ethanologenic strains revealed Saccharomyces cerevisiae 424A(LNH-ST) to be a promising candidate for RS hydrolysate with maximum ethanol metabolic yield of 95.3% and ethanol volumetric productivity of 0.26 g/L/h. The final concentration of ethanol at 37.0 g/L was obtained by S. cerevisiae 424A(LNH-ST) even with low cell density inoculum. A biorefinery combining AFEX pretreatment with S. cerevisiae 424A(LNH-ST) in separate hydrolysis and fermentation could achieve 175.6 g EtOH/kg untreated rice straw at low initial cell density (0.28 g dw/L) without washing pretreated biomass, detoxification, or nutrient supplementation.

A robust Saccharomyces cerevisiae strain has been widely applied in continuous and batch/fed-batch industrial fermentation. However, little is known about the molecular basis of fermentative behavior of this strain in the two realistic fermentation processes. In this paper, we presented comparative proteomic profiling of the industrial yeast in the industrial fermentation processes. The expression levels of most identified protein were closely interrelated with the different stages of fermentation processes. Our results indicate that, among the 47 identified protein spots, 17 of them belonging to 12 enzymes were involved in pentose phosphate, glycolysis, and gluconeogenesis pathways and glycerol biosynthetic process, indicating that a number of pathways will need to be inactivated to improve ethanol production. The differential expressions of eight oxidative response and heat-shock proteins were also identified, suggesting that it is necessary to keep the correct cellular redox or osmotic state in the two industrial fermentation processes. Moreover, there are significant differences in changes of protein levels between the two industrial fermentation processes, especially these proteins associated with the glycolysis and gluconeogenesis pathways. These findings provide a molecular understanding of physiological adaptation of industrial strain for optimizing the performance of industrial bioethanol fermentation.

Winemaking, brewing and baking are some of the oldest biotechnological processes. In all of them, alcoholic fermentation is the main biotransformation and Saccharomyces cerevisiae the primary microorganism. Although a wide variety of microbial species may participate in alcoholic fermentation and contribute to the sensory properties of end-products, the yeast S. cerevisiae invariably dominates the final stages of fermentation. The ability of S. cerevisiae to outcompete other microbial species during alcoholic fermentation processes, such as winemaking, has traditionally been ascribed to its high fermentative power and capacity to withstand the harsh environmental conditions, i.e. high levels of ethanol and organic acids, low pH values, scarce oxygen availability and depletion of certain nutrients. However, in recent years, several studies have raised evidence that S. cerevisiae, beyond its remarkable fitness for alcoholic fermentation, also uses defensive strategies mediated by different mechanisms, such as cell-to-cell contact and secretion of antimicrobial peptides, to combat other microorganisms. In this paper, we review the main physiological features underlying the special aptitude of S. cerevisiae for alcoholic fermentation and discuss the role of microbial interactions in its dominance during alcoholic fermentation, as well as its relevance for winemaking.

Acetic acid fermentation is the biochemical process by which, under strict conditions of aerobiosis, Acetobacter aceti oxidises the ethanol contained in alcoholic substrates into acetic acid. This paper studies the effect of temperature on the specific growth rate of the microorganisms (μC), in particular, the mathematical modelling of this process, with the aim of developing previous studies of the mathematical relationships between μC of A. aceti and the concentrations of substrate (ethanol), product (acetic acid) and dissolved oxygen. Until now this relationship has not been widely studied, and only a few studies have looked at the influence of temperature on growth kinetics of this bacteria. We have developed an extensive experimental system, to determine precisely the influence of temperature on the maximum specific growth rate.

Enzymatic hydrolysis of cellulosic material is an essential step in the bioethanol production process. However, complete cellulose hydrolysis by cellulase is difficult due to the irreversible adsorption of cellulase onto cellulose. Thus, part of the cellulose remains in crystalline form after hydrolysis. In this study, after 96-h hydrolysis of Avicel crystalline cellulose, 47.1 % of the cellulase was adsorbed on the cellulose surface with 10.8 % crystalline cellulose remaining. In simultaneous saccharification and fermentation of 100 g/L Avicel with 1.0 filter paper unit/mL cellulase, a wild-type yeast strain produced 44.7 g/L ethanol after 96 h. The yield of ethanol was 79.7 % of the theoretical yield. On the other hand, a recombinant yeast strain displaying various cellulases, such as β-glucosidase, cellobiohydrolase, and endoglucanase, produced 48.9 g/L ethanol, which corresponds to 87.3 % of the theoretical yield. Higher ethanol production appears to be attributable to higher efficiency of cellulase displayed on the cell surface. These results suggest that cellulases displayed on the yeast cell surface improve hydrolysis of Avicel crystalline cellulose. Indeed, after the 96-h simultaneous saccharification and fermentation using the cellulase-displaying yeast, the amount of residual cellulose was 1.5 g/L, one quarter of the cellulose remaining using the wild-type strain, a result of the alleviation of irreversible adsorption of cellulases on the crystalline cellulose.

This paper reviews the latest developments in microbial products by encapsulated microorganisms in a liquid core surrounded by natural or synthetic membranes. Cells can be encapsulated in one or several steps using liquid droplet formation, pregel dissolving, coacervation, and interfacial polymerization. The use of encapsulated yeast and bacteria for fermentative production of ethanol, lactic acid, biogas, l-phenylacetylcarbinol, 1,3-propanediol, and riboflavin has been investigated. Encapsulated cells have furthermore been used for the biocatalytic conversion of chemicals. Fermentation, using encapsulated cells, offers various advantages compared to traditional cultivations, e.g., higher cell density, faster fermentation, improved tolerance of the cells to toxic media and high temperatures, and selective exclusion of toxic hydrophobic substances. However, mass transfer through the capsule membrane as well as the robustness of the capsules still challenge the utilization of encapsulated cells. The history and the current state of applying microbial encapsulation for production processes, along with the benefits and drawbacks concerning productivity and general physiology of the encapsulated cells, are discussed.

A range of intracellular NADH availability was achieved by combining external and genetic strategies. The effect of these manipulations on the distribution of metabolites in Escherichia coli was assessed in minimal and complex medium under anoxic conditions. Our in vivo system to increase intracellular NADH availability expressed a heterologous NAD+-dependent formate dehydrogenase (FDH) from Candida boidinii in E. coli. The heterologous FDH pathway converted 1 mol formate into 1 mol NADH and carbon dioxide, in contrast to the native FDH where cofactor involvement was not present. Previously, we found that this NADH regeneration system doubled the maximum yield of NADH from 2 mol to 4 mol NADH/mol glucose consumed. In the current study, we found that yields of greater than 4 mol NADH were achieved when carbon sources more reduced than glucose were combined with our in vivo NADH regeneration system. This paper demonstrates experimentally that different levels of NADH availability can be achieved by combining the strategies of feeding the cells with carbon sources which have different oxidation states and regenerating NADH through the heterologous FDH pathway. The general trend of the data is substantially similar for minimal and complex media. The NADH availability obtained positively correlates with the proportion of reduced by-products in the final culture. The maximum theoretical yield for ethanol is obtained from glucose and sorbitol in strains overexpressing the heterologous FDH pathway.

The effects of pH, temperature and concentration of citrate were investigated to achieve an optimal production of diacetyl, acetoin and C2 compounds such as acetaldehyde, acetate and ethanol for free and immobilized cells. The critical conditions of culture, 22°C, pH 4.8, increased the production of C4 compounds (diacetyl, acetoin, 2, 3 butylene glycol), C2 compounds (acetaldehyde, ethanol, acetate) and formate. A higher yield of C2 and C4 compounds was observed for the immobilized cells than for the free cells in continuous culture. At 75 mMol/l of citrate, the citrate bioconversion yield was 42.8% and 80% for free and immobilized cells, respectively. This paper discusses citrate and lactose utilization and NADH2 part on diacetyl reduction.