Anaerobic biotechnological approaches for production of liquid energy carriers from biomass

In recent years, increasing attention has been paid to the use of renewable biomass for energy production. Anaerobic biotechnological approaches for production of liquid energy carriers (ethanol and a mixture of acetone, butanol and ethanol) from biomass can be employed to decrease environmental pollution and reduce dependency on fossil fuels. There are two major biological processes that can convert biomass to liquid energy carriers via anaerobic biological breakdown of organic matter: ethanol fermentation and mixed acetone, butanol, ethanol (ABE) fermentation. The specific product formation is determined by substrates and microbial communities available as well as the operating conditions applied. In this review, we evaluate the recent biotechnological approaches employed in ethanol and ABE fermentation. Practical applicability of different technologies is discussed taking into account the microbiology and biochemistry of the processes.     

Thermophilic,lignocellulolytic bacteria for ethanol production: current state and perspectives

Lignocellulosic biomass contains a variety of carbohydrates, and their conversion into ethanol by fermentation requires an efficient microbial platform to achieve high yield, productivity, and final titer of ethanol. In recent years, growing attention has been devoted to the development of cellulolytic and saccharolytic thermophilic bacteria for lignocellulosic ethanol production because of their unique properties. First of all, thermophilic bacteria possess unique cellulolytic and hemicellulolytic systems and are considered as potential sources of highly active and thermostable enzymes for efficient biomass hydrolysis. Secondly, thermophilic bacteria ferment a broad range of carbohydrates into ethanol, and some of them display potential for ethanologenic fermentation at high yield. Thirdly, the establishment of the genetic tools for thermophilic bacteria has allowed metabolic engineering, in particular with emphasis on improving ethanol yield, and this facilitates their employment for ethanol production. Finally, different processes for second-generation ethanol production based on thermophilic bacteria have been proposed with the aim to achieve cost-competitive processes. However, thermophilic bacteria exhibit an inherent low tolerance to ethanol and inhibitors in the pretreated biomass, and this is at present the greatest barrier to their industrial application. Further improvement of the properties of thermophilic bacteria, together with the optimization production processes, is equally important for achieving a realistic industrial ethanol production.     

Consolidated bioprocessing and simultaneous saccharification and fermentation of lignocellulose to ethanol with thermotolerant yeast strains

Consolidated bioprocessing (CBP), which integrates enzyme production, saccharification and fermentation into a single process, is a promising strategy for effective ethanol production from lignocellulosic materials because of the resulting reduction in utilities, the substrate and other raw materials and simplification of operation. CBP requires a highly engineered microbial strain capable of hydrolyzing biomass with enzymes produced on its own and producing high-titer ethanol. Recently, heterologous production of cellulolytic enzymes has been pursued with yeast hosts, which has realized direct conversion of cellulose to ethanol. Specifically, the development of cell surface engineering, which provides a display of cellulolytic enzymes on the yeast cell surface, facilitates effective biomass hydrolysis concomitantly with ethanol production. On the other hand, the difference in optimum temperature between saccharification and fermentation is a drawback of efficient ethanol production in the simultaneous saccharification and fermentation (SSF). The application of thermotolerant yeast strains engineered to the SSF process would overcome the drawback by performing hydrolysis and fermentation at elevated temperature. In this review, we focus on the recent advances in the application of thermotolerant yeast to CBP and SSF of lignocellulosic material to ethanol. The development of thermotolerant and ethanologenic yeast strains with the ability to hydrolyze lignocellulosic materials is emphasized for high-temperature CBP.     

Bioconversion of Lignocellulose into Bioethanol: Process Intensification and Mechanism Research

Biofuels produced from lignocellulosic biomass can significantly reduce the energy dependency on fossil fuels and the resulting effects on environment. In this respect, cellulosic ethanol as an alternative fuel has the potential to become a viable energy source in the near future. Over the past few decades, tremendous effort has been undertaken to make cellulosic ethanol cost competitive with conventional fossil fuels. The pretreatment step is always necessary to deconstruct the recalcitrant structures and to make cellulose more accessible to enzymes. A large number of pretreatment technologies involving physical, chemical, biological, and combined approaches have been developed and tested at the pilot scale. Furthermore, various strategies and methods, including multi-enzyme complex, non-catalytic additives, enzyme recycling, high solids operation, design of novel bioreactors, and strain improvement have also been implemented to improve the efficiency of subsequent enzymatic hydrolysis and fermentation. These technologies provide significant opportunities for lower total cost, thus making large-scale production of cellulosic ethanol possible. Meanwhile, many researchers have focused on the key factors that limit cellulose hydrolysis, and analyzing the reaction mechanisms of cellulase. This review describes the most recent advances on process intensification and mechanism research of pretreatment, enzymatic hydrolysis, and fermentation during the production of cellulosic ethanol.     

Microbial contamination of fuel ethanol fermentations

Microbial contamination is a pervasive problem in any ethanol fermentation system. These infections can at minimum affect the efficiency of the fermentation and at their worse lead to stuck fermentations causing plants to shut down for cleaning before beginning anew. These delays can result in costly loss of time as well as lead to an increased cost of the final product. Lactic acid bacteria (LAB) are the most common bacterial contaminants found in ethanol production facilities and have been linked to decreased ethanol production during fermentation. Lactobacillus sp. generally predominant as these bacteria are well adapted for survival under high ethanol, low pH and low oxygen conditions found during fermentation. It has been generally accepted that lactobacilli cause inhibition of Saccharomyces sp. and limit ethanol production through two basic methods; either production of lactic and acetic acids or through competition for nutrients. However, a number of researchers have demonstrated that these mechanisms may not completely account for the amount of loss observed and have suggested other means by which bacteria can inhibit yeast growth and ethanol production. While LAB are the primary contaminates of concern in industrial ethanol fermentations, wild yeast may also affect the productivity of these fermentations. Though many yeast species have the ability to thrive in a fermentation environment, Dekkera bruxellensis has been repeatedly targeted and cited as one of the main contaminant yeasts in ethanol production. Though widely studied for its detrimental effects on wine, the specific species–species interactions between D. bruxellensis and S. cerevisiae are still poorly understood.     

Yeasts in sustainable bioethanol production: A review

Bioethanol has been identified as the mostly used biofuel worldwide since it significantly contributes to the reduction of crude oil consumption and environmental pollution. It can be produced from various types of feedstocks such as sucrose, starch, lignocellulosic and algal biomass through fermentation process by microorganisms. Compared to other types of microoganisms, yeasts especially Saccharomyces cerevisiae is the common microbes employed in ethanol production due to its high ethanol productivity, high ethanol tolerance and ability of fermenting wide range of sugars. However, there are some challenges in yeast fermentation which inhibit ethanol production such as high temperature, high ethanol concentration and the ability to ferment pentose sugars. Various types of yeast strains have been used in fermentation for ethanol production including hybrid, recombinant and wild-type yeasts. Yeasts can directly ferment simple sugars into ethanol while other type of feedstocks must be converted to fermentable sugars before it can be fermented to ethanol. The common processes involves in ethanol production are pretreatment, hydrolysis and fermentation. Production of bioethanol during fermentation depends on several factors such as temperature, sugar concentration, pH, fermentation time, agitation rate, and inoculum size. The efficiency and productivity of ethanol can be enhanced by immobilizing the yeast cells. This review highlights the different types of yeast strains, fermentation process, factors affecting bioethanol production and immobilization of yeasts for better bioethanol production.     

Recent advances on conversion and co-production of acetone-butanol-ethanol into high value-added bioproducts

Butanol is an important bulk chemical and has been regarded as an advanced biofuel. Large-scale production of butanol has been applied for more than 100 years, but its production through acetone–butanol–ethanol (ABE) fermentation process by solventogenic Clostridium species is still not economically viable due to the low butanol titer and yield caused by the toxicity of butanol and a by-product, such as acetone. Renewed interest in biobutanol as a biofuel has spurred technological advances to strain modification and fermentation process design. Especially, with the development of interdisciplinary processes, the sole product or even the mixture of ABE produced through ABE fermentation process can be further used as platform chemicals for high value added product production through enzymatic or chemical catalysis. This review aims to comprehensively summarize the most recent advances on the conversion of acetone, butanol and ABE mixture into various products, such as isopropanol, butyl-butyrate and higher-molecular mass alkanes. Additionally, co-production of other value added products with ABE was also discussed.     

High-temperature fermentation: how can processes for ethanol production at high temperatures become superior to the traditional process using mesophilic yeast?

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°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.     

Fuels and industrial chemicals through biotechnology. 2

An overview of research on the biotechnical production of fuels and industrial chemicals during the two-year period of 1983-1984 is presented. Ethanol fermentation has continued to be the subject of major interest. A considerable amount of work has been directed to alternative feedstocks such as pentose sugars and lactose, and to bacterial fermentations. Reports on extrusion cooking as a continuous pretreatment method for subsequent ethanol fermentation, and on novel alternative downstream processing techniques have been published. In addition to ethanol fermentation, much attention has been paid to the biotechnical production of 2,3-butanediol, and of a number of organic and amino acids. In general, there appears to be a growing interest in the application of biocatalysis for the production of specialty chemicals, although only a few examples will be discussed in this paper. The construction of a demonstration plant to produce ethanol from molasses by a two 10 kL bed-volume immobilized yeast bioreactors at the Kyowa Hakko Kogyo Company Hofu plant, the announcement by Nitto Chemical Industries Company to begin the biotechnical production of acrylamide, and the French decision to construct pilot plants for the biotechnical production of acetone-butanol-ethanol cosolvent and of ethanol from renewable resources represent major scale-up developments.     

Continuous SSCF of AFEX™ pretreated corn stover for enhanced ethanol productivity using commercial enzymes and Saccharomyces cerevisiae 424A (LNH‐ST)

High productivity processes are critical for commercial production of cellulosic ethanol. One high productivity process—continuous hydrolysis and fermentation—has been applied in corn ethanol industry. However, little research related to this process has been conducted on cellulosic ethanol production. Here, we report and compare the kinetics of both batch SHF (separate hydrolysis and co‐fermentation) and SSCF (simultaneous saccharification and co‐fermentation) of AFEX? (Ammonia Fiber Expansion) pretreated corn stover (AFEX?‐CS). Subsequently, we designed a SSCF process to evaluate continuous hydrolysis and fermentation performance on AFEX?‐CS in a series of continuous stirred tank reactors (CSTRs). Based on similar sugar to ethanol conversions (around 80% glucose‐to‐ethanol conversion and 47% xylose‐to‐ethanol conversion), the overall process ethanol productivity for continuous SSCF was 2.3‐ and 1.8‐fold higher than batch SHF and SSCF, respectively. Slow xylose fermentation and high concentrations of xylose oligomers were the major factors limiting further enhancement of productivity. Biotechnol. Bioeng. 2013; 110: 1302–1311. © 2012 Wiley Periodicals, Inc.     

Microbial production of ethanol from carbon monoxide

Production of ethanol from fermentation of CO has received much attention in the last few years with several companies proposing to use CO fermentation in their ethanol production processes. The genomes of two CO fermenters, Clostridium ljungdahlii and Clostridium carboxidivorans, have recently been sequenced. The genetic information obtained from this sequencing is aiding molecular biologists who are enhancing ethanol and butanol production by genetic manipulation. Several studies have optimized media for CO fermentation, which has resulted in enhanced ethanol production. Also, new reactor designs involving the use of hollow fiber membranes have reduced mass transfer barriers that have hampered previous CO fermentation efforts.     

酿酒酵母在纤维素乙醇生产中对毒性化合物的耐受机理研究进展

利用木质纤维素生产燃料乙醇的过程中,前期预处理所产生的抑制剂会影响酵母的正常生长和后续的发酵过程。为减小抑制剂的影响所采取的一些脱毒策略往往造成糖的损失和生产成本的增加,这在实际生产与经济上是不可行的。因此,具有强的抑制剂耐受性的酿酒酵母菌株对于提高纤维素乙醇产率是十分重要的。近十年来,对于酿酒酵母胁迫耐受机制的研究取得了一些重要的进展,着重介绍目前酿酒酵母对抑制剂耐受机制的研究现状,包括一些关键性基因的表达及代谢通路过程分析等。同时也介绍一些应对抑制剂提高酵母发酵能力的措施。     

Yeast selection for fuel ethanol production in Brazil

Brazil is one of the largest ethanol biofuel producers and exporters in the world and its production has increased steadily during the last three decades. The increasing efficiency of Brazilian ethanol plants has been evident due to the many technological contributions. As far as yeast is concerned, few publications are available regarding the industrial fermentation processes in Brazil. The present paper reports on a yeast selection program performed during the last 12 years aimed at selecting Saccharomyces cerevisiae strains suitable for fermentation of sugar cane substrates (cane juice and molasses) with cell recycle, as it is conducted in Brazilian bioethanol plants. As a result, some evidence is presented showing the positive impact of selected yeast strains in increasing ethanol yield and reducing production costs, due to their higher fermentation performance (high ethanol yield, reduced glycerol and foam formation, maintenance of high viability during recycling and very high implantation capability into industrial fermenters). Results also suggest that the great yeast biodiversity found in distillery environments could be an important source of strains. This is because during yeast cell recycling, selective pressure (an adaptive evolution) is imposed on cells, leading to strains with higher tolerance to the stressful conditions of the industrial fermentation.     

微生物木糖发酵产乙醇的代谢工程

利用木质纤维素发酵生产乙醇具有广泛的应用前景。而自然界中缺少有效转化木糖为乙醇的微生物是充分利用纤维素水解产物、提高乙醇产率、降低生产成本的关键因素。多年来研究者利用分子生物学技术对微生物菌株进行了代谢工程改造,使其能更有效地利用木糖生产乙醇。以下主要对运动发酵单胞菌、大肠杆菌和酵母等候选产乙醇微生物的木糖代谢工程研究进展进行了概述。     

Utilization of household food waste for the production of ethanol at high dry material content

Background

Environmental issues and shortage of fossil fuels have turned the public interest to the utilization of renewable, environmentally friendly fuels, such as ethanol. In order to minimize the competition between fuels and food production, researchers are focusing their efforts to the utilization of wastes and by-products as raw materials for the production of ethanol. household food wastes are being produced in great quantities in European Union and their handling can be a challenge. Moreover, their disposal can cause severe environmental issues (for example emission of greenhouse gasses). On the other hand, they contain significant amounts of sugars (both soluble and insoluble) and they can be used as raw material for the production of ethanol.

Results

Household food wastes were utilized as raw material for the production of ethanol at high dry material consistencies. A distinct liquefaction/saccharification step has been included to the process, which rapidly reduced the viscosity of the high solid content substrate, resulting in better mixing of the fermenting microorganism. This step had a positive effect in both ethanol production and productivity, leading to a significant increase in both values, which was up to 40.81% and 4.46 fold, respectively. Remaining solids (residue) after fermentation at 45% w/v dry material (which contained also the unhydrolyzed fraction of cellulose), were subjected to a hydrothermal pretreatment in order to be utilized as raw material for a subsequent ethanol fermentation. This led to an increase of 13.16% in the ethanol production levels achieving a final ethanol yield of 107.58 g/kg dry material.

Conclusions

In conclusion, the ability of utilizing household food waste for the production of ethanol at elevated dry material content has been demonstrated. A separate liquefaction/saccharification process can increase both ethanol production and productivity. Finally, subsequent fermentation of the remaining solids could lead to an increase of the overall ethanol production yield.     

Engineering of Saccharomyces cerevisiae for efficient anaerobic alcoholic fermentation of L-arabinose

For cost-effective and efficient ethanol production from lignocellulosic fractions of plant biomass, the conversion of not only major constituents, such as glucose and xylose, but also less predominant sugars, such as l-arabinose, is required. Wild-type strains of Saccharomyces cerevisiae, the organism used in industrial ethanol production, cannot ferment xylose and arabinose. Although metabolic and evolutionary engineering has enabled the efficient alcoholic fermentation of xylose under anaerobic conditions, the conversion of l-arabinose into ethanol by engineered S. cerevisiae strains has previously been demonstrated only under oxygen-limited conditions. This study reports the first case of fast and efficient anaerobic alcoholic fermentation of l-arabinose by an engineered S. cerevisiae strain. This fermentation was achieved by combining the expression of the structural genes for the l-arabinose utilization pathway of Lactobacillus plantarum, the overexpression of the S. cerevisiae genes encoding the enzymes of the nonoxidative pentose phosphate pathway, and extensive evolutionary engineering. The resulting S. cerevisiae strain exhibited high rates of arabinose consumption (0.70 g h(-1) g [dry weight](-1)) and ethanol production (0.29 g h(-1) g [dry weight](-1)) and a high ethanol yield (0.43 g g(-1)) during anaerobic growth on l-arabinose as the sole carbon source. In addition, efficient ethanol production from sugar mixtures containing glucose and arabinose, which is crucial for application in industrial ethanol production, was achieved.     

Immobilized anaerobic fermentation for bio-fuel production by Clostridium co-culture

Clostridium thermocellum/Clostridium thermolacticum co-culture fermentation has been shown to be a promising way of producing ethanol from several carbohydrates. In this research, immobilization techniques using sodium alginate and alkali pretreatment were successfully applied on this co-culture to improve the bio-ethanol fermentation performance during consolidated bio-processing (CBP). The ethanol yield obtained increased by over 60 % (as a percentage of the theoretical maximum) as compared to free cell fermentation. For cellobiose under optimized conditions, the ethanol yields were approaching about 85 % of the theoretical efficiency. To examine the feasibility of this immobilization co-culture on lignocellulosic biomass conversion, untreated and pretreated aspen biomasses were also used for fermentation experiments. The immobilized co-culture shows clear benefits in bio-ethanol production in the CBP process using pretreated aspen. With a 3-h, 9 % NaOH pretreatment, the aspen powder fermentation yields approached 78 % of the maximum theoretical efficiency, which is almost twice the yield of the untreated aspen fermentation.     

利用木质纤维素生产丁醇的研究进展

随着化石燃料的逐年减少,以生物质为原料的生物能源研究近年来成为能源领域的研究热点,充分利用可再生生物质为发展经济的生物燃料生产工艺提供了一个极好的机会。与燃料乙醇和生物柴油相比,生物丁醇更具有优越性,以可再生木质纤维素生物质为原料进行发酵生产丁醇在近年来被广泛的研究。对于利用可再生生物质为原料生产丁醇,需要解决原料的选择、产品收率低、抑制物对生产菌株毒性等问题。本文对以木质纤维素生物质为原料进行生物丁醇发酵过程中的原料预处理、抑制物对丁醇生产菌的影响,以及水解液的脱毒和耐抑制物菌株的选育等方面进行综述,并对以木质纤维素生产燃料丁醇所面临的机遇与问题进行了简要评述。     

Fermentation of whey and starch by transformedSaccharomyces cerevisiae cells

Among the main agro-industrial wastes, whey and starch are of prime importance. In previous work we showed that strains ofSaccharomyces cerevisiae transformed with the episomal plasmid pM1 allow production of yeast biomass and ethanol from whey/lactose. Ethanol production from whey and derivatives has been improved in computer-controlled bioreactors, while fermentation studies showed that the composition of the medium greatly modulates the productivity (g ethanol produced.l in 1 h of fermentation). A yeast strain for the simultaneous utilization of lactose and starch has also been developed. Biotechnological perspective are discussed.     

Ethanol fermentation from biomass resources: current state and prospects

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.