Ethanol fermentation from biomass resources current state and prospects

Ethanol fermentation from biomass resources current state and prospects

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Appl Microbiol Biotechnol (2006) 69: 627–642 DOI 10.1007/s00253-005-0229-x

Yan Lin . Shuzo Tanaka

Ethanol fermentation from biomass resources: current state and prospects

Abstract 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.


With the inevitable depletion of the world’s energy supply, there has been an increasing worldwide interest in alternative sources of energy (Aristidou and Penttila 2000; Jeffries and Jin 2000; John 2004; Kerr 1998; Wheals et al. 1999; Zaldivar et al. 2001). It is now understood that it is important to use biomass energy as a means of providing modern energy to the billions who lack it. It would complement solar, wind, and other intermittent energy sources in the renewable energy mix of the future. One of the most immediate and important applications of biomass energy systems could be in the fermentation of ethanol from biomass.

Biomass is seen as an interesting energy source for several reasons. The main reason is that bioenergy can con- tribute to sustainable development (Van den Broek 2000; Monique et al. 2003). Resources are often locally available, and conversion into secondary energy carriers is feasible without high capital investments. Moreover, biomass energy can play an important role in reducing greenhouse gas emissions; since CO2 that arises from biomass wastes would originally have been absorbed from the air, the use of biomass for energy offsets fossil fuel greenhouse gas emissions (Lynd 1996). Furthermore, since energy plantations may also create new employment opportunities in rural areas, it also contributes to the social aspect of sustainability. In addition, application of agro-industrial residues in bioprocesses not only provides alternative substrates but also helps solve their disposal problem. With the advent of biotechnological innovations, mainly in the area of enzyme and fermentation technology, many new avenues have opened for their utilization.

Nearly all fuel ethanol is produced by fermentation of corn glucose in the US or sucrose in Brazil (MacDonald et al. 2001; Rosillo-Calle and Cortez 1998), but any country with a significant agronomic-based economy can use current technology for fuel ethanol fermentation. This is possible because, during the last two decades, technology for ethanol production from nonfood-plant sources has been developed to the point at which large-scale production will be a reality in the next few years. Therefore, agronomic residues such as corn stover (corn cobs and stalks), sugarcane waste, wheat or rice straw, forestry, and paper mill discards, the paper portion of municipal waste and dedicated energy crops—collectively termed “biomass”—can be converted into fuel ethanol. In this field, although bioethanol production has been greatly improved by new technologies, there are still challenges that need further investigations. A further understanding of the ethanol fermentation needs to be reached.

This review will focus on the current status of ethanol fermentation including biomass resources, microorganisms, technology, the practical examples, and especially the promising prospects of ethanol fermentation.

Y. Lin . S. Tanaka (*) Asian Center for Environmental Research, Meisei University, Tokyo, Japan e-mail:

Biomass resources

There are various forms of biomass resources in the world, which can be grouped into four categories. Wood residues are by far the largest current source of biomass for energy production. It comes from the wood product industry which includes paper mills, sawmills, and furniture manufacturing. Municipal solid waste is the next largest, followed by agriculture residues and dedicated energy crops. Among these biomass resources including short-rotation woody crops and herbaceous crops, primarily tall grasses, dedicated energy crops seem to be the largest, most promising, future resource of biomass. This is because of the ability to obtain numerous harvests from a single planting, which significantly reduces average annual costs for establishing and managing energy crops, particularly in comparison to conventional crops (Monique et al. 2003).

Fermentation processes from any material that contains sugar could derive ethanol. The varied raw materials used in the manufacture of ethanol via fermentation are conveniently classified into three main types of raw materials: sugars, starches, and cellulose materials. Sugars (from sugarcane, sugar beets, molasses, and fruits) can be converted into ethanol directly. Starches (from corn, cassava, potatoes, and root crops) mustfirst be hydrolyzed to fermentable sugars by the action of enzymes from malt or molds. Cellulose (from wood, agricultural residues, waste sulfite liquor from pulp, and paper mills) must likewise be converted into sugars, generally by the action of mineral acids. Once simple sugars are formed, enzymes from microorganisms can readily ferment them to ethanol.

The most widely used sugar for ethanol fermentation is molasses which contains about 50 wt% of sugar and about 50 wt% of organic and inorganic compounds, including water. It is a thick, dark-colored syrup produced during refinement of sugar. Since molasses contains microorganisms which can disturb the fermentation, the molasses is taken first to the sterilizer and then to the fermentor. Then it is diluted with water to the mass fraction of 10±18% to reduce its viscosity in the pipeline. In addition, a very high concentration of sugar can give too much ethanol and results in a prolonged fermentation time and an incomplete sugar conversion. After the pH of the mash is adjusted to about 4–5 with mineral acid, it is inoculated with yeast or bacteria, and the fermentation is carried out nonaseptically at 20–32°C for about 1–3 days.

Most agricultural biomass containing starch can be used as a potential substrate for the ethanol fermentation by microbial processes. These substrates include corn (maize), wheat, oats, rice, potato, and cassava. On a dry basis, corn, wheat, sorghums (milo), and other grains contain around 60–75% (wt/wt) of starch, hydrolyzable to hexose with a significant weight increase (stoichiometrically the starch to hexose ratio is 9:10), and these offer a good resource in many fermentation processes (Jackman 1987).

Fermentation of starch is somewhat more complex than fermentation of sugars because starch must first be converted into sugar and then into ethanol. Starch is first hydrolyzed by adding α-amylase to avoid gelatinization, then cooked at high temperature (140–180°C). Next, the liquefied starch is hydrolyzed to glucose with glucoamylase. The resulting dextrose is fermented to ethanol with the aid of microorganisms producing CO2 as a coproduct. During the process currently employed for industrial-scale ethanol fermentation from starchy materials, high-temperature cooking (140–180°C) is very effective for fermentation of starchy materials because it raises starch saccharification efficiency and achieves high levels of ethanol production under complete sterilization of harmful microorganisms. However, production costs are high due to the high energy consumption in the cooking process and the addition of large amounts of amylolytic enzymes. So processes to reduce the high production costs are required. To resolve these difficulties, noncooking and low-temperature cooking fermentation systems have been developed (Matsumoto et al. 1985).

Industrial ethanol production has been reported using various starchy materials such as corn, wheat, starch and potatoes, cassava root (Lindeman and Rocchiccioli 1979), corn stover (Kadam and McMillan 2003; Wilke et al. 1981), and starch (Maisch et al. 1979). Among many starchy materials, cassava starch is an inexpensive fermentable source. It is a tropical root crop produced in more than 80 countries (Sasson 1990). About 20% of the cassava starch was incorporated into animal feed. A similar amount was converted into starch for industrial use and another portion used for human food in some developing countries. The rest was lost since cassava is perishable after harvest. Harnessing the lost portion in addition to gains from new high-yielding varieties with outputs of 100 tons per hectare could provide the fermentation industry with an abundance of raw material (Anthony et al. 1996). Fresh cassava has a very high starch content, up to 30%. The content of sucrose is about 4%. Dried cassava has 80% fermentable substrate.

However, cassava waste processing is difficult because it is high in toxic materials. The potential toxicity of cassava is due to the presence of cyanogenic glycosides, linamarin, and lotaustralin, which on hydrolysis yield hydrogen cyanide on its peel. Traditional methods of cooking like boiling and decanting remove cyanoglycosides to a certain extent, but even then a certain amount of residual toxicity remains in it (Westley 1980). Moreover, since starch particles in cassava are bigger and there are some branched structures, more glucoamylase has to be added into the reactor. Furthermore, the nitrogen content of the cassava is very low, so during the fermentation, nutrient has to be added into the reactor to maintain the normal growth of the microorganisms.

Among the three main types of raw materials, cellulose materials represent the most abundant global source of biomass and have been largely unutilized. The global production of plant biomass, of which over 90% is lignocellulose, amounts to about 200×109 tons per year, where about 8–20×109 tons of the primary biomass remains potentially accessible. However, the effective utilization of the lignocellulosic feedstock is not always practical because of its seasonal availability, scattered stations, and the high costs of transportation and storage of such large amounts of organic material (Polman 1994). Recently, the enzymatic hydrolysis of biomass cellulose is considered to be the most promising technology available (Ogier et al. 1999; Yu and Zhang 2004). However, despite the work done, the industrial scaleup of this process appears to be still hindered by technological issues or by the lack of a biomass refinery approach in which ethanol is one of several products. In fact, because raw material cost comprises more than 20% of the production cost (Brown et al. 2001;K aylen eta l. 2000;Z huang et al. 2001), the optimization of the cellulose conversion should be accomplished by correct management and utilization of all process streams. A consequence of this situation is that even limited government intervention is still crucial to maintaining ongoing research.

Furthermore, lignocellulose is a more complex substrate than starch. It is composed of a mixture of carbohydrate polymers (cellulose and hemicellulose) and lignin. The carbohydrate polymers are tightly bound to lignin mainly by hydrogen bonds but also by some covalent bonds. The biological process for converting the lignocellulose to fuel ethanol requires the following: delignification to liberate cellulose and hemicellulose from their complex with lignin, depolymerization of the carbohydrate polymers to produce free sugars, and fermentation of mixed hexose and pentose sugars to produce ethanol. Among the key processes described above, the delignification of lignocellulosic raw materials is the rate-limiting and most difficult task to be solved. Another problem is that the aqueous acid used to hydrolyze the cellulose in wood to glucose and other simple sugars destroys much of the sugars in the process. Extensive research has been carried out in this field for decades (Yu and Zhang 2004), and the first demonstration plant using lignocellulosic feedstocks has been in operation in Canada since April 2004 (Tampier et al. 2004). It is expected that the cost of lignocellulosic ethanol can undercut that of starch-based ethanol because low-value agricultural residues can be used.

General process

Besides the initial removal of large and unsuitable items, key components of an integrated residual waste treatment system based on ethanol fermentation include recyclable materials recovery and removal of contaminants via mechanical preprocessing, initial hydrolysis process (conversion to simpler compounds), fermentation of organics, postfermentation purification of ethanol (by distillation or filtration), gasification of solid residuals to provide process heat, and treatment and disposal of wastewater.

Nearly all of the ethanol fermentation technologies use an initial tipping floor removal of large or unsuitable materials, followed by mechanical preprocessing to remove recyclables and contaminants, and shredding of the material. Then the material is processed through vessels using various systems for the purpose of hydrolysis (breaking down to simpler compounds) of the material. Depending on the technology, this may include high temperature, acid treatment, and/or high pressure. Following the initial hydrolysis phase, the slurried material is then fermented to produce alcohol, which is then purified through distillation and/or filtration to produce the desired fuel-grade quality ethanol.

When cellulose was used as the raw material, the cellulase responsible for enzymatic hydrolysis of pretreated cellulosic biomass is strongly inhibited by hydrolysis products: glucose and short cellulose chains. One way to overcome cellulase inhibition is to ferment the glucose to ethanol as soon as it appears in solution. Simultaneous saccharification and fermentation (SSF) combines enzymatic hydrolysis with ethanol fermentation to keep the concentration of glucose low (as shown in Fig. 1). The accumulation of ethanol in the fermentor does not inhibit cellulase as much as high concentrations of glucose, so SSF is a good strategy for increasing the overall rate of cellulose to ethanol conversion. In comparison to the process where these two stages are sequential, the SSF method enables attainment of higher (up to 40%) yields of ethanol by removing end-product inhibition, as well as by eliminating the need for separate reactors for saccharification and fermentation (Bollók et al. 2000; Hari et al. 2001; Stenberg et al. 2000). Other advantages of this approach are a shorter fermentation time and a reduced risk of contamination with external microflora, due to the high temperature of the process, the presence of ethanol in the reaction medium, and the anaerobic conditions (Emert and Katzen 1980; Wyman 1994).

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