**2. Various ethanologenic thermotolerant yeasts and their characteristics**

Increasing global energy demand that exceeds the finite supply of fossil fuel has spurred scientific research to deliver alternative fuels. Microbial fermentation and efficient conversion technologies now allow the extraction of biofuels from biomass, such as wood, crops, and waste materials. Supplies of ethanol have increased tremendously and are expected to continue rising rapidly in both developed and developing countries [41]. A variety of feedstocks from the 1st, 2nd, and 3rd generation have been used in bioethanol production [42]. Firstgeneration bioethanol involves feedstocks rich in sucrose (sugar cane juice, molasses, and sweet sorghum) and starch (corn, wheat, cassava, and potato). Second-generation bioethanol comes from lignocellulosic biomass such as wood, straw, and other agricultural wastes. Third-generation bioethanol is derived from algal biomass including microalgae and macroalgae [43, 44]. The process of ethanol production depends on the types of feedstocks used. Generally, there are three major steps in ethanol production: decomposition of biomass, fermentation, and product recovery. During fermentation, the cooling of fermenters is one of the major energy consuming steps because the metabolism of yeast releases a large amount of heat. Therefore, the application of thermotolerant yeasts can significantly reduce the cooling cost and help prevent contamination [38]. High-temperature ethanol fermentation will also benefit a simultaneous saccharification and fermentation process.

Many thermotolerant yeasts have been isolated from various natural habitats and tested for their capability to produce ethanol at high temperatures (**Table 1**). Many strains of *K. marxianus*, *Pichia kudriavzevii*, and *S. cerevisiae* were often isolated as ethanol-producing yeasts at high temperatures. Of these, *K. marxianus* was found to be the most thermotolerant yeast. Limtong et al. [31] isolated *K. marxianus* DMKU 3-1042 in Thailand and found optimum ethanol production at 40°C. The strain was compared with other *K. marxianus* strains including NCYC587, NCYC1429, and NCYC2791 and found to be the best ethanol producer at 45°C [36]. Kumar et al. [45] isolated *Kluyveromyces* sp. IIPE453 from a soil sample in a sugar mill, which showed high ethanol production rate at 45–50°C. Yanase et al. [46] reported that *K. marxianus* NBRC1777 efficiently produced ethanol corresponding to 92.9% of the theoretical yield. *K. marxianus* DBKKUY-103, that was recently isolated, achieved the maximum ethanol concentration of 83.5 g/L, corresponding to 96.6% of the theoretical yield [47]. Nitiyon et al. [37] reported that *K. marxianus* BUNL-21 is a highly competent yeast for high-temperature ethanol fermentation with lignocellulosic biomass. When compared with the strain DMKU 3-1042, the strain BUNL-21 had stronger ability for conversion of xylose to ethanol and tolerance to various stresses including high temperature and hydrogen peroxide.

Several *S. cerevisiae* strains were also isolated for high-temperature ethanol fermentation. Sree et al. [50] reported a strain VS3 that could grow at 40°C and produced ethanol up to 60 g/L. Auesukaree et al. [51] reported a strain C3867 that produced 38.8 g/L of ethanol at 41°C. Recently, Nuanpeng et al. [52] and Techaparin et al. [53] isolated *S. cerevisiae* DBKKUY-53 and KKU-VN8, respectively, in Thailand. The former strain produced the maximum ethanol concentration and volumetric ethanol productivity of 85.0 g/L and 2.83 g/L h, respectively, at 40°C, and the latter strain produced the maximum ethanol concentration of 89.3 g/L with a productivity of 2.48 g/L h and a theoretical ethanol yield of 96.3% from sweet sorghum juice

**Yeast strain Temp. (°C) P (g/L) Q***p* **(g/L/h) T.Y (%) Refs.**

Potential of Thermotolerant Ethanologenic Yeasts Isolated from ASEAN Countries…

http://dx.doi.org/10.5772/intechopen.79144

125

DMKU 3-1042 40 67.8 1.13 60.4 [31] IIPE453<sup>a</sup> 50 82.0 nd nd [45] NBRC1777 40 47.4 nd 92.9 [46] DBKKUY-103 40 83.5 1.39 96.6 [47]

DMKU 3-ET15 40 78.6 3.28 85.4 [20] KVMP10 42 54.0 2.25 nd [48] RZ8-1 40 33.8 1.41 77.9 [49]

VS3 40 60.0 nd nd [50] C3867 41 38.8 nd nd [51] DBKKUY-53 40 85.0 2.83 — [52] KKU-VN8 40 89.3 2.48 96.3 [53]

P, ethanol concentration; Q*p*, volumetric ethanol productivity; T.Y, fraction of theoretical yield; nd, no data.

**Table 1** shows a number of ethanologenic thermotolerant yeasts. A temperature of 40°C was

Bioethanol significantly contributes to the reduction of crude oil consumption and environmental pollution. Thus, it has been identified as the mostly used biofuel worldwide [42]. Feedstocks for biofuel currently seem to be the option for sustainable development in the

found to be the best condition for most strains to produce ethanol.

**Table 1.** Thermotolerant yeasts used in bioethanol production.

**3. Utilization of various sugars in thermotolerant yeasts**

at 40°C.

a

*Kluyveromyces marxianus*

*Pichia kudriavzevii*

*Saccharomyces cerevisiae*

*Kluyveromyces* sp.

Recently, there have been several reports on ethanol production at high temperatures using *P. kudriavzevii* (formerly known as *I. orientalis*). Several *P. kudriavzevii* strains were reported to grow and produce high levels of ethanol at high temperatures. The strain DMKU 3-ET15 was isolated from traditional fermented pork sausage in Thailand by an enrichment technique in a medium supplemented with 4% ethanol at 40°C. The strain produced 78.6 g/L ethanol from 180 g/L glucose at 40°C [20]. The strain KVMP10 that was isolated from soil located beneath apple trees for ethanol production from orange peel achieved 54 g/L ethanol at 42°C [48]. Strain RZ8-1 that was recently isolated from various samples collected from plant orchards in Thailand produced 33.8 g/L ethanol from 160 g/L glucose at 40°C [49].


a *Kluyveromyces* sp.

technologies now allow the extraction of biofuels from biomass, such as wood, crops, and waste materials. Supplies of ethanol have increased tremendously and are expected to continue rising rapidly in both developed and developing countries [41]. A variety of feedstocks from the 1st, 2nd, and 3rd generation have been used in bioethanol production [42]. Firstgeneration bioethanol involves feedstocks rich in sucrose (sugar cane juice, molasses, and sweet sorghum) and starch (corn, wheat, cassava, and potato). Second-generation bioethanol comes from lignocellulosic biomass such as wood, straw, and other agricultural wastes. Third-generation bioethanol is derived from algal biomass including microalgae and macroalgae [43, 44]. The process of ethanol production depends on the types of feedstocks used. Generally, there are three major steps in ethanol production: decomposition of biomass, fermentation, and product recovery. During fermentation, the cooling of fermenters is one of the major energy consuming steps because the metabolism of yeast releases a large amount of heat. Therefore, the application of thermotolerant yeasts can significantly reduce the cooling cost and help prevent contamination [38]. High-temperature ethanol fermentation will also

Many thermotolerant yeasts have been isolated from various natural habitats and tested for their capability to produce ethanol at high temperatures (**Table 1**). Many strains of *K. marxianus*, *Pichia kudriavzevii*, and *S. cerevisiae* were often isolated as ethanol-producing yeasts at high temperatures. Of these, *K. marxianus* was found to be the most thermotolerant yeast. Limtong et al. [31] isolated *K. marxianus* DMKU 3-1042 in Thailand and found optimum ethanol production at 40°C. The strain was compared with other *K. marxianus* strains including NCYC587, NCYC1429, and NCYC2791 and found to be the best ethanol producer at 45°C [36]. Kumar et al. [45] isolated *Kluyveromyces* sp. IIPE453 from a soil sample in a sugar mill, which showed high ethanol production rate at 45–50°C. Yanase et al. [46] reported that *K. marxianus* NBRC1777 efficiently produced ethanol corresponding to 92.9% of the theoretical yield. *K. marxianus* DBKKUY-103, that was recently isolated, achieved the maximum ethanol concentration of 83.5 g/L, corresponding to 96.6% of the theoretical yield [47]. Nitiyon et al. [37] reported that *K. marxianus* BUNL-21 is a highly competent yeast for high-temperature ethanol fermentation with lignocellulosic biomass. When compared with the strain DMKU 3-1042, the strain BUNL-21 had stronger ability for conversion of xylose to ethanol and tolerance to various stresses including high tempera-

Recently, there have been several reports on ethanol production at high temperatures using *P. kudriavzevii* (formerly known as *I. orientalis*). Several *P. kudriavzevii* strains were reported to grow and produce high levels of ethanol at high temperatures. The strain DMKU 3-ET15 was isolated from traditional fermented pork sausage in Thailand by an enrichment technique in a medium supplemented with 4% ethanol at 40°C. The strain produced 78.6 g/L ethanol from 180 g/L glucose at 40°C [20]. The strain KVMP10 that was isolated from soil located beneath apple trees for ethanol production from orange peel achieved 54 g/L ethanol at 42°C [48]. Strain RZ8-1 that was recently isolated from various samples collected from plant orchards in

Thailand produced 33.8 g/L ethanol from 160 g/L glucose at 40°C [49].

benefit a simultaneous saccharification and fermentation process.

ture and hydrogen peroxide.

124 Fuel Ethanol Production from Sugarcane

P, ethanol concentration; Q*p*, volumetric ethanol productivity; T.Y, fraction of theoretical yield; nd, no data.

**Table 1.** Thermotolerant yeasts used in bioethanol production.

Several *S. cerevisiae* strains were also isolated for high-temperature ethanol fermentation. Sree et al. [50] reported a strain VS3 that could grow at 40°C and produced ethanol up to 60 g/L. Auesukaree et al. [51] reported a strain C3867 that produced 38.8 g/L of ethanol at 41°C. Recently, Nuanpeng et al. [52] and Techaparin et al. [53] isolated *S. cerevisiae* DBKKUY-53 and KKU-VN8, respectively, in Thailand. The former strain produced the maximum ethanol concentration and volumetric ethanol productivity of 85.0 g/L and 2.83 g/L h, respectively, at 40°C, and the latter strain produced the maximum ethanol concentration of 89.3 g/L with a productivity of 2.48 g/L h and a theoretical ethanol yield of 96.3% from sweet sorghum juice at 40°C.

**Table 1** shows a number of ethanologenic thermotolerant yeasts. A temperature of 40°C was found to be the best condition for most strains to produce ethanol.

## **3. Utilization of various sugars in thermotolerant yeasts**

Bioethanol significantly contributes to the reduction of crude oil consumption and environmental pollution. Thus, it has been identified as the mostly used biofuel worldwide [42]. Feedstocks for biofuel currently seem to be the option for sustainable development in the context of economical and environmental considerations. There are various types of feedstocks for ethanol production [54], and accordingly, different processes including biomass pretreatment are required. Feedstock rich in sugar that mainly contains sucrose is readily fermented to ethanol. Feedstock rich in starch must first be hydrolyzed to glucose monomers by the action of enzymes [55]. Lignocellulosic and algal biomass needs further pretreatment and hydrolysis before liberating simple sugars, which can be readily converted to ethanol by microorganisms [56–58]. The resulting hydrolysates of these raw materials contain various sugars depending on the type of biomass [59]. In case of algal biomass, the sugar composition varies largely, based not only on algal species but also on their environmental and nutritional conditions [43, 56]. Lignocellulosic biomass is a complex mixture of carbohydrate polymers, and the biomass hydrolysate mainly contains hexoses (D-galactose, L-galactose, and D-mannose) and pentoses (D-xylose and L-arabinose) [60]. Glucose and xylose are the most abundant monosaccharides in this biomass taking up 60–70% and 30–40% of the total hydrolysate, respectively [61, 62]. Predominant pentose sugars derived from the hemicellulose of most feedstocks are xylose and arabinose. Like in higher plants, algae biomass is comprised of rigid cellulose-based cell walls and various complex polysaccharides, which can be hydrolyzed to sugars and subsequently fermented to ethanol [43, 63]. However, algae biomass contains a low percentage of lignin and hemicellulose compared to other lignocellulosic plants [64].

Microorganisms are the key factor in the conversion of sugars to ethanol. One of their several desired characteristics is thermotolerance. Ethanol production at high temperatures by thermotolerant yeasts has earned much interest due to several advantages as described above [38]. There are several ethanologenic yeasts that have been characterized and classified as thermotolerant yeasts such as *K. marxianus* [31, 37, 47], *P. kudriavzevii* (formally known as *I. orientalis*) [20, 48, 49, 65, 66], *Hansenula polymorpha* [67], and some strains of *S. cerevisiae* [21, 52, 68–70]. However, for cost-effective and efficient ethanol production, not only thermotolerance but also a broad spectrum in sugar assimilation and fermentation capability is beneficial for the conversion of a variety of raw materials containing various sugars to ethanol, especially xylose, which is the most common pentose sugar and the second most abundant after glucose in lignocellulosic biomass and algal biomass [71, 72].

*K. marxianus*'s most important characteristics in this respect are thermotolerance to temperatures between 45 and 52°C, efficient ethanol production at temperatures between 38°C and 45°C, and a rapid growth rate that is twice as high as that of *S. cerevisiae* in rich media. Moreover, it has a broad spectrum of sugar assimilation, which includes glucose, mannose, galactose, fructose, arabinose, xylose, xylitol, sucrose, raffinose, cellobiose, lactose, and inulin [32, 36]. However, there has been little ethanol production from xylose and none from arabinose [32]. This strain can utilize a wide variety of industrially relevant substrates and efficiently converts substrates to ethanol. Especially, with lignocellulosic raw materials, it

NRRLY-6860

**Feedstock Substrate Organism Temp. (°C) P (g/L) T.Y (%) Refs.**

DBKKU-Y102

DBKKUY-103

Starchy materials Taro waste *K. marxianus* K21 40 43.8 94.2 [78] Lignocellulosic biomass Kanlow switchgrass *K. marxianus* IMB3 45 22.5 86 [79]

Palm sap *K. marxianus* TISTR 5925 40 45.4 92.2 [39] Jerusalem artichoke *K. marxianus* PT-1 40 73.6 90 [21]

Potential of Thermotolerant Ethanologenic Yeasts Isolated from ASEAN Countries…

Switchgrass *K. marxianus* IMB4 45 16.6 78 [80] Solka-floc *K. marxianus* L. G. 42 37.6 98 [81]

40 67.8 60.4 [31]

http://dx.doi.org/10.5772/intechopen.79144

127

40 97.5 92 [77]

40 83.5 100 [47]

45 21.5 86 [82]

3-1042

Jerusalem artichoke *K. marxianus*

Sweet sorghum juice *K. marxianus*

Rice straw *K. marxianus*

**Table 2.** Ethanol production of *K. marxianus* from various substrates at high temperatures.

Sugar containing materials Sugar cane juice *K. marxianus* DMKU

**4. Complete genome sequence of thermotolerant yeast** *K. marxianus*

High-temperature fermentation technology with thermotolerant microbes has been expected to reduce the cost of bioconversion of biomass to fuels or chemicals. *K. marxianus* was included in GRAS (FDA) and QPS (EU) lists of safe microorganisms for use in foods [83, 84]. The capacity of *K. marxianus* to utilize a wide variety of sugars reflects its potential for biotechnological applications [29, 84], which has been indicated by many studies with diverse substrates such as whey permeate, crop plants, and lignocellulosic biomass [32, 33, 78, 85, 86]. *K. marxianus* is also distinguished by its thermotolerance [36, 87] and the highest growth rate

resulted in 78–98% of the theoretical ethanol yield (**Table 2**).

**DMKU 3-1042 and transcriptomic analysis**

P, ethanol concentration; T.Y, fraction of theoretical yield.

*S. cerevisiae* is commonly employed in ethanol production due to its high ethanol productivity and high ethanol tolerance [73]. It is capable of converting different types of sugars, such as glucose, mannose, galactose, fructose, sucrose, and maltose to ethanol via the glycolysis pathway under anaerobic conditions [55]. Unfortunately, it is not able to ferment other carbon sources from plant or algal hydrolysates such as D-xylose, L-arabinose, and L-rhamnose [59]. A few types of yeasts can ferment both glucose and xylose but their performance regarding the rate of ethanol production from xylose, and the yield is lower than those from the main hexose sugars (for example, *S.* (*Pichia*) *stipitis* [74], *Scheffersomyces* (*Candida*) *shehatae* [75], *Pachysolen tannophilus* [76], *H. polymorpha* [67], and *K. marxianus* [32, 37]). Among these xylosefermenting yeasts, it seems that *K. marxianus* has the potential for practical application in high-temperature ethanol fermentation because of its thermotolerance and ability to utilize a variety of sugars.


**Table 2.** Ethanol production of *K. marxianus* from various substrates at high temperatures.

context of economical and environmental considerations. There are various types of feedstocks for ethanol production [54], and accordingly, different processes including biomass pretreatment are required. Feedstock rich in sugar that mainly contains sucrose is readily fermented to ethanol. Feedstock rich in starch must first be hydrolyzed to glucose monomers by the action of enzymes [55]. Lignocellulosic and algal biomass needs further pretreatment and hydrolysis before liberating simple sugars, which can be readily converted to ethanol by microorganisms [56–58]. The resulting hydrolysates of these raw materials contain various sugars depending on the type of biomass [59]. In case of algal biomass, the sugar composition varies largely, based not only on algal species but also on their environmental and nutritional conditions [43, 56]. Lignocellulosic biomass is a complex mixture of carbohydrate polymers, and the biomass hydrolysate mainly contains hexoses (D-galactose, L-galactose, and D-mannose) and pentoses (D-xylose and L-arabinose) [60]. Glucose and xylose are the most abundant monosaccharides in this biomass taking up 60–70% and 30–40% of the total hydrolysate, respectively [61, 62]. Predominant pentose sugars derived from the hemicellulose of most feedstocks are xylose and arabinose. Like in higher plants, algae biomass is comprised of rigid cellulose-based cell walls and various complex polysaccharides, which can be hydrolyzed to sugars and subsequently fermented to ethanol [43, 63]. However, algae biomass contains a low percentage of lignin and hemicellulose compared to other lignocel-

Microorganisms are the key factor in the conversion of sugars to ethanol. One of their several desired characteristics is thermotolerance. Ethanol production at high temperatures by thermotolerant yeasts has earned much interest due to several advantages as described above [38]. There are several ethanologenic yeasts that have been characterized and classified as thermotolerant yeasts such as *K. marxianus* [31, 37, 47], *P. kudriavzevii* (formally known as *I. orientalis*) [20, 48, 49, 65, 66], *Hansenula polymorpha* [67], and some strains of *S. cerevisiae* [21, 52, 68–70]. However, for cost-effective and efficient ethanol production, not only thermotolerance but also a broad spectrum in sugar assimilation and fermentation capability is beneficial for the conversion of a variety of raw materials containing various sugars to ethanol, especially xylose, which is the most common pentose sugar and the second most

*S. cerevisiae* is commonly employed in ethanol production due to its high ethanol productivity and high ethanol tolerance [73]. It is capable of converting different types of sugars, such as glucose, mannose, galactose, fructose, sucrose, and maltose to ethanol via the glycolysis pathway under anaerobic conditions [55]. Unfortunately, it is not able to ferment other carbon sources from plant or algal hydrolysates such as D-xylose, L-arabinose, and L-rhamnose [59]. A few types of yeasts can ferment both glucose and xylose but their performance regarding the rate of ethanol production from xylose, and the yield is lower than those from the main hexose sugars (for example, *S.* (*Pichia*) *stipitis* [74], *Scheffersomyces* (*Candida*) *shehatae* [75], *Pachysolen tannophilus* [76], *H. polymorpha* [67], and *K. marxianus* [32, 37]). Among these xylosefermenting yeasts, it seems that *K. marxianus* has the potential for practical application in high-temperature ethanol fermentation because of its thermotolerance and ability to utilize a

abundant after glucose in lignocellulosic biomass and algal biomass [71, 72].

lulosic plants [64].

126 Fuel Ethanol Production from Sugarcane

variety of sugars.

*K. marxianus*'s most important characteristics in this respect are thermotolerance to temperatures between 45 and 52°C, efficient ethanol production at temperatures between 38°C and 45°C, and a rapid growth rate that is twice as high as that of *S. cerevisiae* in rich media. Moreover, it has a broad spectrum of sugar assimilation, which includes glucose, mannose, galactose, fructose, arabinose, xylose, xylitol, sucrose, raffinose, cellobiose, lactose, and inulin [32, 36]. However, there has been little ethanol production from xylose and none from arabinose [32]. This strain can utilize a wide variety of industrially relevant substrates and efficiently converts substrates to ethanol. Especially, with lignocellulosic raw materials, it resulted in 78–98% of the theoretical ethanol yield (**Table 2**).
