**Author details**

The type strains of *C. bescii* typically yield a mixture of lactic and acetic acid in addition to hydro-

have been noted to produce low ethanol titers. Work by Cha [89] deleted the gene coding for lactate dehydrogenase by introducing a non-replicating plasmid via marker replacement. The resultant knockout strain did demonstrate increased biomass yield as well as acetate and hydrogen production with no lactate production when grown on cellobiose and lactose as well as switch grass hydrolysates. Subsequent work by Chung [81] inserted a NADHdependent *adhE* gene (from *Clostridium thermocellum*) into the *ldh* mutant (JWCB018) result-

but produced 14.8 mM of ethanol from 29.2 mM cellobiose or 12.7% of the theoretical yield. It should be noted that this strain only used a small portion (4.4 mM of 29.2 mM cellobiose) provided and not produce ethanol above 65°C. Work by Cha [89] and Chung [93] introduced the alcohol dehydrogenase genes (*adhB* and *adhE*) from *Thermoanaerobacter pseudoethanolicus* into the *ldh* deficient strain. The two resultant strains yielded ethanol at temperatures greater than 65°C although titers were lower than the aforementioned strain JWCB032 (*ldh*<sup>−</sup> *adhE+*

The *C. thermocellum* strain with *adhB* only produced 1.4 mM ethanol on avicel and 0.4 mM on switch grass while a strain with *adhE* gave 2.3 and 1.6 mM of ethanol on avicel and switch grass, respectively. One of the reasons for suggested for the low ethanol titers is the availability of cofactors and it should be noted that *T. pseudoethanolicus* ADHs utilize NADPH while the gene products from *C. thermocellum* use NADH as a source of reducing potential. Additional work is therefore needed to more carefully mimic the complex NAD(P)H system

Overall, efforts to engineer thermophilic anaerobes to increase ethanol titers has resulted in modest gains in yields while minimizing or eliminating the formation of unwanted end products. Future targets for genetic manipulation might include the inclusion of the cellulolytic machinery of *C. thermocellum* into highly ethanologenic *Thermoanaerobacter* and

Bioethanol production from lignocellulosic biomass with thermophilic bacteria needs robust microbes regarding several aspects. One of the main advantages of thermophilic bacteria is their broad substrate spectra with many strains capable of simultaneous pentose and hexose degradations. Additionally, some thermophiles degrade complex carbohydrates like cellulose and hemicellulose although many of these strains are not highly ethanologenic. Recent advantages in genetic engineering have improved ethanol yields, mostly by knocking out pathways of undesired end-products. On the back side is the fact that yields and ethanol tolerance as well as low tolerance for high initial substrate concentrations still limits the use of thermophiles for large scale operations. The use of stable co-cultures where on microbe hydrolyses the sugar polymers and the other one ferments the sugars released to ethanol is an

ing in strain *C. bescii* JWCB032. The resultant *ldh*<sup>−</sup> *adhE*<sup>+</sup>

of multiple ADHs in *Thermoanaerobacter pseduoethanolicus*.

attractive way to go forward but warrants further investigations.

*Thermoanaerobacterium* strains.

**7. Conclusions**

as end-products although other strains within the genus of *Caldicellulosiruptor*

strain yielded less acetate (4.3 mM)

).

gen and CO2

112 Fuel Ethanol Production from Sugarcane

Sean Michael Scully and Johann Orlygsson\*

\*Address all correspondence to: jorlygs@unak.is

Faculty of Natural Resource Sciences, University of Akureyri, Akureyri, Iceland

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**Chapter 7**

**Provisional chapter**

**Potential of Thermotolerant Ethanologenic Yeasts**

**Potential of Thermotolerant Ethanologenic Yeasts** 

**in High-Temperature Fermentation**

**in High-Temperature Fermentation**

Tomoyuki Kosaka, Noppon Lertwattanasakul, Nadchanok Rodrussamee, Mochamad Nurcholis, Ngo Thi Phuong Dung, Chansom Keo-Oudone,

Tomoyuki Kosaka, Noppon Lertwattanasakul, Nadchanok Rodrussamee, Mochamad Nurcholis, Ngo Thi Phuong Dung, Chansom Keo-Oudone,

Constantinos Theodoropoulos, Suprayogi, Jaya Mahar Maligan, Savitree Limtong and

Constantinos Theodoropoulos, Suprayogi, Jaya Mahar Maligan, Savitree Limtong and

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

Masayuki Murata, Peter Götz,

Masayuki Murata, Peter Götz,

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

Mamoru Yamada

**Abstract**

Mamoru Yamada

**Isolated from ASEAN Countries and Their Application**

**Isolated from ASEAN Countries and Their Application** 

Thermotolerant ethanologenic yeasts receive attention as alternative bio-ethanol producers to traditionally used yeast, *Saccharomyces cerevisiae*. Their utilization is expected to provide several benefits for bio-ethanol production due to their characteristics and robustness. They have been isolated from a wide variety of environments in a number of ASEAN countries: Thailand, Vietnam, Laos, and Indonesia. One of these yeasts, Kluyveromyces marxianus has been investigated regarding characteristics. Some strains efficiently utilize xylose, which is a main component of the 2nd generation biomass. In addition, the genetic basis of *K. marxianus* has been revealed by genomic sequencing and is exploited for further improvement of the strains by thermal adaptation or gene engineering techniques. Moreover, the glucose repression of *K. marxianus* and its mechanisms has been investigated. Results suggest that *K. marxianus* is an alternative to *S. cerevisiae* in next-generation bio-ethanol production industry. Indeed, we have succeeded to apply *K. marxianus* for bio-ethanol production in a newly developed process, which combines high-temperature fermentation with simultaneous fermentation and distillation under low pressure. This chapter aims to provide valuable information on thermotolerant ethanologenic yeasts and their application, which may direct the economic bioproduction of ethanol and other useful materials in the future.

**Keywords:** thermotolerant yeast, high-temperature fermentation, genomic aspects

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

DOI: 10.5772/intechopen.79144


#### **Potential of Thermotolerant Ethanologenic Yeasts Isolated from ASEAN Countries and Their Application in High-Temperature Fermentation Potential of Thermotolerant Ethanologenic Yeasts Isolated from ASEAN Countries and Their Application in High-Temperature Fermentation**

DOI: 10.5772/intechopen.79144

Tomoyuki Kosaka, Noppon Lertwattanasakul, Nadchanok Rodrussamee, Mochamad Nurcholis, Ngo Thi Phuong Dung, Chansom Keo-Oudone, Masayuki Murata, Peter Götz, Constantinos Theodoropoulos, Suprayogi, Jaya Mahar Maligan, Savitree Limtong and Mamoru Yamada Tomoyuki Kosaka, Noppon Lertwattanasakul, Nadchanok Rodrussamee, Mochamad Nurcholis, Ngo Thi Phuong Dung, Chansom Keo-Oudone, Masayuki Murata, Peter Götz, Constantinos Theodoropoulos, Suprayogi, Jaya Mahar Maligan, Savitree Limtong and Mamoru Yamada

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

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

#### **Abstract**

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Thermotolerant ethanologenic yeasts receive attention as alternative bio-ethanol producers to traditionally used yeast, *Saccharomyces cerevisiae*. Their utilization is expected to provide several benefits for bio-ethanol production due to their characteristics and robustness. They have been isolated from a wide variety of environments in a number of ASEAN countries: Thailand, Vietnam, Laos, and Indonesia. One of these yeasts, Kluyveromyces marxianus has been investigated regarding characteristics. Some strains efficiently utilize xylose, which is a main component of the 2nd generation biomass. In addition, the genetic basis of *K. marxianus* has been revealed by genomic sequencing and is exploited for further improvement of the strains by thermal adaptation or gene engineering techniques. Moreover, the glucose repression of *K. marxianus* and its mechanisms has been investigated. Results suggest that *K. marxianus* is an alternative to *S. cerevisiae* in next-generation bio-ethanol production industry. Indeed, we have succeeded to apply *K. marxianus* for bio-ethanol production in a newly developed process, which combines high-temperature fermentation with simultaneous fermentation and distillation under low pressure. This chapter aims to provide valuable information on thermotolerant ethanologenic yeasts and their application, which may direct the economic bioproduction of ethanol and other useful materials in the future.

**Keywords:** thermotolerant yeast, high-temperature fermentation, genomic aspects

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **1. Introduction**

Worldwide economic growth with the related increase in CO2 emissions from fossil fuels causes global warming. Utilization of renewable energy with low CO2 emission therefore has been getting increased attention. Renewable energy is generated from renewable natural resources, such as sunlight, wind, rain, tides, waves, geothermal heat, as well as biomass. One such important source of renewable energy, *bio-ethanol*, has been highlighted due to the characteristics of its production from biomass, which is generated by plants using sunlight for CO2 fixation, resulting in carbon neutrality. Bio-ethanol is the name for ethanol produced from biomass by fermentation. This bio-process is thoroughly researched and well-established, and to-date, it produces the most prominent and cost-effective biofuel [1]. Although bio-ethanol production is increasing worldwide and the production of biofuels including ethanol in 2022 is forecasted to be more than 126 billion L [2], biofuels are still more costly than fossil fuels [3]. Therefore, several industrial companies and researchers aim to develop new technologies, enabling the cost-effective production of bio-ethanol from biomass. Since microorganisms are essential for material production through bio-processing, their characteristics and traits are crucial for the production process efficiency. Ethanologenic yeast, *Saccharomyces cerevisiae*, has been traditionally and widely utilized for the production of alcoholic beverages and bioethanol [4, 5]. Industrially common problems in bio-ethanol production related to *S. cerevisiae* strains are temperature level (35–45°C) and high ethanol concentration (over 20%) [6]. These two factors inhibit yeast proliferation and fermentation activity if they reach the upper limit. In addition, for cost-effective bio-ethanol production, the production source must be changed from 1st generation biomass (sugarcane, corn, and wheat, which are important food sources) to 2nd generation biomass (lignocellulosic biomass or woody crops, which are agricultural residues or waste) [7]. Lignocellulosic biomass is composed of hemicellulose, cellulose, and lignin, and the first consists of six (e.g., glucose) and five (e.g., xylose) carbon sugars. However, the low efficiency of ethanol production by *S. cerevisiae* from lignocellulosic biomass hydrolyzates is mainly due to its little ethanol productivity from xylose [8]. Although the *S. cerevisiae* genome encodes all components necessary for xylose utilization, most of them are rarely expressed [9]. In addition, *S. cerevisiae* preferably utilizes glucose while repressing the uptake and catabolism of alternate carbon sources by a mechanism such as glucose repression [10]. This results in the reduction of ethanol production rates from several kinds of biomass. For economically feasible bioethanol production from lignocellulosic biomass, the efficient co-fermentation of glucose and other sugars is also necessary. Therefore, genetic engineering of *S. cerevisiae* strains has been extensively performed, and metabolically engineered strains were developed [11], which have showed higher stress tolerance and/or improved xylose utilization [12, 13]. However, the utilization of genetically recombinant strains in industry has been very limited, especially due to the instability of the desirable phenotype and the necessary confinement to a closed system to prevent their leakage into the environment, which can eventually endanger public health or biodiversity. Therefore, the development of new feasible strains for next-generation bio-ethanol production is under way, and new yeast strains have been isolated that may have advantages compared to *S. cerevisiae*.

belonging to the same genus or even to the same species [14]. These thermotolerant mesophiles are mainly and widely distributed in foods, plants, soils, and waters from tropical environments in ASEAN countries [15]. In these environments, relatively high temperature presumably becomes a selective pressure to enrich thermotolerant strains. These thermotolerant strains are expected to provide a benefit for the industries because they are more robust and resistant to many stressors [14]. In addition, some of these thermotolerant microorganisms can produce distinctive enzymes that function under relatively high temperature conditions [16– 18]. Thermotolerant yeasts have been found and isolated from a number of countries [19–28]. Of these, *K. marxianus* is a haploid, homothallic, thermotolerant, and hemiascomycetous yeast [29, 30]. One such yeast, *K. marxianus* DMKU 3-1042 isolated in Thailand, shows relatively high ethanol productivity and fermentation ability at high temperatures [31], assimilates various sugars including xylose and/or arabinose [32], and exhibits relatively weak glucose repression on utilization of some sugars including sucrose [33]. Therefore, *K. marxianus* is, in comparison to *S. cerevisiae,* a promising candidate for next-generation bio-ethanol production. In addition, the genomic sequences of *K. marxianus* are available [34, 35], and genetic technology and tools have also been developed [36]. Moreover, *K. marxianus* has been a platform for next-generation protein production for structural and biochemical studies [18, 29]. However, it is possible that unidentified and more beneficial thermotolerant yeasts exist in ASEAN countries, especially, thermotolerant high xylose-utilizing and ethanol-producing yeasts, which are needed for 2nd generation biomass utilization. None of the isolated *K. marxianus* strains, however, are able to more efficiently convert xylose to ethanol than strains of

Potential of Thermotolerant Ethanologenic Yeasts Isolated from ASEAN Countries…

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

123

other xylose-utilizing yeasts, such as *Pichia stipitis* (*Scheffersomyces stipitis*) [32, 37].

as thermotolerant strains, leads to lowest energy consumption and emission of CO2

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

production [41].

**characteristics**

for ethanol production are presented.

Thermotolerant strains allow the development of high-temperature fermentation (HTF) technology, which enables fermentation at 5–10°C higher than the traditional fermentative process [38, 39]. HTF is thus expected to reduce cooling costs, running costs at the simultaneous saccharification and fermentation (SSF) stage, and contamination risks [6, 31, 38–40], therefore offering a promising technology for bio-ethanol production. Moreover, thermotolerant yeast can also be applied for temperature-uncontrolled fermentation, hence offering another economical advantage. A combination of efficient bioreactors and robust hosts, such

In this chapter, we outline a number of thermotolerant yeasts including *K. marxianus* species isolated in Thailand and their characteristics, including utilization of various sugars, glucose repression, and genetic information, that are beneficial for high-temperature fermentation. In addition, new strains of thermotolerant yeasts that have been isolated in Indonesia, Vietnam, and Laos are summarized. Subsequently, the trial results of HTF with some of these strains

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

in biofuel

Recently, thermotolerant microorganisms were found among mesophiles with optimum growth temperatures that are 5–10°C higher than those of the typical mesophilic strains belonging to the same genus or even to the same species [14]. These thermotolerant mesophiles are mainly and widely distributed in foods, plants, soils, and waters from tropical environments in ASEAN countries [15]. In these environments, relatively high temperature presumably becomes a selective pressure to enrich thermotolerant strains. These thermotolerant strains are expected to provide a benefit for the industries because they are more robust and resistant to many stressors [14]. In addition, some of these thermotolerant microorganisms can produce distinctive enzymes that function under relatively high temperature conditions [16– 18]. Thermotolerant yeasts have been found and isolated from a number of countries [19–28]. Of these, *K. marxianus* is a haploid, homothallic, thermotolerant, and hemiascomycetous yeast [29, 30]. One such yeast, *K. marxianus* DMKU 3-1042 isolated in Thailand, shows relatively high ethanol productivity and fermentation ability at high temperatures [31], assimilates various sugars including xylose and/or arabinose [32], and exhibits relatively weak glucose repression on utilization of some sugars including sucrose [33]. Therefore, *K. marxianus* is, in comparison to *S. cerevisiae,* a promising candidate for next-generation bio-ethanol production. In addition, the genomic sequences of *K. marxianus* are available [34, 35], and genetic technology and tools have also been developed [36]. Moreover, *K. marxianus* has been a platform for next-generation protein production for structural and biochemical studies [18, 29]. However, it is possible that unidentified and more beneficial thermotolerant yeasts exist in ASEAN countries, especially, thermotolerant high xylose-utilizing and ethanol-producing yeasts, which are needed for 2nd generation biomass utilization. None of the isolated *K. marxianus* strains, however, are able to more efficiently convert xylose to ethanol than strains of other xylose-utilizing yeasts, such as *Pichia stipitis* (*Scheffersomyces stipitis*) [32, 37].

**1. Introduction**

122 Fuel Ethanol Production from Sugarcane

Worldwide economic growth with the related increase in CO2

causes global warming. Utilization of renewable energy with low CO2

strains have been isolated that may have advantages compared to *S. cerevisiae*.

Recently, thermotolerant microorganisms were found among mesophiles with optimum growth temperatures that are 5–10°C higher than those of the typical mesophilic strains

has been getting increased attention. Renewable energy is generated from renewable natural resources, such as sunlight, wind, rain, tides, waves, geothermal heat, as well as biomass. One such important source of renewable energy, *bio-ethanol*, has been highlighted due to the characteristics of its production from biomass, which is generated by plants using sunlight for CO2 fixation, resulting in carbon neutrality. Bio-ethanol is the name for ethanol produced from biomass by fermentation. This bio-process is thoroughly researched and well-established, and to-date, it produces the most prominent and cost-effective biofuel [1]. Although bio-ethanol production is increasing worldwide and the production of biofuels including ethanol in 2022 is forecasted to be more than 126 billion L [2], biofuels are still more costly than fossil fuels [3]. Therefore, several industrial companies and researchers aim to develop new technologies, enabling the cost-effective production of bio-ethanol from biomass. Since microorganisms are essential for material production through bio-processing, their characteristics and traits are crucial for the production process efficiency. Ethanologenic yeast, *Saccharomyces cerevisiae*, has been traditionally and widely utilized for the production of alcoholic beverages and bioethanol [4, 5]. Industrially common problems in bio-ethanol production related to *S. cerevisiae* strains are temperature level (35–45°C) and high ethanol concentration (over 20%) [6]. These two factors inhibit yeast proliferation and fermentation activity if they reach the upper limit. In addition, for cost-effective bio-ethanol production, the production source must be changed from 1st generation biomass (sugarcane, corn, and wheat, which are important food sources) to 2nd generation biomass (lignocellulosic biomass or woody crops, which are agricultural residues or waste) [7]. Lignocellulosic biomass is composed of hemicellulose, cellulose, and lignin, and the first consists of six (e.g., glucose) and five (e.g., xylose) carbon sugars. However, the low efficiency of ethanol production by *S. cerevisiae* from lignocellulosic biomass hydrolyzates is mainly due to its little ethanol productivity from xylose [8]. Although the *S. cerevisiae* genome encodes all components necessary for xylose utilization, most of them are rarely expressed [9]. In addition, *S. cerevisiae* preferably utilizes glucose while repressing the uptake and catabolism of alternate carbon sources by a mechanism such as glucose repression [10]. This results in the reduction of ethanol production rates from several kinds of biomass. For economically feasible bioethanol production from lignocellulosic biomass, the efficient co-fermentation of glucose and other sugars is also necessary. Therefore, genetic engineering of *S. cerevisiae* strains has been extensively performed, and metabolically engineered strains were developed [11], which have showed higher stress tolerance and/or improved xylose utilization [12, 13]. However, the utilization of genetically recombinant strains in industry has been very limited, especially due to the instability of the desirable phenotype and the necessary confinement to a closed system to prevent their leakage into the environment, which can eventually endanger public health or biodiversity. Therefore, the development of new feasible strains for next-generation bio-ethanol production is under way, and new yeast

emissions from fossil fuels

emission therefore

Thermotolerant strains allow the development of high-temperature fermentation (HTF) technology, which enables fermentation at 5–10°C higher than the traditional fermentative process [38, 39]. HTF is thus expected to reduce cooling costs, running costs at the simultaneous saccharification and fermentation (SSF) stage, and contamination risks [6, 31, 38–40], therefore offering a promising technology for bio-ethanol production. Moreover, thermotolerant yeast can also be applied for temperature-uncontrolled fermentation, hence offering another economical advantage. A combination of efficient bioreactors and robust hosts, such as thermotolerant strains, leads to lowest energy consumption and emission of CO2 in biofuel production [41].

In this chapter, we outline a number of thermotolerant yeasts including *K. marxianus* species isolated in Thailand and their characteristics, including utilization of various sugars, glucose repression, and genetic information, that are beneficial for high-temperature fermentation. In addition, new strains of thermotolerant yeasts that have been isolated in Indonesia, Vietnam, and Laos are summarized. Subsequently, the trial results of HTF with some of these strains for ethanol production are presented.
