**6. Thermotolerant and ethanologenic yeasts in Vietnam**

one group of 2-DOG-resitant mutants with intragenical insertion of *Kan*MX4. This group also exhibits enhanced utilization of xylose in the presence of glucose, presumably due to a defect

On the other hand, Zhou et al. focused on the function of Mig1 in *K. marxianus* and showed that the *MIG1* mutation increased hydrolysis of lactose [133] and production of inulinase [134]. Nevertheless, information on the function of Rag5 as a transcriptional regulator is hardly available, and thus construction of the complete disrupted mutation of *RAG5* and its analysis become a challenge. Thus, disrupted mutants of genes for Mig1 and Rag5 were constructed, and their characteristics were compared with those of the corresponding mutants of *S. cerevisiae*. *MIG1* and *RAG5* mutants exhibited more resistance to 2-DOG in YP plates containing sucrose. *RAG5* and *HXK2* mutants showed more resistant to 2-DOG than the cor-

Several attractive characteristics of *MIG1* and *RAG5* mutants of *K. marxianus* DMKU 3-1042 were uncovered. *MIG1* mutants consumed almost two times faster xylose and accumulated glycerol and xylitol much more than those of the parental strain and the *RAG5* mutant in the liquid media YPX (containing 20 g/L of xylose) and YPDX (containing 20 g/L of glucose and 20 g/L of xylose) at 30°C. The accumulation of glycerol and xylitol may be due to accumulation of NADH. *RAG5* mutants exhibited very slow utilization of glucose in the liquid media of both YPD (containing 20 g/L of glucose) and YPDS (containing 20 g/L of glucose and 20 g/L of sucrose). However, with this mutant, high amounts of fructose (about 11.9 g/L in YPDS at 30°C for 96 h) were accumulated. *MIG1* and *HXK2* mutants of *S. cerevisiae* also accumulated high amounts of fructose in the same medium, but after 12 h, fructose was

The fructose accumulation in *RAG5* mutants is probably due to the inability of this mutant to uptake fructose or the lack of kinase activity. To further analyze this phenom-

*RAG5*-disrupted mutants and the parental strain were measured (**Table 3**) [135]. *RAG5* mutants showed very high activities of inulinase, about 77 times higher than those of the parental strain, but almost no activities of hexokinase and glucokinase that are encoded by *RAG5* and *GLK1*, respectively. The inulinase activity in *RAG5* mutant was consistent with the gene expression level of *INU1*, being about 22 times higher than that of the parental strain. However, the expression level of *GLK1* in this mutant was higher, which was inconsistent with glucokinase activity. It is thus likely that there is a post-transcriptional regulation for glucokinase. *MIG1* mutants showed no significant increase in inulinase activity, but *INU1* transcriptional expression was eight times higher than that of the parental strain. This inconsistence may also be due to post-transcriptional regulation for inulinase. These results suggest that Mig1 and Rag5 are related to the glucose repression mechanism in *K. marxianus* and share some functions with Mig1 and Hxk2, respectively,

In conclusion, Mig1 and Rag5 in *K. marxianus* share some functions with Mig1 and Hxk2, respectively, in *S. cerevisiae*. Mig1 and Rag5 in *K. marxianus* may form a complex similar to that

and gene expression levels of inulinase and kinase in *MIG1*- and

in the glucose-repression mechanism [131].

134 Fuel Ethanol Production from Sugarcane

responding *MIG1* mutants [135].

consumed.

in *S. cerevisiae*.

consisting of Mig1 and Hxk2 in *S. cerevisiae.*

enon, Enzyme activities<sup>a</sup>

In Vietnam, ethanol is a compound in many different products from fermentation technology including alcoholic drinks and biofuel. In the national strategy with a vision to 2025 designed by the government, the technology of biofuel production in Vietnam using the various raw material resources that are abundantly available, e.g., pineapple, cassava, sugarcane, etc., will reach the advanced worldwide level. For the scheme on the development of Vietnam's alcoholic beverages with a vision to 2025, the Mekong Delta is one of the top national areas for the improvement of such products. In addition, nowadays due to global warming, the exploration of thermotolerant yeasts for ethanol fermentation at high temperature also falls in the potential priorities in Vietnam.

#### **6.1. Characteristics of thermotolerant and ethanologenic yeasts**

Recent research studies under international programs, such as the Asian Core Program (2008–2012) and the Core-to-Core Program (2014–2018), have addressed the exploration of useful thermotolerant ethanologenic yeasts isolated from Vietnam and their applications for fermentation technology at high temperature. The diversity of yeast isolates with high capacities and stability for the controlled processing of alcoholic winemaking and ethanol production from cheap and available raw materials in the region has been studied.

A total of 712 yeast isolates were purified from many different kinds of raw material sources in the Mekong Delta, Vietnam, such as ripe fruits, flowers of fruit-tree, cocoa, fermented products, alcoholic fermentation starters, sugarcane, molasses, sawdust, agricultural by-products, and soil samples. All of these yeast isolates could grow well at 37°C and about 80, 45 and 10% of these yeasts could grow at 40, 43 and 45°C, respectively. More than 80% of yeasts were able to grow in a medium containing 9% (v/v) of ethanol, this number decreased to about 40% of yeasts growing in a medium supplemented with 12% (v/v) of ethanol. For conservation, all pure yeast isolates have been stored at −20 and −80°C in stock culture of glycerol freezing broth.

A bank collection of genetically diverse yeasts with thermotolerant ethanologenic capacity at high temperatures was developed and systemized. The full data of morphological, physiological, and biochemical characteristics, as well as the nucleotide sequencing analyses of the 88 selected yeasts, have been established. Some predominantly abundant identified species include *Candida tropicalis*, *S. cerevisiae*, *P. kudriavzevii*, and *C. glabrata* (**Table 4**). Besides, a number of other species was also characterized, such as *Torulaspora globosa*, *Candida nivariensis*, *Pichia manshurica*, *C. lusitaniae*, *Hanseniaspora opuntiae*, and *Meyerozyma caribbica*.

The research findings on the diversified collection of thermotolerant ethanologenic yeasts isolated from Vietnam and the high ethanol yields as well as and fermentation efficiencies by using the selected yeast isolates indicate the promising application of such newly isolated functional thermotolerant yeasts for the controlled ethanol production at high temperatures from agricultural by-products and the winemaking manufacture from different available fruit resources in the region. Further advanced research on the expression levels of the selected genes and the metabolic pathways will be performed to explore the regulation of these genes to get maximum benefits of the superior thermotolerant yeasts for high-temperature ethanol production.

Potential of Thermotolerant Ethanologenic Yeasts Isolated from ASEAN Countries…

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

137

Ethanol production in Lao PDR is generally used for human consumption and household use, rather than for small or large-scale industries. Until now, no ethanol as a substitute of energy in Lao PDR is produced in the industry. The raw material used to make ethanol for drinking is mostly sticky rice and the starter culture used for fermentation contains sticky rice and many other herbs. Drinking alcohol in Lao PDR is available in all provinces, mainly for consumers in their own province. Currently, alcoholic beverages are still very productive and the most popular products to customers are produced in the Saravan province in Meuangkhong district. High quality ethanol used for medicine, hospitals or laboratories are imported from neighboring countries. The National Economic Research Institute under the Ministry of Planning and Investment reported that production of ethanol in 2010–2011 was increased 3.2 times compared to 2001. Lao government plans to develop other sources of renewable energy, which have been investigated by the private sector. Demonstration projects including a bio-diesel oil from Jatropha plant and biofuel (bio-gasoline and bioethanol) from Palm and Carmelina plants have been developed. In 2011, the Savannakhet sugar factory has been established by a Thai company to produce biogas and biomass energy. In 2013, a Vietnam company started a biomass power

Isolation of yeasts was first attempted from fruits, vegetables, leaves and soils in four provinces, Louang Phrabang, Xayaburi, Xiengkhouang, and Vientiane of Lao PDR. The attempt was carried out at 37°C by an enrichment culture. Samples (5–10 g) of fruits pressed in small pieces, leaves cut in small portions, and mashed soil were transferred into 100-mL Erlenmeyer flasks containing 10 mL of YPD (1% yeast extract, 2% peptone and 2% glucose) medium and incubated at 37°C for 3 days with occasional shaking. The cultures were then streaked on YPD agar plates and incubated at 37°C for 24–48 h. As a result, 43 strains were isolated, and their ethanol fermentation ability was characterized under various conditions including different sugars and different temperatures. A second isolation was attempted from similar kinds of samples described above in four provinces, Bolikhamxay, Champasak, Louang Phrabang, and Oudomxay, and 116 strains were obtained after enrichment culture as described above except that 4% ethanol was added in YPD medium. Of a total of 159 strains, 89 were identified by nucleotide sequencing of D1/D2 domains and analysis on MALDI-TOF/ MS [28]. Fermentation experiments allowed to classify them into two groups: the first bears

**7. Thermotolerant and ethanologenic yeasts in Laos**

and ethanol production plant in Phouwong District, Attapeu Province.

**7.1. Characteristics of thermotolerant and ethanologenic yeasts**

With the aim to pave the way for the application of useful thermotolerant ethanologenic yeasts toward industrial fermentation technology, ethanol production, and winemaking by using the selected thermotolerant yeasts, investigations at laboratory-scale and pilot-scale were performed. The optimum fermentation conditions at different temperatures (37, 40, and 43°C) were also tested in a factorial design with three factors including yeast inoculum, initial sugar concentration, and fermentation time. For wine manufacture, different kinds of fruits were employed as raw materials such as: pineapple, watermelon, dragon fruit, guava, jackfruit, rambutan, tangerine, and three-leaved wild vine. The highest ethanol concentration of the final wine product reached about 12% (v/v) and up to 7% (v/v) during the fermentation at 37 and 40°C, respectively. For ethanol production, a number of raw materials were tested including molasses, sugarcane juice, sugarcane waste, and pineapple waste hydrolysate. The highest ethanol concentration could be found at about 7% (v/v) and up to 4% (v/v) during the fermentation at 37 and 40°C, respectively.


**Table 4.** Isolated yeast strains from Vietnam, Laos, and Indonesia.

The research findings on the diversified collection of thermotolerant ethanologenic yeasts isolated from Vietnam and the high ethanol yields as well as and fermentation efficiencies by using the selected yeast isolates indicate the promising application of such newly isolated functional thermotolerant yeasts for the controlled ethanol production at high temperatures from agricultural by-products and the winemaking manufacture from different available fruit resources in the region. Further advanced research on the expression levels of the selected genes and the metabolic pathways will be performed to explore the regulation of these genes to get maximum benefits of the superior thermotolerant yeasts for high-temperature ethanol production.
