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

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*,

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.

**No Isolated yeast species Vietnam Laos Indonesia**

1 *Blastobotrys adeninivorans* 2 2 *Candida glabrata* 7 2 3 *Candida manshurica* 2

5 *Candida stellimalicola* 1

8 *Cyberlindnera rhodanensis* 2

16 *Saccharomyces cerevisiae* 19 1

6 *Candida tropicalis* 16 26 16

10 *Issatchenkia orientalis* 1 11 *Kluyveromyces marxianus* 6 3

13 *Meyerozyma guilliermondii* 2 14 *Pichia kudriavzevii* 35 47 1

> Not identified 624 70 56 Total 712 159 79

4 *Candida nivariensis* 4

136 Fuel Ethanol Production from Sugarcane

7 *Clavispora lusitaniae* 1

9 *Hanseniaspora opuntiae* 1

12 *Meyerozyma caribbica* 1

15 *Pichia manshurica* 2

17 *Torulaspora globosa* 2

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

*Pichia manshurica*, *C. lusitaniae*, *Hanseniaspora opuntiae*, and *Meyerozyma caribbica*.

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 and ethanol production plant in Phouwong District, Attapeu Province.

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

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 an ethanol-fermenting ability at high temperature (116 strains) and the second the converting ability of xylose to ethanol at 37°C or more (43 strains). In fermentation of ethanol, the first group can use glucose, sucrose, sugar cane juice, and molasses as carbon sources, producing a maximum of ethanol concentrations of 7.9% (w/v), 6.7% (w/v), 7.3% (w/v), and 4.0% (w/v) from 16% sugar concentration, respectively. The second group produced 1.2–1.7% (w/v) ethanol from 4% xylose at 37°C. Species identification revealed that isolates include nine species including *C. tropicalis, P. kudriavzevii*, and *K. marxianus* (**Table 4**).

some obstacles such as limitation of fuel grade ethanol market, inconsistency supply, insufficient

Potential of Thermotolerant Ethanologenic Yeasts Isolated from ASEAN Countries…

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

139

In international programs including the e-ASIA Joint Research Program, yeast strains were isolated from various samples such as soils, waters, flowers, fruits, vegetables, and fermented foods. The isolation method for thermotolerant and ethanol-producing yeast was similar to that applied in Lao PDR. The enrichment culture was carried out in YPD medium without the addition of ethanol. Most of the isolates can grow at relatively high temperatures ranging from 37 to 48°C. Of those, 52 yeast isolates grow well at 37°C on agar plates containing different types of sugar, such as glucose, xylose, and sucrose. Some can produce around 6% ethanol in a rich medium containing 16% (w/v) glucose at 40°C. These prominent characteristics are

Most yeast strains isolated from Indonesia are able to grow at relatively high temperatures not only in glucose medium but also in xylose and sucrose. However, their growth gradually decreases as temperature increases and is very weak at more than 45°C. Indonesian yeast isolates from fruits and fermented foods seem to be more thermotolerant than those from soils and waters. Most of the isolates grow very well at 40°C. These isolates include *C. tropicalis, K.* 

**9. High-temperature fermentation technologies with thermotolerant** 

**9.1. Temperature-noncontrolled fermentation with thermotolerant yeast**

Currently, biofuel-aimed ethanol fermentation in industry is performed at around 30°C because the most frequently applied yeast is nonthermotolerant *S. cerevisiae*. In the fermentation process, the temperature in the fermenter increases close to a nonpermissible level for the yeast by metabolic and mechanical heat sources. A cooling system with a large amount of water and/or by a cooling unit is equipped for effective fermentation. The cooling cost tends to be higher in tropical countries or increases in summer time in other many countries, and the electricity problem largely affects productivity of ethanol. The HTF using a thermotolerant microbe is expected to provide several advantages. First, it can reduce the cooling cost. Second, the amount of enzyme used for saccharification can be reduced in the simultaneous saccharification and fermentation at higher temperature. Third, higher temperature causes lower contamination by various germs. Fourth, when the distillation under reduced pressure is applied at around 40°C, fermentation and distillation can be performed by one tank, which reduces the manufacturing time and the cost of equipment. Here, we introduce a fundamental research for an energy-saving fermentation technology using thermotolerant yeast.

For development of the fermentation technology, *K. marxianus* DMKU 3-1042 was used, which efficiently produces ethanol at high temperatures as mentioned above [32, 33]. The utilization

demand, and price volatility, there is almost no fuel ethanol production since 2010 [136].

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

important for the development of bioethanol production in Indonesia.

*marxianus* and *P. kudriavzevii* (**Table 4**).

**yeast**

#### **7.2. Characteristics of newly isolated** *K. marxianus* **strains**

Out of six isolated *K. marxianus* strains, BUNL-17 was found to be the most efficient ethanol producer at high temperature [28]. Comparison with DMKU 3-1042, which is one of most thermotolerant *K. marxianus* strain isolates from Thailand, revealed that BUNL-17 possesses an efficient conversion activity of xylose to ethanol, resistance to 2-deoxyglucose and tolerance to various stresses including temperature, high sugar concentration, and hydrogen peroxide [37]. Compared to *S. stipitis* the fermentation activity toward xylose of BUNL-21 is slightly lower at around 30°C and much higher at higher temperatures. BUNL-21 is thus a highly competent yeast for high-temperature ethanol fermentation with lignocellulosic biomass. Interestingly, the fermentation activity was shown to be significantly enhanced by over-expression of *KmADH2* for alcohol dehydrogenase 2 [37].
