**8. Thermotolerant and ethanologenic yeasts in Indonesia**

Ethanol production in Indonesia is generally performed for medical, industrial processes, and beverages. Several potential biomass resources for bioethanol production in Indonesia are (1) sugar-based materials including sugar cane (molasses), (2) starch-based including root (cassava and sweet potato) and grain (corn and sorghum), and (3) lignocellulosic-based including bagasse, straw, stalk, wood waste, corn cob, and sap of several plants or trees. The main biomass used for bioethanol production in Indonesia is molasses [136] probably because Indonesia is one of the largest sugarcane producers in the world. Annual cane production in Indonesia is about 32–35 million tons with an average cane productivity of 70–85 ton/ha. Sugar production is about 2.2–2.7 million tons, including molasses with about 1.3–1.5 million tons. Molasses are mainly used for monosodium glutamate production in the ethanol industry and for export to other countries [137].

Bioethanol development for fuel in Indonesia was started from 2006. Its road map until 2010 showed production of 99.5% ethanol as a fuel grade ethanol (FGE), which can be mixed with petroleum for gasohol E10 (10% ethanol and 90% petroleum). For the first period, biomass used for bioethanol production was molasses and cassava and bioethanol supply was about 1.48 mil kL (million kiloliters) or equal to 10% of total gasoline consumption. In the period 2011–2015, bioethanol supply was estimated to increase to 2.78 mil kL or equal to 15% of total gasoline consumption. Until 2025, bioethanol supply is predicted to be 6.28 mil kL or 20% of total gasoline consumption [138]. The application of bioethanol for fuel in Indonesia is E5, and only two bioethanol filling stations are operating in two cities, Malang and Semarang [139]. However, because of some obstacles such as limitation of fuel grade ethanol market, inconsistency supply, insufficient demand, and price volatility, there is almost no fuel ethanol production since 2010 [136].

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

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

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

Ethanol production in Indonesia is generally performed for medical, industrial processes, and beverages. Several potential biomass resources for bioethanol production in Indonesia are (1) sugar-based materials including sugar cane (molasses), (2) starch-based including root (cassava and sweet potato) and grain (corn and sorghum), and (3) lignocellulosic-based including bagasse, straw, stalk, wood waste, corn cob, and sap of several plants or trees. The main biomass used for bioethanol production in Indonesia is molasses [136] probably because Indonesia is one of the largest sugarcane producers in the world. Annual cane production in Indonesia is about 32–35 million tons with an average cane productivity of 70–85 ton/ha. Sugar production is about 2.2–2.7 million tons, including molasses with about 1.3–1.5 million tons. Molasses are mainly used for monosodium glutamate production in the ethanol indus-

Bioethanol development for fuel in Indonesia was started from 2006. Its road map until 2010 showed production of 99.5% ethanol as a fuel grade ethanol (FGE), which can be mixed with petroleum for gasohol E10 (10% ethanol and 90% petroleum). For the first period, biomass used for bioethanol production was molasses and cassava and bioethanol supply was about 1.48 mil kL (million kiloliters) or equal to 10% of total gasoline consumption. In the period 2011–2015, bioethanol supply was estimated to increase to 2.78 mil kL or equal to 15% of total gasoline consumption. Until 2025, bioethanol supply is predicted to be 6.28 mil kL or 20% of total gasoline consumption [138]. The application of bioethanol for fuel in Indonesia is E5, and only two bioethanol filling stations are operating in two cities, Malang and Semarang [139]. However, because of

including *C. tropicalis, P. kudriavzevii*, and *K. marxianus* (**Table 4**).

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

138 Fuel Ethanol Production from Sugarcane

over-expression of *KmADH2* for alcohol dehydrogenase 2 [37].

try and for export to other countries [137].

**8. Thermotolerant and ethanologenic yeasts in Indonesia**

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 important for the development of bioethanol production in Indonesia.

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