Introduction

• Bioethanol was oneof the first fuels used in automobile engine. It was broadly used inGermany during World War 2 and also inBrazil, thePhilippines, and theUnited States. It was replaced by gasoline but soon it was reissued. In recent day, approximately 12 countries use a lot of bioethanol.
• There are a lot of benefits of suing bioethanol. First, it is more efficient than petroleum because it also can produce high octane rating with low cost and it is not a harmful fuel additive.[i] Second, it is biodegradable and the mixture of itself and petroleum can reduce CO₂ emission amount. Third, while we use bioethanol and produce CO₂, the plants which are the main source of bioethanol absorb CO₂ to make cellulose and glucose. Fourth, they burns at low temperature; it is more efficient. Fifth, it will reduce the country’s dependency on high cost petroleum ethanol.
• Basically, theprocess of producing bioethanol is almost similar to the production of eatable alcohol. The bioethanol can be obtained from glucose by fermentation process. There are two way of producing bioethanol. The old version is ethanol produced from grains such as corn or wheat and fermented and distilled into usable grain alcohol. And then, denatured and mixed with gasoline to make E85 or E10. The other new version is bioethanol can be produced from lignocellulosic agricultural waste. Also, this process includes different steps: pre-treatment such as wet-oxidation, acidic hydrolysis, and steam explosion and fermentation. The new way is still developing in order to be competitive to ordinary fuel.  Thegeneral equation of ethanol is C₆H₁₂O₆ → 2C₂H₅OH + 2CO_2.
• However,grass contains cellulose which cannot be fermented by yeast. There are some bacteria that can ferment cellulose. In this project, there will be two bacteria: one is mesophilic celluloytic clostridia and the other is glycolytic clostridia.[ii]
•              Basically, Clostridium (plural, Clostiridia) is a large genus of Gram-positive bacteria that belonging to the Firmicutes which is endobacteria. They are obligate anaerobes capable of producing endospores. It also includes common free-living bacteria as well as important pathogens.[iii]
• Also, Clostridiumacetobutylicum and many other Clostridia can digest whey, sugar, starch, lignin, cellulose fiber and other biomass directly into butanol, propionic acid, ether, and glycerin.[iv]