In the industry whereby ethanol is produced from starch, temperature around 140°C-180°C is applied to cook the starch during hydrolysis using a-amylase prior to liquefaction. This high-temperature completely sterilizes harmful microorganisms and increases the efficiency of saccharification for high ethanol yield (Shigechi et al, 2004a, b). Consequently, this resulted in high energy consumption and added cost to amylolitic enzymes used in the process which ultimately increased the overall production cost. Several methods have been developed to reduce the energy consumption by applying milder liquefaction and/or saccharafication temperatures (Kolusheva and Marinova, 2007; Majovic et al., 2006; Montesinos and Navarro, 2000; Paolucci-Jeanjean et al, 2000) and also by exercising non­cooking fermentation (Shigechi et al., 2004b; Zhang et al, 2010). However, these types of fermentation usually required longer process time and sometimes may demand for additional volume of enzyme to maintain same productivity. The cost of enzyme will upset the total process cost.

To overcome this shortcoming, an alternative method of direct fermentation from starch may be employed to reduce the cost of enzyme. However, there are relatively few fermentation microorganisms that are capable of converting starch directly to ethanol since they do not produce starch-decomposing enzymes. One of the attempts to resolve this problem is by constructing recombinant microbes to coproduce a-amylase and glucoamylase with incorporating low temperature cooking of starch prior to fermentation by many research teams as shown in Table 3.

Several investigators reported that direct fermentation of starch using amylolytic microorganism offers a better alternative to the conventional multistage using commercial amylases for liquefaction and saccharification followed by fermentation with yeast (Abouzied and Reddy, 1986; Verma et al., 2000; Knox et al., 2004). By using this amylolytic microorganism in direct fermentation, the ethanol production cost can be reduced via recycling some of the microorganism back to fermentors, thereby maintaining a high cell density, which facilitates rapid conversion of substrate into ethanol. Furthermore by using cell exhibiting amylolytic activities, unlike using liquid enzyme that needs to be replenished or recycled unless if it is in immobilized system, the cell can multiply and reproduce with the enzymes. Fermentation using recombinant microbes, the starch medium can be prepared at low temperature cooking or uncook as a raw starch.

Another attempt of the direct fermentation without utilising any enzyme is by using co­culture microbes in the process. Instead of having enzyme separated and purified in different processes and subsequently to be used for hydrolysis in another separated process

which contribute to higher expense, co-culture fermentation is worth to be considered as it might reduce the cost by omitting the unnecessary steps. While recombinant microorganism is constructed to provide the amylase activities, co-culture is simply selecting the microorganisms that naturally possess these amylase activities and combine them to work together to produce ethanol from starch.

Not many research works dedicated and related to co-culture fermentation for direct bioconversion of starch to ethanol. From just a few, same conclusions were drawn on the fermentation yield of the co-culture was better compared to mono-culture with improvement in the ethanol fermentation process. For instance study done by Verma et al. (2000), the co-culture fermentation of liquefied starch to ethanol can be carried out effectively with fermentation efficiency up to 93% compared to 78% and 85% when two-step bioconversion process using a-amylase and glucoamylase were applied to hydrolyze starch. Abuzied and Reddy (1986) observed that higher cell mass was produced in monoculture than in co-cultures which suggesting that substantially more carbon is used for cell production in monoculture, whereas in the co-culture most of the substrate carbon is utilized for ethanol production. Studies on co-culture microorganisms and systems are summarized in Table 4. The co-culture fermentation can either be simultaneous or subsequent mode for direct fermentation of low-temperature-cooking starch.

Strains for co-culture fermentation can also be obtained inexpensively from dry starter such as Ragi Tapai or Ragi Tape. This is similar to other oriental starter such as Ragi in Malaysia and Indonesia, Bubod in Philipine, Loog-pang in Thailand, Nurok in Korea, Koji in Japan, Banh Men in Vietnam, Chinese yeast in Taiwan and Hamei and Marcha in India. It is a dry — starter culture prepared from a mixture of rice flour and water or sugar cane juice/extract (Merican and Yeoh, 2004, Tamang et al., 2007). Clean rice flour is mixed with water or sugar cane juice to form thick paste. Sometime spices such as chilies, pepper, ginger and garlic which are assumed to carry desirable microorganism or may inhibit the development of undesirable microorganism are added to the paste (Basuki et al., 1996; Merican and Yeoh, 2004). Then the thick paste is shaped into hemispherical balls. Ragi from previous batch is inoculated either on thick paste before or after it is shaped into hemispherical balls. Hesseltine et al. (1988) reported that at least one yeast and one Mucoraceous mold (Mucor, Rhizopus, and Amylomyces) were present with one or two of cocci bacteria in every sample of the dry starter. Apart from the Rhizopus sp. which capable of producing lactic acid besides fermentable sugar and ethanol (Soccol et al., 1994), lactic acid bacteria are among the integral of the dry starter such as Pediococcus pentosaceus, Lactobacillus curvatus, Lactobacillus plantarum and Lactobacillus brevis (Sujaya et al., 2002; Tamang et al., 2007).

The traditional fermented food of tapai or tape’ usually contains ethanol at concentration of 1.58% with high sugar content at concentration of 32.06%. Microaerophilic condition is required for the fermentation condition since fungi are unable to grow under anaerobic conditions and will result in unhydrolyzed starch. At lower temperature of 25°C, higher alcohol content will be produced after 144 h whereas at temperature of 37°C the tapai produces higher sugar content and becomes sweeter. (Merican and Yeoh, 2004). Tapai may contain up to 5% (v/v) of ethanol concentration (Basuki et al., 1996).

The benefit of using strains from dry starter such as ragi is that its application to produce fermented food such as tapai, is proven edible. Moreover, with addition of S. cerevisiae into the medium, the residue from ethanol recovery will contain yeast extract which can be processed as animal feed since it is edible and contain valuable nutrient that suitable for animal consumption as compared to fermentation using microbe such as Escherichia coli. Direct fermentation has several advantages. First, to have multistage processes carried out in one reactor in which the glucose is produced during saccharification and simultaneously is fermented to ethanol can reduce contaminations and process handling cost. Second, direct fermentation reduces energy consumption. The starch medium can be prepared either at low-cooking temperature or by using the raw starch (uncooked starch). Even though some aseptic chemical or method may be required especially in raw starch fermentation, the cost incurred is still lower than the cost of energy consumption used in conventional fermentation.

Third, by applying direct fermentation, it is able to reduce inhibition of reducing sugar on fermenting yeast. In conventional fermentation, when starch is hydrolyzed using enzyme or mineral acid, certain amount of reducing sugar will be produced depending on the starch concentration. High level of reducing sugar in the fermentation medium (above 25% (w/ v)) exerts osmotic pressure to the cells and limits their fermenting activity. This value may vary with different fermenting yeasts. However in direct fermentation, the osmotic pressure can be reduced by simultaneous converting starch to sugar and sugar to ethanol. This is particularly true in the recombinant clone which can co-express both the degrading enzymes. In the case of co-culture fermentation, the suitable inoculation time for the second microorganism needs to be determined. This is to avoid high yield of reducing sugar in

medium before the second inoculation. When reducing sugar inhibition is avoided, fermentation of high starch concentration can be achieved for high ethanol yield and thus it reduces the water use. Subsequently this will reduce energy consumption in ethanol-water separation.

Direct fermentation is not limited to starch as it had been reported that different sugars from lignocellulosic hydrolysates such as mixture of glucose and pentose sugar for instance; xylose (Murray and Asther, 1984; Kordowska and Targonski, 2001; Qian et al., 2006) were fermented by glucose and pentose-fermenting microorganisms.