Various microorganisms are capable of hydrolyzing starch, though a preliminary process called gelatinization is needed to ensure an efficient hydrolysis. During this preliminary process, the starch granules swell, particularly rupturing the hydrogen bonds in the crystalline regions. The long glucose chains comprising the starch must be converted into fermentable sugars by means of a process called the "hydrolysis technique", during which the starch reacts with the water normally used to break down the starch into its fermentable sugars. There are numerous microorganisms capable of hydrolyzing starch, but those involved in the starch degradation process are generally amylase, a-amylase, P-amylase and isoamylase. The most important for the purposes of the SSF process are certainly the first two. a-amylase is an endo-amylase that randomly attacks the a-1,4 bonds, rapidly reducing the starch molecule’s dimensions and consequently also its viscosity, i. e. it liquefies the starch. a-amylase can be obtained by means of heat-resistant bacteria such as Bacillus licheniformis, or by means of new strains of Escherichia coli or Bacillus subtilis, used on the starch suspensions during the first hydrolysis stage. For amylase to succeed in attacking these suspensions, they must be brought up to high temperatures (90-110°C) to rupture the starch cell nuclei. The products of this preliminary hydrolysis phase, called liquefaction, is a solution containing dextrins and a small amount of glucose.

At this point, the liquefied starch undergoes saccharification at low temperatures (60-70°C), induced by the action of glycoamylase generally obtained from Aspergillus or Rhizopus species. This enzyme is an exo-amylase capable of producing units of glucose from amylose and amylopectin chains.

The factors that influence starch hydrolysis include the substrate, enzyme activity and the reaction conditions (temperature, pH and other parameters) (Prasad et al., 2007). The microorganisms take effect more easily on gelatinized starch, but this process demands large amounts of energy so on an industrial level there has been a tendency to focus on using microorganisms capable of growing on ungelatinized starch. Various studies on this issue have considered certain species of fungi for producing enzymes capable of degrading starch in its natural state (Soccol et al., 1994). Liquefaction is followed by a saccharification stage under the effect of glycoamylase.

1.11.1 Milling

The milling phase enables the starch to be extracted from the biomass and it is very important for the purposes of analyzing the bioethanol production process as a whole because it strongly influences not only the subsequent stages but also the co-products obtained at the end of intermediate stages, which also vary according to the specific raw material entering the process (wheat, barley, corn, oats). The two main options are wet milling and dry milling.

Wet milling is the standard procedure generally used in the starch-based foodstuffs industry. Though this procedure demands more energy and more economic resources, and it delivers a smaller quantity of ethanol, it is still preferred at industrial level because its capacity to separate the grain into its components enables a purer form of starch to be obtained, along with more valuable byproducts. Wet milling can be used to obtain not only ethanol, but also products such as corn oil, gluten-based foods and flour, and corn steep liquor (CSL).

Dry milling means there is no need to pre-treat the raw material, which simply has to be ground before going through the other processing stages (hydrolysis, fermentation, distillation), which are identical to those following the wet milling process. Because dry milling does not break down the cereals into their various components, the unfermentable residue leaving the process that extracts the ethanol from the fermentation broth is rich in proteins, fibers, fats and sugars.