Fermentation of Sugars

A widely used form of sugar for ethanol fermentation is blackstrap molasses, which contains about 30-40 wt% sucrose, 15-20 wt% invert sugars such as glucose and fructose, and 28-35 wt% of nonsugar solids. The direct fermenta­tion of sugarcane juice, sugarbeet juice, beet molasses, fresh and dried fruits, sorghum, whey, and skimmed milk have been considered, but none of these could compete economically with molasses. From the viewpoint of industrial ethanol production, sucrose-based substances such as sugarcane and sug — arbeet juices present many advantages, including their relative abundance and renewable nature. Molasses, the noncrystallizable residue that remains after sucrose purification, has additional advantages: it is a relatively inex­pensive raw material, readily available, and already used for industrial etha­nol production. Molasses is used in dark brewed alcoholic beverages such as dark ales and also for rum. Bioethanol production in Brazil uses sugarcane as feedstock and employs first-generation technologies based on the use of the sucrose content of sugarcane. The enhancement potential for sugarcane ethanol production in Brazil was discussed by Goldemberg and Guardabassi [17] in the two principal areas of productivity increase and area expansion.

Park and Baratti [18] studied the batch fermentation kinetics of sugarbeet molasses by zymomonos mobilis, a rod-shaped gram-negative bacterium that can be found in sugar-rich plant saps. Z. mobilis degrades sugars to pyru­vate using the Entner-Doudoroff pathway [19]. The pyruvate is then fer­mented to produce ethanol and carbon dioxide as the only products. This bacterium has several interesting and advantageous properties that make it competitive with the yeasts and, in some aspects, superior to yeasts; impor­tant examples include higher ethanol yields, higher sugar uptake, higher ethanol tolerance and specific productivity, and lower biomass production.

When cultivated on molasses, however, Z. mobilis generally shows poor growth and low ethanol production as compared to cultivation in glu­cose media [18]. The low ethanol yield is explained by the formation of by-products such as levan and sorbitol. Other components of molasses such as organic salts, nitrates, or the phenolic compounds could also be inhibi­tory for growth [20]. As such, its acceptable and utilizable substrate range is restricted to simple sugars such as glucose, fructose, and sucrose. Park and Baratti [18] found that in spite of good growth and prevention of levan formation, the ethanol yield and concentration were not sufficient for the development of an industrial process.

In a study by Kalnenieks et al., potassium cyanide (KCN) at submillimolar concentrations (20-500 цМ) inhibited the high respiration rates of aerobic cultures of Z. mobilis but, remarkably, stimulated culture growth [21]. Effects of temperature and sugar concentration on ethanol production by Z. mobilis have been studied by scientists. Cazetta et al. [22] investigated the effects of temperature and molasses concentration on ethanol production. They used factorial design of experiments (DOE) in order to study varied conditions concurrently; the different conditions investigated included varying com­binations of temperature, molasses concentration, and culture times. They concluded that the optimal conditions found for ethanol production were 200 g/L of molasses at 30°C for 48 hours and this produced 55.8 g ethanol/L.

Yeasts of the "saccharomyces genus" are mainly used in industrial pro­cesses for ethanol fermentation. One well-known example is Saccharomyces cerevisiae, which is most widely used in brewing beer and wine. However, S. cerevisiae cannot ferment D-xylose, the second most abundant sugar form of the sugars obtained from cellulosic materials. One micro-organism that is naturally capable of fermenting D-xylose to ethanol is the yeast Pichia sti — pitis, however, this yeast is not as ethanol — and inhibitor-tolerant as tradi­tional ethanol-producing yeast, that is, Saccharomyces cerevisiae. Therefore, its industrial application is impractical, unless significant advances are made. There have been efforts that attempt to generate S. cerevisiae strains that are able to ferment D-xylose by means of genetic engineering [23]. Scientists have been working actively to ferment xylose with high productivity and yield by developing variants of Z. mobilis that are capable of using C5-sugars (pen­toses or xyloses) as a carbon source [24]. Advances with promising results are being reported in the literature.

As a significant advance in metabolistic changes brought about by genetic engineering, Tao [25] altered an Escherichia coli B strain, which is an organic acid producer, to E. coli strain KO11, which is an ethanol producer. The altered KO11 strain yielded 0.50 g ethanol/g xylose using 10% xylose solu­tion at 35°C and pH of 6.5. This result provides an example of how the output of a microbe can be altered.

Utilizing a combination of metabolic engineering and systems biology techniques, two broad methods for developing more capable and more toler­ant microbes and microbial communities are the recombinant industrial and native approaches [26]. The two methods differ as follows:

1. Recombinant industrial host approach: Insert key novel genes into known robust industrial hosts with established recombinant tools.

2. Native host approach: Manipulate new microbes with some complex desirable capabilities to develop traits needed for a robust industrial organism and to eliminate unneeded pathways.

The research on yeast fermentation of xylose to ethanol has been very actively studied; particular emphasis has been placed on genetically engi­neered Saccharomyces cerevisiae. S. cerevisiae is a safe micro-organism that plays a traditional and major role in modern industrial bioethanol produc­tion [27]. Saccharomyces cerevisiae has several advantages including its high ethanol productivity as well as its high ethanol and inhibitor tolerance. Unfortunately, this yeast does not have the capability of fermenting xylose. A number of different strategies based on genetic engineering and advanced microbiology have been applied to engineer yeasts to become capable of efficiently producing ethanol from xylose. These novel strategies included: (a) the introduction of initial xylose metabolism and xylose transport, (b) changing the intracellular redox balance, and (c) overexpression of xylulo — kinase and pentose phosphate pathways [27]. One of the pioneering studies involves the development of genetically engineered Saccharomyces yeasts that can co-ferment both glucose and xylose to ethanol by Sedlak et al. [28]. Even though their recombinant yeast Saccharomyces cerevisiae with xylose metabolism added was found to be the most effective yeast, they still utilized glucose more efficiently than xylose.

According to their experimental results, following rapid consumption of glucose in less than 10 hours, xylose was metabolized more slowly and less completely. In fact, xylose was not totally consumed even after 30 hours. Ideally, xylose should be consumed simultaneously [26] with glucose at a similar efficiency and speed; however, the newly added capability of co­fermentation of both glucose and xylose was a ground-breaking discovery. Furthermore, they found that although ethanol was the most abundant product from glucose and xylose metabolism, small amounts of the meta­bolic by-products of glycerol and xylitol also were obtained [28]. The above
two issues, viz. higher efficiency for xylose fermentation and optimization and by-product control, are the subjects of intense research investigation.