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Ethanol is produced when yeast cells break down and metabolise sugar to produce ethanol and carbon dioxide as waste products. The fermentation process is a main role in production of many different commercial items including beer, lagers, wine, spirits, biofuel and bread.
Fermentation occurs when yeast anaerobically metabolises sugars to produce ethanol and carbon dioxide as waste products. Mono-saccharide sugars are most commonly used in the fermentation process as these are easiest to break down. Sucrose, a di-saccharide sugar can be used in the fermentation process but relies on an additional step converting it to fructose and glucose.
The Di-saccharide sugar must first be broken down into a mono-saccharide sugar by the enzyme invertase;
C12H22O11 + H2O + Invertase â†’ 2(C6H12O6)
(Disaccharide sugar) + (water) + Invertase â†’ 2 (mono-saccharide sugars)
The sugar is converted to pyruvate using the hydrogen carrier NADH which is present in the yeast;
C6H12O6 + 2NAD+ â†’ 2CH3-CO-COOH + 2NADH + 2H+
(Sugar) + 2NAD+ â†’ (Pyruvate) + 2NADH + 2H+
The enzyme zymase, which can be found in the yeast cell, catalyses the conversion of pyruvate into ethanol and carbon dioxide using the hydrogen carries NADH
CH3-CO-COOH + NADH + H+ + Zymase â†’ C2H5OH + CO2 + NAD+
(Pyruvate) + NADH + H + + Zymase â†’ (Ethanol) + (Carbon Dioxide) + NAD+
If oxygen is present during the metabolism of sugar, most species will completely convert the sugar to carbon dioxide and water by the following process;
C6H12O6 + 6 O2 â†’ 6CO2 + 6 H2O
(Sugar) + (oxygen) â†’ (Carbon Dioxide) + (water)
Batch and Continuous Fermentation
Traditionally, industrial fermentation for the production of alcoholic beverages, particularly beer, is carried out in a closed bioreactor. The reactor vessel is filled with water, yeast cell nutrients, a carbon source and immobilised or suspended yeast cells. Batch fermentation is costly, time consuming and labour consuming, with low production rates particularly at large scale, industrial levels (Baptista, et al. 2007). Although batch fermentation is the preferred method for beer fermentation, it is unsuitable for producing high ethanol concentrations in large scale fermentations, such as, fermenting ethanol for bio fuel (Montealergre, et al 2012).
In continuous fermentations, an open reactor system is used. There is a constant flow of nutrients, carbon sources and water into the bioreactor and an equal amount of fermentation products removed from the system.
Continuous fermentation is a great deal quicker than batch fermentation, with an end product being achieved after only three or four days compared with thirty days as required with batch fermentation (Branyik T. et al. 2006). The continuous fermentation processes is not yet widely used in large scale industry as is yet to achieve the characteristics enabling it to outperformed batch fermentation. There is a main focus in current research is being carried out towards find a continuous fermentation method which has a simple design, low costs and effective process control methods for use in large scale industry. (Branyik T. et al. 2006).
Many research groups are currently working towards finding a continuous fermentation process which can be used in a large scale. One problem which has been experienced in continuous fermentation systems which use freely suspended yeast cells is the decrease or complete loss of yeast cells in the flow of end products out of the reactor. As a result of this, continuous fermentation systems are limited to low flow rates to prevent a loss of cells, reducing the process efficiency. (Montealergre, et al. 2012).
By introducing the use of immobilised yeast cells, continuous fermentation systems will be able to operate at higher flow rates which will reduce the time required for fermentation. Immobilisation is also thought to reduce cell sensitivity to fermentation factors such as pH, oxygen concentration and toxin levels; this depends on which method of immobilisation is used (Montealergre, et al. 2012).
There are many different immobilisation methods which are currently used in fermentation processes. These methods can be categorised into either active methods or passive methods...
Active Cell Immobilisation
Active methods of immobilisation are those which involve the use of a chemical agent including methods which involve; covalent bonding, cross linking and entrapment of
Covalent bonding requires chemical modification of the immobilization medium which requires chemicals and additional cost of immobilization.
. In crosslinking, chemical agents are used to bind the cells and the immobilization medium. These chemical agents, such as glutaraldehyde, find limited applications in cell immobilization due to their toxicity
In matrix encapsulation, cells are trapped in a polymer matrix of materials such as alginate and carrageenan. Sodium alginate forms a viscous solution with water which hardens when reacted with some metal salts like calcium
Entrapment in CA is commonly used in studies with ethanol fermentation due to "the requirement for mild conditions and the simplicity of the used procedure" (Verbelen, et al. 2006)
Passive Cell Immobilisation
Passive methods of cell immobilisation are those which involve no chemical agents, such as; flocculation, colonization and adsorption
flocculation, colonization and adsorption. Thus chemical reagents are unnecessary and the cost of immobilization is low but there is a possibility for cell detachment with changes in the environment of the cell and the immobilization medium.
Flocculation of cells occurs for several reasons including growth, interaction with other cells and as a response to their surroundings. Flocculation may be induced by chemically modifying the cells' immediate environment.
In colonization, porous biomass support particles are used to provide interconnecting voids within an open network of matrix support materials.
In surface adsorption, cells adhere to surfaces due to electrostatic forces hence immobilized.
Nata de Coco Biocellulose
Sparkling wine is usually produced by "method Champenoise" - fermentation in the bottle of white wine to which sugar (up to 24g/L) and yeast are added. Once the fermentation is complete yeast cells are removed from the bottle by mechanically twisting and inclining the bottle until all the yeast cells are in the neck of the bottle. The bottleneck is iced and the portion containing yeast is ejected by internal gas pressure.
The final step - removing the yeast- can take several weeks, involves a considerable amount of labour and needs a significant number of cellars devoted to it. Several alternatives are being studied; chemical additives, use of flocculent yeast strains and immobilised yeast are some of these.
the use of immobilised cells has been studied in depth in recent years with only a few publications on this. A main drawback was the release of the cells from the beads causing turbidity. One way to avoid this is to cover the beads with an external coating which is free from microorganisms.
Apparent enhancement of final wine quality from immobilised beads should be deeply characterised
Industrial application of this technology would need different new developments, eg. Scaled up of bead making devices. Bead dosage machines, and complete absence of yeast in the base wine as well as sterility in some other areas of the process. (Godia. F, et al. 1991)
Application of cheap carrier materials in a suitably designed bioreactor will substantially lower the investment costs of continuous fermentation. Brewers spent grains- usually used as animal feed- are a cheap food grade by-product of the brewing industry which can be used as a carrier for immobilised cell technologies.
Corncobs are large volume solid waste that results from sweet corn processing - used as animal feed or land application. Less commonly for production of value-added products eg. Reducing sugars, ethanol. Adsorbent for wastewater treatment, microbial carriers in bioremediation, additives in thermoplastic polymers, source of biodegradable plastics. - Potential carrier for microbial fermentation process. (Branyik T. et al. 2006)
Yeast directly ferments glucose and fructose to produce ethanol. To ferment sucrose, yeast must use the enzyme invertase to convert sucrose to glucose and fructose. (Eiadpum. A, et al. 2012)
Tropical countries which usually have high day-time temperatures, for example Thailand which is between 30degrees and 36 degrees pay a great deal of money on cooling systems for their fermentation process. Therefor ethanol fermentation at higher temperature has received much attention. High temperatures have advantages of increased productivity, reduced cooling costs and reduced risk of contamination.
Previous studies suggest Saccharomyces cerevisiae is capable of producing ethanol in blackstrapmolasses medium at operating temperatures of 30-35 degrees however activity is supressed at temperatures above 37 degrees.
For kluyveromyces marxianus, it has been found effective in producing ethanol at elevated temperatures of up to 45 degrees when sugarcane was used as a raw material.
Cell immobilisation is used to increase the rate of ethanol production and to protect cells from inhibiting factors.
Efforts for improved fermentation with co-cultures have been reported. Co-cultures have been reportedly used in ethanol fermentation from low cost materials such as cellodextrins, hemicellulose dextrins, sorghum carbohydrates and starches which are not easily converted into ethanol by monocultures.
Cells and co-cultures of these cells were immobilised using trisuccinimidyl citrate (TSC and loofa-reinforced alginate matrix ALM.
Suspended and immobilised cells were used in continuous and batch fermentation processing with traditional industrial raw materials, blackstrap molasses and sugarcane juice.
Therefor a co-culture can increase production of ethanol over a wider range of temperatures in both sugarcane and blackstrap molasses.
Typical fuel fermentation is done using mesophilic yeast under controlled condition of temperatures varying between 30-35 degrees. Fermentation cooling costs impact fuel price. With a higher fermentation temp, fuel production costs can be reduced . this study showed that co-culture enhanced thermal stability and extended operating temperature ranges which would help reduce heating and cooling costs. (Eiadpum. A, et al. 2012)
Well known technical and economic advantages of immobilised cells compared to free cell systems.
Industrial use is limited to production of sparkling wine
Recent research has been focused on supports of food grade purity due to demand for 'clean; and natural products. Foods such as gluten pellets, brewers spent grains quince, dried raisin berries, potatoes, corn starch gel, and wheat grains have been proposed for supports for immobilised yeast.
Immobilised cells have been found to be suitable for rapid fermentation at ambient or extremely low temperatures producing wine with fine and distinct characteristics.
Immobilised cells on starchy supports in winemaking, especially at low temperatures, led to wines with improved tastes and aromas, while the reduced activation energy and the higher reaction rate are constant. Supports may behave as catalysts or promoters of the enzyme involved in the process.
Using alcohol-resistant and cryotolerant Saccharomyces cerevisiae AXAZ-1, isolated form a greek vineyard plantation.
First use of whole grain corn as supports for yeast cell immobilisation, examining their feasibility for repeated batch fermentations of glucose and grape at various temperatures. - available area of immobilisation is increased making it possible to immobilise more cells per grain.
Corn grain provided a suitable support for the immobilisation of yeast cells. Corn-supported biocatalyst proved suitable for wine-making over a wide range of temperatures producing quality wines with great aroma as was proved by GC-MS analysis. (Kandylis. P, et al. 2012)