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 (1) 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 mono-saccharide sugar is converted to pyruvate using the hydrogen carrier NADH which is present in the yeast;
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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)
In the presence of oxygen, ethanol will not be produced and yeast will multiply in the solution.
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 (Montealergre, et al. 2012). As a result of this, continuous fermentation systems are limited to low flow rates to prevent a loss of cells. This limitation reduces the overall efficiency of the process by the decrease in flow rate which has an effect on the speed of the process and subsequently many industrial factors such as energy consumption, and labour costs.
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, giving a more efficient process. The use of immobilised cells presents many benefits including increased stability and 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).
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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 cell immobilisation are those which involve the use of a chemical agent, most commonly those involving; covalent bonding, cross linking and entrapment of cells.
Covalent bonding occurs between the proteins present on the cell wall and the chemically treated support. The use of covalent bonding for immobilisation of cells is a widely used technique which produces a very stable bond between the cell and the support used; therefore the cells will not be released into the solution. However, this requires chemical modification of the support which may be costly dependant on the method used.
An example of covalent bonding immobilisation can be seen in a 2012 study by T.H Tran which demonstrates the use of the modified polymer, polytetrafluoroethylene for immobilising Saccharomyces cerevisiae. This was carried out using a plasma immersion ion implantation technique in which the polymer is immersed in nitrogen plasma to which a radio frequency was applied. The method creates active radicals on the surface on the polymer by use of accelerated ions from the plasma. Immobilisation of proteins occurs from a covalent bond which occurs between the proteins in the cells and the unpaired, mobile electrons on the polymer surface. (Tran, et al. 2012)
In a study by Fahmy, et al.(1997) demonstrates the use of diethylaminoethyl cellulosetreated which has been with cyanuric chloride in cell immobilisation. The cyanuric chloride depolymerised the cellulose structure, breaking down the long chain polymers into shorter hydrocarbons which allows it to covalently bond with proteins on the cell membranes (Fahmy A.S, et al. 1998). This method is used in several different studies where yeast cells are covalently bound to depolymerised cellulose (Van Iersel, et al. 2000).
Cross linking is a covalent bond immobilisation method has the additional use of chemical agents such as gluteraldehyde or diazonium salt, to encourage the bonds. Cross linking is a cheap and simple technique however, is not often used due to the severe environment required for the process producing toxic conditions and significantly reduced cell activity.
A 2004 study by Kabul demonstrated the immobilisation of whole cell catalase by cross linking catalase from yeast cells with the white of a hen egg with the use of gluteraldehyde with aims of maintaining enzyme activity. In this study, production of catalase in Saccharomyces cerevisiae was induced before treating the cells with toluene to permeabilise them for release of the catalase enzyme. Saccharomyces cerevisiae cells were mixed with the hen egg white then treated with gluteraldahyde and left to harden at 4Â°C. The results of this study showed potential for further research in the use of hen egg white for immobilisation as the non-toxicity of the egg white allowed for immobilisation with little loss of enzyme activity. (Kabul & D'Souza. 2004)
The cell entrapment technique used for the immobilisation of cells involves the entrapment of cells within a porous matrix. The main way this technique is carried out is by synthesising the matrix around the cells. Often used matrixes in this process are polysaccharide gels such as calcium alginate, Ðº-carrageenan and chitosan (Ramakrishna & Prakasham. 1999), other polymeric matrices including gelatine and collagen are also used (Kourkoutas et al, 2004). This method of cell entrapment is not frequently used in industry due to several drawbacks, including; cell viability due to the diffusion limitations of nutrients, metabolites and oxygen, the chemical and physical instability of the gel beads and the lack of renewability making the process fairly expensive (Verbelen, et al. 2006).
In recent years, research has been aimed at finding new techniques which will solve most of these drawbacks, such as techniques which use hydrogels with adjusted size and shape (Nedovic et al. 2005(b)). A study by Kong et al, in 2003 suggested that by adjusting the molecular weight of the polymers used to form gels would allow formation of hydrogels while maintaining cell viability. The investigation carried out used alginate as a model system to examine the effect of lowering the molecular weight. The results indicated that by altering the molecular weight of alginate increased the viability of cells due to the ability of nutrients, metabolites and oxygen to diffuse through the medium.
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Another problem with immobilisation of cells in polysaccharide beads is the potential for cells located on the surface of the bead to be released from the gel (Kourkoutas et al, 2004). One way to avoid this is to cover the beads with an external coating which is free from microorganisms (Godia et al. 1991). In a 1991 study Godia, et al, investigated the release of cells from the beads surface and the use of beads which had been externally coated in a material which was free from organisms for use in the fermentation of sparkling wine.
Saccharomyces cerevisiea cells were immobilised in calcium alginate beads, using a double flux needle device to control the bead diameter. Half of these beads were externally coated by dropping calcium alginate beads which were wet with calcium chloride solution into a 0.5% alginate solution. The beads were continuously stirred in the solution as a thin layer of calcium alginate was promoted by the calcium ions on the bead surface. The results showed that the use of immobilised cells in sparkling wine production was feasible by both approaches. In cells which were not externally coated, there was little cell release from the beads, suggesting that the amount of sugar available was not sufficient for ell division and release. The use of externally coated beads imposes a physical barrier to cell release and also required up to half as many beads for fermentation of the wine. The preparation of externally coated beads required an additional step, which show future prospects, however, should be further investigated to improve efficiency for use in large scale industries as they would require bead making machines, bead dosage control and an absence of yeast on the base wine. (Godia, et al. 1991)
Passive Cell Immobilisation
Passive cell immobilisation is any method of immobilisation which do not involve the use chemical agents. This includes flocculation and adsorption. The lack of requirement for chemical agents keeps the cost of immobilisation low however there is potential for cell detachment from the medium in the case of environmental changes.
Flocculation is the process where the yeast cells clump together as a result of random collisions. This can occur due to cell growth and increased mass, cell interaction and as a response to the surroundings. This process may be induced by modifying the cells' immediate environment. Flocculation usually occurs when the cells no longer have access to fermentable sugars. It is suggested that flocculation is the cell's stress response to starvation in an attempt to produce a sheltered environment to increase the chance of survival. Cells will return to their normal state when exposed to a source of fermentable sugars (Mamvura et al. 2012). Flocculation is a widely used technique in the ethanol production industry due to the advantages of the simple and inexpensive procedure which requires no costly materials or technical machinery compared to some other methods (Zhao & Bai, 2009).
In a study by Mamvura, et al, Carbon nanotubes were tested as possible flocculation surfaces in the immobilisation of Saccharomyces cereviciae. The investigation involved the use of positively charged carbon nanotubes to attract the negatively charged viable yeast cells. It was found that the nanotubes were able to be used to increase the flocculation rates of yeast cells in the absence if fermentable sugars. This process has potential to be applied in the ethanol industry to remove suspended yeast cells after the fermentation process to reduce turnaround time. (Mamvura, et al. 2012)
Surface adsorption of yeast cells onto a support occurs by electrostatic interactions between the positively charged support and the negatively charged cell. A great deal of research is under way in this area with an aim of finding cheap, readily available supports. In studies by Montealergre, et al, in 2012, comparisons are made between immobilisation of Saccharomyces cerevisiae by surface adsorption onto NATA De Coco and entrapment by calcium alginate beads. The process which used calcium alginate for immobilisation reached a higher concentration of ethanol than the process carried out with adsorbed cells on NATA de Coco. The results from the inverstigation show thathe cells adsorbed onto NATA de Coco were more stable during the fermentations, further research in this area would allow for optimisation of the use of Nata de Coco, such as the potential for increasing the cell loading capacity and subsequently the ethanol production rate. (Montealergre et al. 2012)
In a 2009 study by Nguyen et al , the use of bacterial cellulose as a cell immobilisation support for Saccharomyces cereviciae. Bacterial cellulose is synthesised from bacteria, particularly Acetobacter xylinum, and does not require any special treatment before use. Bacteria cellulose is considered to be a good support for cell immobilisation as it So BC can be considered as a good support for yeast cell immobilization as it holds a great deal of water and retains its degree or polymerisation (Serafica, 1997). The results for this investigation show that Saccharomyces cereviciae can be immobilised on bacterial cellulose successfully through the use of a very simple and inexpensive method. (Nguyen et al. 2009)
Current research in the area of ethanol production is mainly aiming to find effective methods of lowering the costs of fermentation. The application of cheap support materials for immobilising yeast cells has the potential to substantially reduce the investment costs of the continuous fermentation process. Researchers have proposed the use of many materials for yeast immobilisation, including; porous glass, fruit pieces, and alginates. Despite the many advantages of inorganic carriers. the inorganic supports were considered inconvenient for food production most of the industrial applications are performed with organic matrices (Kourkoutas et al. 2004).
One particular study by Branyik, et al. explored the possible use of two cellulose-based carriers for immobilisation of Saccharomyces carlsbergensis. The two carriers used in this investigation are corncobs and Brewers spent grains. Corncobs are large volume waste products from sweet corn processing which are often used for animal feed or land application. Corncobs were identified as potential carriers for immobilised cells due to their ability to be used as a carrier for microbes involved in wastewater treatment and bioremediation. Brewers spent grains are also generally used as animal feed, are a by-product of the brewing industry (Branyik, et al. 2006).
The results of this study showed the use of corncobs and brewers spent grains would be a cheap and simple carrier material for use in cell immobilisation. The materials are readily available and can be washed and reused. Further research would be required in this area to investigate the use of these carriers in continuous fermentation (Branyik, et al. 2006).
Another study carried out in 2012 by Vucurovic and Razmovski, investigated the use of sugar beet pulp as a carrier for Saccharomyces cerevisiae immobilisation along with the use of the extracted sugar as a carbon source. The sugar beet was cut into slices and the fermentable sugars were extracted before being used for immobilising Saccharomyces cerevisiae cells in a batch fermentation. The results confirmed that the sugar beet pulp is an acceptable carrier material for immobilising cells. This method is a cheap and simple method for the immobilisation of cells with sugar beet being readily available. Further research could allow for this method to be developed for use in continuous fermentation or for application in industry fermentations (Vucurovic & Razmovski. 2012).
In recent years, research has paid a great deal of attention to finding a suitable carbon source for use in the fermentation process. In particular, one study by Vucurovic and Razmovski, in 2012 investigated the use of sugar beet as a source of carbon for Saccharomyces cerevisiae in the production of bioethanol and also the use of the sugar beet pulp as an immobilisation carrier for these cells. Sugar beet has been considered more advantageous in fermentation in comparison to lignocellulose or starch crops due to their high content of monosaccharides which are more readily fermented.
The sugar beet is processed by introducing sliced beet to hot water, around 70â-¦C, to extract the raw sugar juice. The raw sugar juice is processed by purifying and then concentrating to produce a thick juice with a sugar content of 67%. This sugar solution was crytalised to form molasses which was used in a batch fermentation using immobilised Saccharomyces cerevisiae as previously discussed. The use of molasses was found to be less appropriate for ethanol fermentation than sugar solution due to the metabolism of the yeast being affected by accumulations of coloured compounds. Further research could develop this process to allow for the use of the thick sugar solution in continuous fermentation processes (Vucurovic & Razmovski. 2012).
The research discussed in this review all has potential for further development. Main areas in which development is currently under way include research into possible methods of cell immobilisation, different carriers of immobilised cells and possible sugar sources of the fermentation process. Further research mainly aims to find cheap, simple and effective methods to improve fermentation processes, in particular continuous fermentation processes which would greatly advance the large scale fermentation industries.