Ethanol fermentation is a complex biological process for the production of alcohol from sugar. Alcohol is widely used for many different purposes. Yeast, under anaerobic conditions, convert glucose to ethanol. The stoichiometric equation for the production of alcohol by fermentation is given below:
C6H12O6 ——————> C2H5OH + 2CO2 -265kcal /kg cal
Glucose ethyl alcohol carbon dioxide
It is a non-sterile process. The presence of undesirable microorganisms in the process can not be completely avoided. The raw material used for fermentation itself shows the presence of contaminating microorganisms.Normally 60-70% of the production cost is contributed by the feedstock in alcohol production. Owing to the increasing demand and cost of molasses, improving and maintaining the quality of the fermentation process has become a crucial factor.
What is Wild Yeast?
Yeasts, other than the elliptical culture yeast, are called as wild yeast. Wild yeast occurs naturally in the raw material and produce small amounts of alcohol (Webster’s, 2004). Report suggests that they can be harder to detect and control than bacteria (Fal, 1994). Generally the wild yeast Dekkera and Brettanomyces are observed inhabiting molasses source. They are genetically identical but Dekkera is the sporulating form of this yeast (Goode, 2003). Dekkera species can also be fermentative yeast capable of producing alcohol. Brettanomyces grows and ferments slowly and can ferment in low levels of fermentable sugars (Kelly, 2003; Lansing, 2004).
Sources of Contamination
Wild yeast is considered to be indigenous and found in the air and on surfaces (Tessier, 2004). Wild yeast primarily comes through molasses in the fermentation process because of the presence of fermentable sugars. Solid addition as well as liquid streams entering the process can be a source of wild yeast contamination (Ingledew, 2001).
Problems Caused by Wild Yeast Contamination
A. Scum Formation: What is Scum?
Wild yeast contamination is commonly observed in wine fermentation. Little research has been performed on the topic of yeast contamination in molasses based fermentation. Controversy surrounds the subject of scum formation as microbiologists and engineers debate whether the wild yeast itself is responsible or morphological changes in the cultured yeast under unfavorable conditions.In the fermentation process, yeast multiplies in the form of heavy branched structure. This branching yeast comes at the top of the fermentor. The sparging of air in the fermentor helps the branched yeast to rise to the top and form a thick layer called the ‘scum’.The scum formation in the fermentor traps gas bubbles inside the branched structure thereby building pressure in the fermentation vessel. Excessive and uncontrolled scum in the fermentation process is the major problem caused by wild yeast. This scum cannot be reduced by the addition of anti-foaming agents. The process needs to be stopped after a few weeks due to a loss of efficiency and excessive overflow of mash from the fermentors. There is washout of culture yeast and a gradual drop in the alcohol concentration in the fermentation process. According to Lorenz et al. (2000), the morphological changes in the culture yeast (Saccharomyces cerevisiae) leads to filament formation under unfavorable fermentation conditions. This causes scum formation and foaming. The budding yeast S. cerevisiae, starved for nitrogen, differentiates into a filamentous growth form. In nitrogen poor conditions leucine, the precursor of iso-amyl alcohol (a chief constituent of fusel oil) can also induce elongation of cells. Ceccato-Antonini and Paula Christina (2002) suggested genetically controlled morphological changes and filamentous growth in response to Iso-amyl alcohol.
B. Reduced Efficiency
According to Lorenz et al, (2000), the reduction in efficiency is attributed to the presence of wild yeast and bacterial contamination in the input to the fermentor. Due to heavy foaming, juice losses result in the loss of alcohol. The efficiency reduction depends upon the extent of contamination.
Isolation and Identification of Wild Yeast
There are various methods to identify and differentiate wild yeast, two of them are:
- Heat resistance method: Suspend the yeast in sterile water and heat to 53 °C for 10 minutes. Then take a viability test to measure the survival rate.Normal cultured yeast will not survive this test. The wild yeast isolated is heat resistant.
- Microscopic examination: Since detection and isolation of wild yeast is a problem, the common method is plating followed by microscopic evaluation. The presence of branched structure under the microscope reveals the presence of wild yeast (Lansing, 2004).
Differentiation and identification of various yeast strains along with contaminated wild yeasts has been done using selective culturing methods based on selective chemicals, fermentation tests, and other methods. A synthetic medium, such as lysine, is used for the isolation and enumeration of wild yeasts (Oxoid, 2004). Normal S. cerevisiae or carlsbergensis strains cannot utilize lysine whereas many other wild types of yeast do (Morris, 1957). Differential media such as SDM (Schwartz Differential Medium) and WL (Wallerstein Laboratory nutrient agar, Green, 1950), Lysine medium (LYS), Lin Wild Yeast Medium (LWYM) and UBA (Universal Beer Agar) can be used to isolate yeast strains from different sources (Ceccato-antonini et al., 2004; brewingtechniques, 2004). Crystal violet in the medium will inhibit the growth of cultured yeast and allow the growth of Saccharomyces spp. which will not be detectable on lysine medium (Fal, 1994). The uses of selective chemicals like actidione (cycloheximide), lysine, or cupric sulfate (LCSM) are commonly used for isolating wild yeast. Recently modern techniques like DNA fingerprinting and RT-PCR analysis have been applied to successfully detect and enumerate lower levels of wild yeast in a much shorter time (Morris, 1994; Cocolin et al., 2004; Lansing, 2004).
Wild Yeast Management
A. Killer Yeast Strains
There are widespread occurrences of killer phenotypes in yeasts in alcoholic fermentation. Many of these fermentative processes use non-pasteurized medium, which can enhance the predominance of wild yeast. These contaminants can contribute to the fermentation rate decrease or blockage, increases in acidity, fusel oil production, and an overall decrease in ethanol production. Isolation of killer yeast strains from the ethanol process is imperative for good yields. The presence of cultured yeast with “killer” tendencies against wild yeast will help overcome the problem of excess growth of wild yeast contamination during the process (Ceccato-Antonini et al., 2004). Sugar cane molasses are normally pasteurized or decontaminated in order to reduce the amount of microorganisms appearing during its production, transportation, or storage. Usually, heating the molasses above 76.6°C reduces lactic bacteria but also decreases viscosity and precipitates calcium. High concentrations of suspended solids also makes pasteurization inefficient. Furthermore, the availability of good quality molasses depends on the efficiencies of the specific sugar refineries, weather conditions, and harvesting techniques. Unfortunately, these variables lead to an inconsistent supply of good quality sugar cane molasses and a constant dilemma for sugar cane molasses distillers (IFT, 2003). It is not altogether necessary to sterilize molasses. However, there are advantages in terms of alcohol yield and purity when pretreating the molasses. Treatment of the molasses solution by means of heat and sulfuric acid will precipitate undesirable salts and help to improve the purity of alcohol obtained and reduce the amount of scaling.
B. Sulfur Dioxide and Sulfite Solutions
The principle source of SO2 is sodium bisulfite or potassium metabisulfite. Sulfite is available in powdered form but because of its elevated level of toxicity, its use is limited. Potassium metabisulfite contains approximately 55% SO2 by weight. This free SO2 kills the microorganisms (Kelly, 2003). Culture yeast is generally tolerant to SO2, but at higher concentrations it shows loss of viability. SO2 is toxic and requires appropriate handling. SO2 binds to various compounds present in molasses or juice and pulp or the compound formed during fermentation. Hence the dose should be optimized so that the deleterious effect on wild yeast is achieved without harming the culture yeast. The concentration should be sufficient to overcome the efficiency loss caused by binding to the compounds during fermentation. Generally the dose should be optimized in order to achieve the proper results. The total SO2 added to any wine is under 100 PPM. Sodium bisulfite and potassium metabisulfite are corrosive and will degrade upon exposure to oxygen and moisture. Purchasing should be done depending upon the climatic conditions. The storage should be done in airtight containers preferably glass containers. Precaution should be taken while handling sulfites as it destroys mucus membranes including lung tissues (Nashwoodwinery, 2004).
C. Process Parameters
Control of Infection
As discussed earlier, pasteurization of molasses can help control wild yeast and bacteria. Contaminants generally occupy surfaces covered with deposits of starch, sugar, protein or mineral rich materials. Contaminants thrive in places that are hard to clean such as, porous surfaces, cracks, sharp angles, corners, gaskets, valves, pressure gauges, in-place thermometers, and pump packing. Deposits in these areas protect the organisms in them from heat and sterilizing solutions.
Adequate Yeast Cell Population
According to Abbott et al., (2004) the growth rate of Dekkera wild yeast is lower than culture yeast S. cerevisiae and hence their ability to compete with S. cerevisiae is hindered in batch fermentation. Due to different reaction rates Dekkera will produce different end product as they ferment the juice sugars to alcohol (Aris, 2002). Considering this fact, the cell concentration of the culture yeast can be higher but suppressed if wild yeast outnumbers the culture yeast. Recycle of yeast builds up higher population of yeast in the fermenting mash, thus giving higher productivity as well as robustness of operation (Deshpande, 2002). There are processes that would allow the yeast cell concentration in the fermentation process to be increased. Yeast recycling is one of the most common methods. This process maintains desirable cell concentration for healthy fermentation. However, maintaining the population of these contaminants at lower levels and the use of proper operating procedures is key to the fermentation process.
Online cooling and mixing of molasses is important to avoid the caramelization of sugars. Molasses, if not cooled properly, helps the growth of heat stable microbes that affects fermentation. At higher temperatures, generally in the summer, wild yeast is produced in high quantities. The processing of fresh molasses usually results in a high microbial count in distillery fermentation. Sulfurous gases can inhibit yeast. Similarly fresh molasses has a high foaming tendency as well as a high buffering capacity. It also contains high levels of suspended sludge. This is why fresh molasses is stored for at least a month before use in a distillery. Cooling and frequent mixing by recirculation during storage helps to avoid internal combustion and caramelization that can deteriorate quality. Excessive storage time (more than six month) on the other hand can reduce the fermentable sugar content slowly. Molasses is stored in steel tank to prevent contamination. In order to control the effects of aging, recording the production span, life, and operating with “first in first out” principle are normally standard procedure. The sugar factory process along with handling, storage and transportation of molasses is vital to managing the contamination of wild yeast. As discussed earlier, the presence of sugar in the medium favors growth of wild yeast along with nutrients. Contact with water and pockets of dilution in bulk stock can give rise to high microbial flora. Contact between soil with molasses also should be avoided. It is necessary to handle and store via a protective environment (Bhutto, 2004; Lansing, 2004).
The vital risk factors for Dekkera growth include residual sugar and nitrogen present in the fermentation medium (Goode, 2003). Nitrogen is supplemented in the form of Diammonium Phosphate (DAP) or urea to control fermentation. This can lead to the excess growth of Dekkera due to the presence of excess nitrogen left over during fermentation. Hence the quantity of nitrogen added to the fermentation process should be adequate only to help growth of culture yeast. According to Lorenz (2000) S. cerevisiae can change its morphology due to an inadequate nutrient supply. In this case, the proper supply of nitrogen should be used to overcome the morphological changes in culture yeast.
Airborne dust can vector microorganisms (Fal, 1994). The most efficient means of reducing wild yeast coming from the air is to use a filtration membrane of 0.45 microns.
Wild yeast contamination can lead to serious problems including scum, foaming, and the loss of yield in fermentation. The widespread occurrence of these problems challenges the industries and scientific community to look forward and contributes to the management of wild yeast contamination. Further study of this topic is obviously warranted.
“Managing Wild Yeast Contamination in Fermentation for Alcohol Production,” Che Plus, accessed on april 10 2013,http://www.cheresources.com/wild_yeast_contamination.shtml