Latest Content
Latest Community Postings
Recent Blog Entries
Community Downloads
ChExpress Blog
Ankur's Tech Blog
Community Admin Blog
Energy Efficient Hot and Cold Water
Electrical Process Tomography
Biodiesel: The Road Ahead
Methanol Plant Capacity Enhancement
Plate and Frame Heat Exchangers: Preliminary Design
Compressor Surging Under Control
Plant and Equipment Wellness, Part 1: Observing Variability


Share this topic:



cheplusbannerbig.gif (7470 bytes)

Managing Wild Yeast Contamination in Fermentation for Alcohol Production

Can wild yeast contaminate the fermentation process?  They sure can!  Here, you'll learn about how yeast get contaminated in alcohol fermentation.  We'll examine the sources, isolation methods, and how to identify contamination.  Also, learn about the problems caused and how to take preventative measures to prevent contamination.

Introduction

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.  Additionally, the distillery must consistently achieve the maximum possible alcohol yields from a given feedstock.  It therefore becomes important to monitor and achieve the proper quality of raw material and fermentation parameters.

Most distilleries in India are using cane molasses-c as a feedstock for alcohol production.  Molasses-c differs from other feed stocks for alcohol production such as corn, sorghum, and potatoes etc. which have their carbohydrate content stored as starch which is usually precooked and hydrolyzed into fermentable sugars.  Molasses doesn’t require pretreatment as the carbohydrates are already in the form of sugars (Gunjal, 1999).  During the sugar process, one of the important by-products is molasses-c.  Molasses is used to produce ethanol, citric acid, yeast and cattle feed.  Over 20 billion litres of ethanol is produced annually from molasses (Praj, 2004).

Table 1: Typical composition of cane molasses-c  

Sr. no

Analytical Parameters

Units

Normally observed values

A. Chemical Analysis:

1

Total solids

% by wt.

65 to 80

2

Total Reducing Sugars

% by wt.

45 to 50

3

Unfermentable Sugars

% by wt.

4 to 5

4

Fermentable Sugars

% by wt.

40 to 46

5

F/N ratio

-------

1 to 2.5

6

Total inorganics

% by wt.

8 to 12

7

Calcium as CaO

% by wt.

1.5 to 3.5

8

Total settlable sludge at 4.5 pH

% by wt.

5 and below

9

Specific gravity

-------

1.38 to 1.55

10

Titrable volatile acidity in terms of acetic acid & acetate salts

M1 of 0.1 N NaOH

29 ml

11

pH at 40o Bx dilution

----------

4.5 to 5.2

12

Suspended particles >100 microns

% by wt.

Up to 0.05

13

Color in terms of Optical Density (OD) at 375 nm with 0.01%w/v dilution

OD

0.15 to 0.4

14

Soluble sulphates as SO4

--------

1.5 to 5

B. Microbiological:

1

Total bacterial count

CFU/gm

10 exp 3  and below

2

Wild yeast and fungal count

CFU/gm

10 exp 2 and below

(Adopted from Konchady, 2003)

The major cause of concern in molasses based fermentation for ethanol production is the presence of wild yeast along with bacterial contamination.

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:

  1. 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.

  2. 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 76.6C 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.

Temperature

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).

Nutrient Supplement

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.

Air Filtration

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.

Conclusions

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.

Acknowledgement

The authors would like to dedicate this article to Praj Industries Ltd., Pune, India.  The authors also wish to show their gratitude to Dr Vipin Kumar and Dr Deepak Acharya, Natural Product Laboratory, SRISTI Ahmedabad, for their constant encouragement and support.


References

1. Abbott, DA, Hynes, SH, Ingledew, WM. 2004. Growth rates of Dekkera / Brettanomyces yeasts hinder their ability to compete with Saccharomyces cerevisiae in batch corn mash fermentation. Appl Microbiol Biotechnol. Nov 6[Pubmed] PMID: 15538553.

2. Aris. 2002. http://www.thewinemerchantinc.com/educational/yeast.html (Viewed on 24/12/2004)

3. Bhutto, AW.http://www.pakistaneconomist.com/page/issue38/i&e3.htm (Viewed on 10/12/2004)

4. Brewingtechniques. 2004. http://www.brewingtechniques.com/library/backissues/issue2.6/allentable.html (Viewed on 12/12/2004)

5. Ceccato-Antonini, SR, Christina da silva, P. 2002. Hyphal-like extension and pseudohyphal formation in industrial strains of yeasts induced by iso-amyl alcohol. Braz. J. Microbiol.  33(3): 209-212. ISSN. 1517-8382

6. Ceccato-Antonini, SR, Tosta, CD, Claudia da Silva, A. 2004. Determination of yeast killer activity in fermenting sugarcane juice using selected ethanol-making strains. Braz. Arch. Biol. Technol. 47(1): 13-23. ISSN 1516-8913.

7. Cocolin, L, Rantsiou, K, Lacumin, L, Zironi, R, Comi, G. 2004. Molecular Detection and Identification of Brettanomyces / Dekkera bruxellensis and Brettanomyces / Dekkera anomalus in Spoiled Wines. Appl Environ Microbiol. 70(3): 1347-1355.

8. Deshpande, GB. 2002. Overview of continuous alcohol fermentation and multipressure distillation technology. Proc S Afr Sug Technol Ass. 76: 999-1007.

9. Fal, 1994. http://www.brewingtechniques.com/library/backissues/issue2.6/allen.html (Viewed on 24/12/2004)

10. Goode, J. Brettanomyces. http://www.wineanorak.com/brettanomyces.htm (Viewed on 18/12/2004)

11. Green, SR, Greay, PP. 1950. Differential Procedure applicable to investigating brewing. Wallerstein Lab. Comm. 13: 357

12. Gunjal, BB. 1999. Managing fermentation for alcohol production with special reference to contamination problem. DSTA bulletin. 5-7.

13. IFT, 2003. http://ift.confex.com/ift/2003/techprogram/paper_16150.htm (Viewed on 24/12/2004)

14. Ingledew, WM. 2001. Alcohol production by Saccharomyces cerevisiae: a yeast primer. Nottingham university press. Nottingham, UK. Vol. 3, Pg. 49-87

15. Kelly, J. 2003. Oak in winemaking. http://www.grapeandwine.com.au/2003/july/06.htm (Viewed on 10/12/2004)

16. Konchady, S. 2003. Alcohol Palm Book. Praj Industries Ltd. Pg. 22.

17. Lansing, R. Recent Developments in Brett Management and Monitoring. http://winebusiness.com/html/monthly article.cfm (Viewed on 9/12/2004)

18. Lorenz, MC, Cutler, NS, Heitman, J. 2000. Characterization of Alcohol-induced Filamentous Growth in Saccharomyces cerevisiae. Mol. Biol. Cell. 11(1): 183-199.

19. Morris, EO, Eddy AA. 1957. J. inst. Brew. 63(1): 34-35

20. Morris, RL. 1994. Simple detection of wild yeast and yeast stability.

http://www.brewingtechniques.com/library/backissues/issue2.3/morris.html (Viewed on 17/12/2004)

21. Nashwoodwinery, 2004. http://www.nashwoodwinery.com/intro_to_sulfites.pdf (Viewed on 12 /12/2004)

22. Oxoid. 2004. http://www.oxoid.com/uk/index.asp (Viewed on 12/12/2004)

23. Praj, 2004. http://www/praj.net/article8.htm (Viewed on 23/12/2004)

24. Tessier, J. 2004. http://www.grtc.ca/articles/2001/baker04.cfm (Viewed on 9/12/2004)

25. Webster’s. 2004. http://www.websters-online-dictionary.org/definition/english/wi/wild+yeasts.html (viewed on 12/12/2004)


About the Authors

Devendra Dusane (microbesinaction@yahoo.co.in)

Devendra works as a microbiologist at Praj Industries Ltd. in Pune, India.  His research interests include microbiology, biotechnology, and environmental sciences.  Learn more at http://devendradusane.tripod.com.  

Ghanshyam Deshpande (ghanshyamdeshpande@praj.net)

Ghanshyam is Vice President (ESD Division) of Praj Industries Ltd. in Pune, India.  He has years of experience with various fermentation processes and has participated in various national and international events in these fields.   Deshpande has contributed research articles to various scientific journals and magazines. 

 

 


smalllogo.gif (4001 bytes)
JOIN OUR COMMUNITY

  • Stay up to date on new content
  • Post questions and answers in our forums
  • Access downloads and attachments
  • Read member blogs and start your own blog
  • Connect with members via our friends feature
  • Receive and post status updates