Identifying, Controlling the Most Common Microbial Contaminants

Microbial contamination is a significant concern for fuel ethanol producers. Proper cleaning guidelines and control strategies will help avoid potential yield losses and the production of unwanted organic acids.
By Patrick Heist | March 05, 2009
Bacteria that contaminate fuel ethanol plants are referred to as lactic acid bacteria as they produce various organic acids including lactic and acetic acids the primary indicators of bacterial contamination. Lactic acid bacteria are further subdivided based on their ability to produce one or more organic acids (homo- versus heterofermentative).

Bacterial contamination often occurs due to inadequate cleaning or impediments to process flow (heat exchanger blockages, tools and other items in the piping, and biofilms, among others). Because fermentation is an anaerobic process, these bacteria are most often either anaerobic or facultative anaerobes, although there are some aerobic bacteria that can cause problems in other areas such as the propagation tank or the beer well. These bacteria enjoy lower pH levels (less than 5) than most other bacteria, grow well under fermentation conditions and compete with yeast for nutrients conditions and fermentable sugars resulting in reductions in ethanol yield, among other things.

These bacteria are typically gram positive rods. However, gram positive cocci as well as various gram negative bacteria also have the potential to cause contamination of grain-based fermentations. Proper sanitation and implementation of a rotational antibacterial control strategy are important for preventing bacterial contamination and emergence of resistant bacterial strains. It is also important to remember that certain species of yeast can also lead to contamination problems and yield losses. Here we discuss some of the most common microbial contaminants of the fuel ethanol production process as well as any unique features and methods for control and prevention.

Lactobacillus Species
Lactobacillus species are gram positive, rod-shaped bacteria and are among the most prevalent bacteria known to contaminate fuel ethanol plants. They are found throughout the environment and are present on various grains including corn. Several species of Lactobacillus have been isolated from contaminated fermentors. Some of the most common Lactobacillus species include L. fermentum, L. brevis and L. plantarum. These bacteria generally respond well to each of the different antibacterial products on the market, but can lose their sensitivity if cleaning and other facets of sanitation are not strictly followed. Weissella species, formerly classified in the genus Lactobacillus, are also commonly isolated from grain-based fermentations and include Weissella confusa among others.

Pediococcus Species
Pediococcus species are gram positive cocci (spherical) and often arranged in pairs or tetrads (packets of four). These bacteria are significant contaminants of grain-based fermentations and have the potential to cause major yield losses. Due to their unique cell morphology compared to more prevalent gram positive rods such as Lactobacillus species, Pediococcus can be easily differentiated and identified using simple microscopy. Pediococcus species are resistant to most commonly used antibacterial products such as virginiamycin and penicillin, but are controlled at low concentrations using other products developed specifically for their control. Pediococcus acidilactici and P. pentosaceus are the most commonly isolated Pediococcus species from fuel ethanol plants.

Other Gram Positive Bacteria
Various other gram positive bacteria have been isolated from grain-based fermentations and include Enterococcus species such as E. faecium, E. faecalis and E. durans. These bacteria are found normally in the intestinal tracts of humans and other mammals and likely get into the fuel ethanol production process through sump water. Keeping the floors clean and the plant free of rodents helps prevent introduction of these bacteria. Enterococcus species are notoriously resistant to antibacterial products, but there are options for their control available to fuel ethanol plants.

Acetobacter Species
Acetobacter and related bacteria such as Gluconobacter species are gram negative rods and are commonly found in grain-based fermentations. Because these bacteria are strictly aerobic they are often limited in ethanol plants to areas like the propagation tank and beer well (although the beer well is often anaerobic due to high levels of carbon dioxide, it is a common place for isolating these bacteria).

Acetobacter and related species usually have a mucoid appearance when growing on agar and characteristic tan- to brown-colored translucent colonies. A common misconception is that elevated acetic acid levels are an indicator of Acetobacter contamination. Although Acetobacter species are known producers of acetic acid, elevated acetic acid levels without corresponding lactic acid is more likely to be an indication of yeast stress (Saccharomyces cerevisiae, the yeast used for fuel ethanol production also produces acetic acid). These bacteria are resistant to several commonly used antibacterials, but there are options available to ethanol plants for controlling these microbes. Another interesting fact about
Acetobacter species is that they can use ethanol as a carbon source, resulting in acetic acid production.

Other Gram Negative Bacteria
Other gram negative bacteria commonly isolated from grain-based fermentations include members of the family Enterobacteriaceae. These bacteria are rod shaped and are typically longer than Acetobacter and related species. These bacteria are facultative anaerobes meaning they can grow either aerobically or anaerobically (with or without oxygen).

These bacteria are known to ferment glucose and are negative for the enzyme cytochrome oxidase, a test used to differentiate them from other morphologically similar bacteria. Examples of bacteria in the family Enterobacteriaceae isolated from contamination events at fuel ethanol plants include Klebsiella, Escherichia coli and Shigella species. As normal inhabitants of the intestinal tracts of humans, other mammals and birds, these bacteria gain access to the system through sump water. For this reason it is important to keep the floors clean and the grounds free of small rodents and birds to prevent introduction of these bacteria.

Wild Yeast
Wild yeast refers to any yeast species including S. cerevisiae that was not intentionally put into the fermentation process. These organisms cause problems primarily in ethanol plants utilizing the continuous process, but are also found in batch plants. Wild yeasts such as Dekkera species can produce organic acid byproducts including acetic acid and have the potential to cause significant yield losses. Because wild yeasts are eukaryotic organisms and different from bacteria, they are not affected by antibacterial products available for bacterial control. Thorough cleaning is the best way to prevent introduction and proliferation of wild yeast.

Detection, Prevention and Control
High performance liquid chromatography is one of the best ways to detect bacterial contamination. Elevated lactic and/or acetic acid (0.3 percent and 0.05 percent, respectively) is the most obvious sign of a bacterial contamination event. Increased residual glucose and decreased ethanol yields may also occur.

The first line of defense for preventing bacterial contamination is through intensive cleaning. Making sure your caustic solution is at the proper concentration, temperature, and flowing through the system at the appropriate pressure is imperative for adequate cleaning. Another requirement for proper cleaning is to make sure the caustic flows through the system for the proper amount of time. Together these different components are referred to as the four Ts of cleaning: time, temperature, turbulence and titration.

Sulfamic acid is commonly used as a solvent to clean heat exchangers and for removal of biofilms. Routine review of all cleaning practices and associated equipment (e.g., pumps and sprayballs) is recommended for maximum effectiveness. Secondary to cleaning is the use of antibacterial products, developed specifically to control bacteria in fuel ethanol plants. These products, each with a different site of action and spectrum of activity, are very selective for bacterial toxicity at very low concentrations and do not affect the yeast at recommended doses. Modes of action for antibacterial products used in fuel ethanol production include inhibition of cell wall (peptidoglycan) synthesis and inhibition of protein synthesis at the level of the bacterial ribosome.

Conclusion
In summary, microbial contamination is a significant concern for fuel ethanol producers due to potential yield losses and production of unwanted organic acids. By following proper guidelines for cleaning and sanitation as well as using a rotational antibacterial control strategy, contamination can be kept to a minimum while maximizing ethanol yields and return on investment.

Patrick Heist, Ph.D., is chief scientific officer for Ferm Solutions Inc. Reach him at eheist@ferm-solutions.com or (859) 402-8707