Antimicrobials 101

Antimicrobials play an important role in achieving consistent ethanol yield, warding off bacteria that negatively impact plant performance. Ethanol Producer Magazine speaks with Phibro Ethanol, BetaTec Hop Products and Lallemand to learn more.
By Katie Schroeder | March 18, 2022

“An ethanol plant is never going to be sterile,” says Jenny Forbes, Phibro’s vice president of sales and service in North America, as she explains the essential role of antimicrobials. Forbes describes how the ambient process temperatures in certain areas of a biorefinery provide a great environment for the growth of bacteria, how sugars become a food source, and how the dark, warm piping and process equipment give bacteria places to build up, vie for sustenance and cause fouling.

Bacteria ultimately compete with yeast for the same nutrients within an ethanol plant’s ecosystem, says Rachel Murdy, antibiotics category manager with Lallemand Biofuels & Distilled Spirits. “So any glucose that goes to bacterial growth is glucose that is no longer available to yeast, thereby reducing your yield,” she explains “Another negative impact of bacteria is that they produce organic acids that can inhibit yeast growth.”

Mike Karavolos, lab manager at BetaTec Innovation Center in Malvern, United Kingdom, further explains the impact bacteria can have on ethanol plants, specifically on yeast health. He explains that the bacteria will also create a biofilm to protect it from antimicrobials, making it harder to get rid of and “very, very detrimental to any fermentation.”

Bacterial infections can also reduce yeast viability, decreasing fermentation yields. As it multiplies, the bacteria produce lactic and acetic acid which is toxic for the yeast. “The yeast must constantly work to adapt and overcome contamination stress and continue the fermentation,” Karavolos explains. “But eventually—if the infection is left uncontrolled—it will lead to stuck fermentations, and the plant will suffer negative impacts.” Fortunately, there are a variety of antimicrobial products available to help regulate bacteria.

Prevalence and Prevention    
Karavolos outlines common problem spots within an ethanol plant where bacteria often start growing. “Bacteria are constantly sensing their environment trying to survive,” he says, listing several problem areas: crevices where bacteria can attach and be protected from antimicrobials; the heat exchanger, due to the increase in food source and lack of antimicrobials; dead legs where there isn’t any liquid movement and bacteria can multiply, continuing to infect future batches; and finally, bottom-of-the-pile corn which has an increased bacterial load due to higher humidity and a variety of other issues.

“We have high temperatures in most plants on the front end that help mitigate bacterial growth, but it’s never sterile,” Forbes says. “There’s always some bacteria population present, and you’ll find that if you don’t clean adequately (properly or frequently enough), the bacteria just thrives, multiplies and causes problems downstream.”

Karavolos also affirms the importance of clean-in-place procedures, which can help lower the initial amount of bacteria present. “Prevention is better than a cure,” he says, explaining that consistent cleaning procedures will help slow the multiplication of bacteria.

Murdy says that monitoring high performance liquid chromatography (HPLC) profiles early in fermentation can help plants monitor for infections and yeast health. Any early increase in organic acids, such as acetic acid or lactic acid, can help you [determine] when to add more antimicrobials if corrective measures are needed. “The HPLC will give them a profile of, not only baseline organic acid concentrations, but also of the breakdown of the starch and the production of ethanol. So, it’s a really good way of checking for yeast health,” Murdy says.

Forbes outlines several ways that bacteria can negatively impact the profit margin of an ethanol plant. The two main negative impacts are “loss of starch” due to the bacteria consuming it, and yeast damage from the acid produced by the bacteria. “Ultimately, you want every molecule of starch or sugar converted to ethanol, because that’s where your profit is,” she says. “Bacteria will consume that starch, and then it’s not going to ethanol. The bacteria will also make off-products—lactic and acetic acid are the two most predominant.”

Forbes explains that the presence of lactic and acetic acid, which are toxic to yeast, cause problems with fermentation. “There are a lot of new yeast products in the market that are genetically modified to do amazing things, but a lot of these products tend to be more sensitive to organic acid,” she says. “Infection risk and associated impact becomes more severe than it would have [been] ten years ago with conventional yeast.”

Antimicrobials: Antibiotics
Forbes explains that Phibro recommends using a maintenance dose of antibiotic in every fermentation batch added as early in the process as possible. As soon as mash enters a vessel (propagator or fermenter), antibiotic should be present. If there is increased risk of bacterial contamination, it is suggested to increase the antibiotic dose.

Murdy emphasizes that antibiotic dosing is not a “one-size-fits-all” situation. “A single antibiotic system may only target certain bacteria, while allowing other bacteria to proliferate in fermentation. So we often recommend combinatory antibiotic systems and antibiotic rotations that will be dependent on the natural flora that’s in those plants. Sometimes a single antibiotic mode of action can be quite effective, but other times you need to hit them with more than one type of antibiotic to target different populations in the process and prevent antibiotic resistance. This is the mechanism behind our new BioSustain combinatory antibiotic platform.”

Phibro offers two main antibiotic products: virginiamycin and penicillin, with the respective trademarked product names of Lactrol and PhibroPen. Forbes explains that both products accomplish the same thing but work slightly differently. Virginiamycin—an antibiotic only manufactured by Phibro—binds to the bacterial ribosome, keeping the bacteria from producing protein, rendering the bacteria static and unable to complete any cellular processes. Penicillin binds to the bacterial cell wall and causes the cell to rupture. According to Forbes, the two antibiotics complement each other well when used together.

Phibro helps its customers determine an effective maintenance dose amount and product actives by having them gather samples post liquefication. The company asks plants to sample the mash before and during propagation and fermentation so they can evaluate the facility’s contamination risk. “We ask plants to systematically walk process lines and pull mash from every sample port they have until the start of fermentation. Ideally, you would see no measurable contamination in hot liquefact through the start of the mash cooling system and then be able to identify the 'hotspots,' or the direct location of bacterial residence. You would be able to verify that the process is contaminant free to a certain place in the process line, and then pinpoint the spot where the contamination is located,” Forbes says.

This allows them to tailor the recommended maintenance dose to the bacterial infection risk and dose antibiotic products in an informed manner. There is an upper limit for antimicrobial dosing in order to comply with generally recognized as safe (GRAS) guidelines.

“There is a maximum dose that’s been established, but we rarely see customers add products at the upper dosing levels, Forbes says, explaining the testing methods used to help producers understand their normal bacterial load and the best antibiotic dosing to manage it. “We like to be stewards of responsible product use.”

Murdy outlines the risks of using more antibiotic than necessary in an ethanol plant, including the possibility of creating antibiotic resistance. “The presence of resistant bacteria would have a negative impact to your process because you would actually need either more antibiotics for antibacterial control, or you might require a different antibiotic to now target those resistant bacteria.” She explains.

“That’s why we recommend having a combination of antibiotics with differing modes of action or rotating antibiotics to prevent [the establishment of a] resistant population, and to always follow the products recommended dosage rate.”

Antimicrobials: Hop Extracts
Natural hop extracts work differently from antibiotics by having a multitargeted effect on the bacteria. Karavolos explains that while antibiotics may target a specific part of the bacteria, such as the ribosomes or DNA, hop extracts “will act on the total membrane potential and will inhibit the vast array of proteins that are involved in many processes in that regular bacteria physiology and behavior,” Karavolos says. “For example, it could be pH homeostasis, it could be membrane transport, cell division and even environmental sensing.” The extracted hop acids can penetrate the bacterial cell wall and throw the pH of the bacterial cytoplasm out of balance by acidifying it. “The membrane potential is lost and that leads to the inability to function properly, and the inability to uptake nutrients, which eventually leads to starvation and death,” he explains.

 BetaTec offers several different antimicrobial hop extracts to the ethanol industry. FermaHop Pro is very active against lactic acid bacteria in a broad pH range from about 4.5 to 6. The company also offers IsoStab, LactoStab and VitaHop Silver Liquid. Plants using hops typically introduce them into the propagator or fermenter depending on many factors, Karavolos says. Dosing hops earlier when the microbial load is lower gives producers the ability to monitor and adjust fermentation if needed later, he explains. BetaTec creates custom product trials for each plant using hops, optimizing dosage as much as possible. The company also provides application development, consulting services and technical support for its global customers. “We also evaluate other naturally derived substances in combination with hops to create new and improved hop products with added functionalities,” Karavolos says.

There are several issues ethanol producers must closely consider when utilizing antimicrobials, Karavolos explains. Foremost, he says, they should be keenly aware of how and where their coproducts are used. “It is important to consider what is added in fermentation ... [to ensure] compliance with food and safety regulations.”

Another potential issue is heavier use of antimicrobials, which has become slightly more prevalent since the introduction of genetically modified yeasts, according to Karavolos. He also finds that overlooking yeast health is a common mistake. “The yeast is the heart of fermentation, you have to take care of it, and you have to be more cautious on how the yeast perceives the antimicrobials itself,” he says. The more bacterial stress that the yeast can handle, the better the fermentation; leading to improved ethanol yields overall.

Innovation is important for any industry to keep moving forward, and the antimicrobial industry is no different. To go along with the ethanol industry’s focus on automation, Phibro developed an automatic dosing system, trademarked PhibroMat. “We provide automated dosing systems for antibiotics; if plants want to operate fully automated, we now can offer them a way to do that,” Forbes says.

BetaTec has actively worked on developing new applications for existing products while creating “new breakthrough products and technologies,” according to Karavolos. He says BetaTec is studying the impacts of its hop extract products on yeast health. “Yeast that is healthier can overcome more stress over longer periods of time, which is a benefit for the fermentation,” he explains. The experiment was conducted using 6,000 yeast variants divided up into different categories such as ethanol production, metabolic processes and more. According to the study conducted in partnership with the University of Manchester, Institute of Biotechnology, the hop-based antimicrobials provide a 21 percent benefit across all categories compared to non-hop antimicrobials.

Author: Katie Schroeder
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