2019 March Ethanol Producer Magazine by BBI International

MARCH 2019

EVOLUTIONARY

EDGE

Yeast Modiﬁcation Techniques Advance Continuously Page 16

ALSO

State Programs Fund Ethanol Curriculum Page 24

The Model for Bioﬁlm Control Page 32

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Customer Service Please call 1-866-746-8385 or email us at service@bbiinternational.com. Subscriptions to Ethanol Producer Magazine are free of charge to everyone with the exception of a shipping and handling charge for anyone outside the United States. To subscribe, visit www.EthanolProducer.com or you can send your mailing address and payment (checks made out to BBI International) to: Ethanol Producer Magazine Subscriptions, 308 Second Ave. N., Suite 304, Grand Forks, ND 58203. You can also fax a subscription form to 701-746-5367. Back Issues, Reprints and Permissions Select back issues are available for $3.95 each, plus shipping. Article reprints are also available for a fee. For more information, contact us at 866-746-8385 or service@bbiinternational.com. Advertising Ethanol Producer Magazine provides a specific topic delivered to a highly targeted audience. We are committed to editorial excellence and highquality print production. To find out more about Ethanol Producer Magazine advertising opportunities, please contact us at 866-746-8385 or service@bbiinternational.com. Letters to the Editor We welcome letters to the editor. Send to Ethanol Producer Magazine Letters to the Editor, 308 2nd Ave. N., Suite 304, Grand Forks, ND 58203 or email to lgibson@bbiinternational.com. Please include your name, address and phone number. Letters may be edited for clarity and/or space.

4 | ETHANOL PRODUCER MAGAZINE | MARCH 2019

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PUBLISHING & SALES

MARCH 2019 VOLUME 25

FEATURES

CONTENTS

DEPARTMENTS YEAST

‘The Right Eﬀect’

Genetic engineering accelerates yeast development By Susanne Retka Schill

MASCOMA LLC

EDUCATION

Planting the Seeds

Programs, funding encourage ethanol curriculum By Matt Thompson

ISSUE 3

KANSAS CORN GROWERS ASSOCIATION

BIOFILM

The Big Picture

AD INDEX

EDITOR'S NOTE

EVENTS CALENDAR

DRIVE

GLOBAL SCENE

Clariﬁcation and Education By Lisa Gibson

Ethanol’s Global Growth By Craig Willis Campaigning on Climate By Andrea Kent

GRASSROOTS VOICE

Will 2019 Finally be the Year of E15? By Ron Lamberty

BUSINESS BRIEFS

MARKETPLACE

Prevention brings direct, indirect benefits By Matt Thompson

CHIPPEWA VALLEY ETHANOL CO.

CONTRIBUTION

38 FERMENTATION

Consequences of a Lower pH

Common solution to bacteria might not be the best By Dennis Bayrock Ethanol Producer Magazine: (USPS No. 023-974) March 2019, Vol. 25, Issue 3. Ethanol Producer Magazine is published monthly by BBI International. Principal Oﬃce: 308 Second Ave. N., Suite 304, Grand Forks, ND 58203. Periodicals Postage Paid at Grand Forks, North Dakota and additional mailing oﬃces. POSTMASTER: Send address changes to Ethanol Producer Magazine/Subscriptions, 308 Second Ave. N., Suite 304, Grand Forks, North Dakota 58203.

ON THE COVER

Emily Stonehouse, research and development manager at Mascoma LLC, a division of Lallemand Inc.

ETHANOLPRODUCER.COM | 5

EDITOR'S NOTE

Clarification and Education I have to laugh when I see labels that say “non-GMO” on food products that haven’t been genetically modified anyway. Peanuts and

Lisa Gibson

Editor lgibson@bbiinternational.com

sunflower seeds, for example. There aren’t any GM peanuts or sunflower seeds on the market, so the label is a feel-good tactic aimed at people of the mindset that GM food is tainted, unhealthy or somehow harmful. It showed up on potatoes before any GM potatoes were available. Many people don’t quite understand what genetic modification actually is and how it’s done. In my days as the editor of an agricultural journal, I read and published multiple letters from readers, clarifying that GM was the practice of coaxing evolution in organisms, using traits from other naturally occurring organisms. Cold and pest tolerance in crops, for example. It’s not artificial food. It doesn’t involve chemicals. Anyway, I digress. My point is that genetic modification is largely misunderstood. In the ethanol industry, we have a better handle on it because yeast is genetically modified. It’s amplified to make sure it’s tolerant of high temperatures, solids and organic acids; it’s altered to inhibit glucoamylase production. Yeast for ethanol production is tailored to produce ethanol efficiently. It’s the focus of the cover story, starting on page 16. Freelancer Susanne Retka Schill delves into the specific tactics used by yeast providers in modifications of their products. The practice has been polished by those who do it, shaving off time and money. One source even says widespread genetic modification of yeast could help educate the public on the process, prompting its acceptance. I really hope so. Education is crucial, whether it’s about GMOs, or ethanol itself and its many benefits. In Kansas and Ohio, state corn groups have teamed up to offer workshops to train teachers on ethanol education. Then, they provide materials and funding so those teachers can educate their students, offering labs and visits to ethanol plants. It’s an incredibly useful and effective strategy in those states. Turn to page 24 to find out more. Our last feature in this issue focuses on biofilm. Bob Jewell from Chippewa Valley Ethanol Co. is vocal about the measures his plant takes to prevent and control it. From grime to gleam, a biofilm-free cooling system can affect many facets of a plant’s performance. It starts on page 32. Back to yeast, the contribution at the end of the magazine focuses on health, laying out some problems caused by standard bacteria-control strategies. It seems the common themes through this issue are clarification and education. In our industry, those concepts are crucial and, rightfully, are dedicated much time and attention. Now, if only we could communicate our knowledge to the peanut and sunflower seed sectors.

FOR INDUSTRY NEWS: WWW.ETHANOLPRODUCER.COM OR FOLLOW US: 6 | ETHANOL PRODUCER MAGAZINE | MARCH 2019

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EVENTS CALENDAR

2019 International Fuel Ethanol Workshop & Expo June 10-12, 2019 Indiana Convention Center Indianapolis, Indiana

From its inception, the mission of this event has remained constant: The FEW delivers timely presentations with a strong focus on commercial-scale ethanol production—from quality control and yield maximization to regulatory compliance and fiscal management. The FEW is the ethanol industry’s premier forum for unveiling new technologies and research findings. The program covers cellulosic ethanol while remaining committed to optimizing existing grain ethanol operations. 866-746-8385 www.fuelethanolworkshop.com

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2019 ACE Conference August 14-16, 2019 Omaha Marriott Downtown Omaha, Nebraska The ACE Conference is a must-attend event for industry leadership. Relaying timely updates on public policy, market development, board of director training, and much more, this event combines the detail of high-level training courses with all the fun of a family reunion. (605) 334-3381 www.ethanol.org/events/conference

Please check our website for upcoming webinars www.ethanolproducer.com/pages/webinar

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DRIVE

Ethanol’s Global Growth By Craig Willis

While trade disputes and regulatory challenges have had a significant impact on demand, U.S. biofuels have still seen unprecedented popularity on the global front in recent years. In fact, ethanol exports reached record highs in 2018—expected to be the most successful calendar year for biofuels exports in history. With 2018 export figures— reported through October—reaching a record 1.41 billion gallons, we have already eclipsed the 1.37 billion gallons exported in 2017. Just last year, my colleagues and I logged 19 international trips to promote trade and forge relationships with key industry officials and stakeholders. We also submitted comments on five separate biofuelsrelated regulations abroad in places like Canada, the European Union, the United Kingdom and Japan. As we look to 2019, one of our key priorities at Growth Energy is to ensure that American producers are well-positioned to take advantage of burgeoning markets as countries seek to leverage ethanol’s environmental and economic benefits.

Growth in the Americas

In 2018, North and South America made up over half of our total ethanol exports at 840 million gallons. The bulk of these exports stemmed from large markets in Canada and Brazil, with Brazil alone making up over half of the region’s total intake. Export success in these primary markets is a result of the blending standards they have in place, with Brazil having the highest standard globally at a 27 percent minimum blend and reaching an overall blend rate of 47.8 percent. Canada has also consistently committed to blending biofuels to keep fuel prices low and meet its environmental goals, and last year considered two major pieces of biofuels-related legislation. In particular, the province of Ontario, historically a leader among Canadian provinces and the country’s largest transportation market, is at the leading edge for biofuels in the country and recently announced a commitment to implement an E15 requirement by 2025 to meet its emissions reduction goals. Over the years, Canada has remained a consistent importer of U.S. ethanol and could therefore see even higher growth in exports in the coming years to fulfil its new blending requirements.

The steady intake of U.S.-produced ethanol by North and South America reflects the growing popularity among foreign governments and consumers to make the move toward more affordable and environmentally friendly biofuels. While Brazil’s ethanol demand tends to be more volatile because of its domestic sugar and ethanol industries, the consistency we see from Canada provides a level of certainty for U.S. growers and producers as they forecast the years ahead.

Potential in the East

We are also seeing tremendous export potential and growth outside of the Americas. Asia ranks second in terms of exports, importing 452 million gallons, and many countries within Asia have begun implementing their own biofuels legislation. China, in particular, is in the process of implementing a nationwide biofuels requirement that aims to achieve a 10 percent blend rate for ethanol by 2020. At the start of 2018, China was on pace to be a regional leader in ethanol imports, importing over 52 million gallons in just the months of February and March. By comparison, in 2016, American producers exported nearly 200 million gallons to China alone. But trade tensions between the two countries have meant a near halt in exports to China in 2018, depriving American producers and farmers from access to a key export market and slowing the pace at which China can meet its climate goals. Trade negotiations and disputes must be resolved in a timely manner, not only to meet China’s needs, but also to open back up a critical market for U.S. agriculture. Despite significant setbacks, the global ethanol export outlook reached unprecedented heights in 2018 and should only continue growing in 2019, as more countries begin to blend ethanol into their fuel supplies. The untapped potential across the Americas, Asia and other continents is tremendous. By building biofuels markets abroad and forging strong trade relations, our industry can ensure stable markets for America’s producers and farmers. As we head into 2019, Growth Energy remains committed to supporting burgeoning ethanol markets to promote the benefits of biofuels both at home and abroad.

Author: Craig Willis Senior Vice President, Growth Energy 217.358.6182 cwillis@growthenergy.org

8 | ETHANOL PRODUCER MAGAZINE | MARCH 2019

GLOBAL SCENE

Campaigning on Climate By Andrea Kent

2019 is an election year in Canada and campaigning is already underway. Taking early center stage

is climate change. Whether it’s a policy to eliminate it, reduce it or price it, carbon is the new currency and political parties are battling over who will pay. First, we have the Liberal Party seeking re-election. Under the leadership of Prime Minister Justin Trudeau, the Liberals ran and won on an environmental agenda in 2015. At the top of their list was putting a price on carbon pollution. Three years into their four-year term, the government delivered a national carbon price and is developing a Clean Fuel Standard. Both policies are ambitious and (either via tax or life cycle carbon analysis) are designed to curtail emissions. They also prove politically contentious when it comes time to explain to the public and industry what the price will be and who will bear the cost. With almost 10 months before Canadians go to the polls, the Liberals are facing projections from their own environment department showing Canada will fall far short of its greenhouse gas (GHG) commitments, as well as allegations from political opponents of a skyrocketing carbon price. This time around, Trudeau’s climate action is coming with a price tag. Second are the Conservatives, who so far have not released their environmental plan. They are, however, very clear in their opposition to pricing as a way to reduce GHG emissions. While we don’t yet know what climate policies the federal Conservatives would implement, it most certainly will not be a price on carbon. And for some voters, just knowing that could be enough. Consider the new Conservative government in Ontario as a good and recent example. Ontario Premier Doug Ford promised to repeal Ontario’s carbon pricing Cap and Trade Program if elected; he won, and he did. Different parties bring different principles and values to public policy including carbon pricing; that’s no surprise. But even the broadest spectrum has some potential for common ground. Politics aside and a carbon price notwithstanding, every candidate wants to find solutions that will help the environment without hurting the economy. And that’s where ethanol comes in, strong. Want a carbon price? Choose ethanol. Transportation continues to be one of the heaviest emitters of GHGs, and it is near impossible to meet any reduction target without addressing fossil fuel use. An

10 | ETHANOL PRODUCER MAGAZINE | MARCH 2019

effective carbon pricing system puts a visible price on the life cycle carbon emissions of all fuels. Simply put, the higher the carbon intensity of a fuel, the higher the price will be. Consumers and businesses will choose to use cleaner fuels to avoid paying a higher carbon price. Ethanol reduces GHG emissions by as much as 60 percent compared to gasoline, so the potential for increased biofuels use, including ethanol, can be especially large in jurisdictions that have a carbon price. Oppose a carbon price? Choose ethanol. Carbon pricing might be in the spotlight, but regulations still do most of work (i.e., phasing out coal and biofuels mandates). Every year, Canada’s federal mandate reduces GHG emissions by 4.2 megatonnes, the equivalent of taking 1 million cars off the road. And now, Canada’s new CFS will use life cycle carbon analysis to incentivize the use of low-carbon fuels and technologies. Properly designed, the CFS will recognize the value of low-carbon fuels like ethanol, and establish market conditions to capture and return that value to producers, blenders and consumers. As for GHGs, analysis by Renewable Industries Canada shows that upwards of 20 megatonnes of annual GHG reductions could come from biofuels under the CFS, due mainly to higher ethanol blends (e.g., E15 to E25). And, for economic growth, choose ethanol. It’s rare for an industry to increase production while at the same time reducing emissions, but that’s precisely what ethanol producers do. Ethanol facilities support agriculture and are anchor tenants in rural communities. Canadian renewable fuel producers create $3.7 billion in economic activity every year. In Ontario, local biofuels producers employ 733 people, buy approximately 145 million bushels of corn, and deliver $1.75 billion in economic activity. It is no coincidence the province recently announced its intention to expand ethanol blending to 15 percent by 2025. Come October, Canadians will return to the polls and face a range of climate policies. Some will think politicians are going too far while others not far enough. But if anything is sure, it’s that political indifference on climate isn’t a viable option for winning elections anymore. And while carbon has become highly political, any environmental policy wins with ethanol, whether you price carbon or not. Author: Andrea Kent Vice President of Government and Public Relations, Greenfield Global Board Member, Renewable Industries Canada 1.833.476.3835 andrea.kent@greenfield.com

GRASSROOTS VOICE

Will 2019 Finally be The Year of E15? By Ron Lamberty

Retailer interest in E15 increased over the past few months, with many retailers believing President Donald Trump “signed something” making E15 legal year-round at an event in Council Bluffs, Iowa, in October. We’ve explained

there wasn’t any law or rule put into place at the rally, and there’s a process to be completed before the E15 summer RVP “fix” is a reality, but most retailers we’ve talked to seem confident E15 yearround will be the law because the president said it would be the law. We expect increased interest from other retailers (who don’t follow politics as closely) when the U.S. EPA submits the rule the president promised (soon, we hope), and petroleum and convenience store industry press covers the rule and process between now and Memorial Day. Again, assuming the rule is submitted soon, this new curiosity about E15 should coincide nicely with the half-dozen or more petroleum marketer conferences and trade shows ACE is attending this spring. Station owners and operators who can’t attend conferences or workshops can get the most current information available on flexfuelforward.com, where retailers who have already “been there and done that” with E15 are available to share their realworld experiences adding and marketing E15. Everything from regulations to incentives to RIN-less ethanol suppliers to fuel marketer war stories, as well as links to other ethanol information sources, can be found at flexfuelforward.com. One of our most pressing challenges is correcting a mixed message we’ve unintentionally put out to the fuel retailing community, especially the single-store and small-chain owners and operators. When we encouraged retailers to try E15 after EPA granted its partial waiver in 2011, we said it would cost little or nothing to add this new fuel grade. Ethanol opponents threw around outrageous cost numbers for adding E15, usually obfuscating the truth using E85-compatible equipment costs, and always assuming station

12 | ETHANOL PRODUCER MAGAZINE | MARCH 2019

owners would have to add new pumps, tanks and lines to offer a new grade of fuel. Apparently, oil companies can’t understand why station owners would be willing to give up premium sales making up a few percent of their fuel volume, or the midgrade infrastructure that is as good as the day Big Oil forced stations to install it, since nobody ever buys midgrade. Changing one of those products to E15 costs next to nothing and 90 percent of cars on the road can use it. What’s holding everyone up? Unfortunately, we muddied that message ourselves when the U.S. Department of Agriculture’s Biofuels Infrastructure Partnership (BIP) program was announced. Suddenly we were touting $200 million in grants from USDA, states and ethanol industry initiatives for pumps, tanks and other equipment for blends above 10 percent ethanol. That confused people we had recently told there was very little cost to add E15. After all, why would they need millions of dollars to offer a product we can sell with existing equipment? And while touting E15’s introduction to the market by large, respected retail convenience store chains captured the attention of most of the c-store industry, it also created backlash among the single and small chains mentioned earlier. First, there was the perception big retailers put in E15 because only big retailers could, reinforcing the “high cost” propaganda. Then, when press reports showed their “rich” competitors got all the BIP money and other funds, single-store and small-chain owners—the people who were first to move ethanol in the past—had enough of E15. That perception is not reality, and we must correct it if E15 is ever going to spread nationwide. ACE looks forward to continuing to do our part in the coming months and beyond.

Author: Ron Lamberty Senior Vice President American Coalition for Ethanol 605.334.3381 rlamberty@ethanol.org

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BUSINESS BRIEFS

People, Partnerships & Projects

Katzen promotes Vigil to president Tara Vigil has been promoted to president of Katzen International Inc. Phil Madson, who had been the president since 1993, will remain chairman and CEO. Vigil joined Katzen as a chemical engineer in 2002. After a succession of engineering and management positions, she was named vice president of business development in 2012. In her role as president, Vigil is responsible for leadership and direction of Katzen’s engineering, technology development and business development operations.

“We are proud to announce Tara’s promotion to president and it is in recognition of the leadership that she has demonstrated progressively during her career,” Madson says. “Her management competency, expertise and strong business acumen have contributed to the solid execution of our operational strategies and substantial growth.” Madson says the move will provide him with a greater opportunity to support the executive team, engage in corporate strategic planning, and become more directly involved

in project development with clients. “Working together, and with the support of our talented and dedicated associates, this will ensure Katzen’s continued Vigil commitment to our clients, which is the hallmark of our company,” Madson says.

Phibro hires tech service rep Jim Ekenstedt has joined Phibro’s Ethanol Performance Group as senior technical service representative. Ekenstedt’s duties will include supporting EPG’s sales team, ensuring field qualification of products and providing overall technical support for customers.

Ekenstedt joins Phibro with a depth of hands-on industry experience from working within ethanol plants and as a provider of technical services to the ethanol market. He received a bachelor’s degree in microbiology from South Dakota State University in Brookings.

“We are excited to have someone with Jim’s level of expertise join Phibro EPG,” says Jenny Forbes, vice president of products and services for EPG. “The knowledge and experience that Jim brings to Phibro will provide great leadership and continued top-notch technical support for our customers.”

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14 | ETHANOL PRODUCER MAGAZINE | MARCH 2019

BUSINESS BRIEFSÂŚ

FuelIowa recognizes fuel marketing success Iowa Secretary of Agriculture Mike Naig presented biofuel marketing awards to two central Iowa-based companies. Quick Oil Co. in Perry was winner of the 2019 Biodiesel Marketing Award, and Caseyâ&#x20AC;&#x2122;s General Stores, headquartered in Ankeny, received this yearâ&#x20AC;&#x2122;s Ethanol Marketing Award. The annual awards, initiated 11 years ago by FuelIowa in partnership with the Iowa Department of Agriculture and Land Stewardship, recognize fuel marketers going above and beyond to promote and sell home-grown biofuels. â&#x20AC;&#x153;We commend Caseyâ&#x20AC;&#x2122;s General Stores for their outstanding efforts to educate consumers about higher blends of ethanol,â&#x20AC;? says Dawn Carlson, FuelIowa president and CEO. â&#x20AC;&#x153;Caseyâ&#x20AC;&#x2122;s has committed to offering E15 at more than 500 locations over the

next four years and currently has 13 stores with E15 and E85. In addition, they have participated in educational campaigns to drive consumer interest in these products and have provided significant discounts to draw attention to the new alternative fuels.â&#x20AC;? Naig presented the awards during the FuelIowa Annual Membership Meeting in Des Moines, Iowa. The Marketing Awards are designed to honor exceptional efforts to market ethanol and biodiesel through commitment and belief in the product. Winners are selected based upon five core criteria: product offerings, consumer education, hosting special events highlighting Iowa Secretary of Agriculture Mike Naig ethanol, initiation of new advertisements presents the Ethanol Marketing Award to Doug Beech, of Casey's General Stores. or marketing efforts, and commitment to increase biofuel availability. FuelIowa is the state trade association representing fuel marketers.

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YEAST

‘The Right Effect’ Yeast can be genetically modified through multiple techniques, all offering advancements for the ethanol industry. By Susanne Retka Schill

Genetically modified (GM) yeasts have found their place in the ethanol industry with more than half of producers routinely using them. All yeast providers,

plus enzyme providers expanding into yeasts, now offer GM yeasts in their portfolios. DuPont Industrial Biosciences has Synerxia, Lallemand Biofuels & Distilled Spirits has TransFerm, Leaf-LeSaffre has ER-Xpress, DSM has eBoost, Novozymes has Innova, Taurus Energy has XyloFerm. “The fuel ethanol market was a logical place to introduce these types of yeast,” says Emily Stonehouse, research and development manager at Mascoma LLC, a division of Lallemand Inc. “This industry has allowed us to demonstrate the safety and build trust in the use of a GM yeast and I believe this will have an effect on the acceptance of GM yeast outside of fuel ethanol.”

“Yeast research has been going on for a very long time,” says Pauline Teunissen, principal scientist at Dupont Industrial Biosciences. “We do know a lot about yeast, but to be honest, every time you make a change, if you do genetic engineering, you will need to do some research to figure out what the effects are because it can have multiple reactions in a yeast cell.” The research on yeast metabolism took off when it was sequenced in 1996—the first organism with a nucleus and DNA organized in chromosomes to be fully sequenced. Earlier work had been done with viruses and bacteria with simpler genetic structures. It took 92 laboratories around the world working for three years to sequence Saccharomyces cerevisiae. The work opened the way to learning how genes impact cell function and how metabolic pathways work, and led to yeast becoming the focus of multiple genetic engineering studies. Sixteen years later, in 2012, the first GM yeasts entered the ethanol

16 | ETHANOL PRODUCER MAGAZINE | MARCH 2019

PORTABLE SEQUENCER: Kayla Eudy, Mascoma research associate, uses the sequencer attached to her laptop to determine the genetic changes in a newly engineered yeast strain. DNA sequencing technology has advanced to the point where portable sequencers can be used to rapidly sequence and analyze a sample. PHOTO: MASCOMA LLC

ETHANOLPRODUCER.COM | 17

market when LBDS introduced TransFerm, a glucoamylase-expressing strain engineered with a single fungal gene, and added a glycerol-reducing pathway the following year. DuPont introduced Synerxia in 2013, and other companies quickly followed. Today, the speed and cost of genome sequencing has improved to the point where any strain can be sequenced in less than a week at a cost around $3,000. Plus, new genetic en-

gineering tools are promising to speed up the process of modifying and testing new strains. â&#x20AC;&#x153;Genetic engineering has changed rapidly,â&#x20AC;? Stonehouse says. â&#x20AC;&#x153;Techniques used to be much less precise.â&#x20AC;? Work to add enzyme expression, for example, involved adding DNA extracted from an enzyme-producing organism into the chromosome of yeast and then screening the strains for high

expression of the enzyme. The early techniques often used antibiotic markers that later had to be removed. Introducing foreign genes is one technique, Teunissen says. Other techniques could be considered an acceleration of evolution. â&#x20AC;&#x153;The techniques involve putting yeast in a certain environment where you can show certain genes are expressed more than others, and then you actively work

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YEAST

MODIFIED AND ISOLATED (FAR LEFT): The modified yeast are isolated on selective growth media. Researchers test these prototype strains to ensure the correct genetic changes are present and to determine if their design caused the intended outcome, such as new enzyme activity or higher ethanol production. PHOTO: MASCOMA LLC

MASH MODIFICATIONS (LEFT): A corn mash fermentation is used to evaluate the performance of various modified yeasts. PHOTO: MASCOMA LLC

on those genes to turn them on or off, as needed.” To do the work, researchers rely on yeast genome libraries and their own collections of yeast strains that can number in the thousands. “Because the genome has been sequenced for yeast, and we know each gene that is present, we can compare our own new yeast strain with the publicly known yeast genome,” Teunissen says. “We

may see 50 differences, and then we zoom in to find out which are most important. And in some cases, it’s not just one gene, but multiple genes that work together to get the right effect.”

How it Works

Yeast genetics involve about 6,000 genes comprised of combinations of adenine, thymine, cytosine and guanine

(ATCG), sugar-phosphate molecules with a different amine group hanging off each one that become the basic building blocks of DNA. In sequencing, colored dyes enable a computer to read the ATCG sequence. In genetic engineering, enzymes are used to copy or chemically synthesize the DNA strands from a related yeast, fungi or bacteria with a desired trait. Those DNA fragments are introduced to the yeast chro-

ETHANOLPRODUCER.COM | 19

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20 | ETHANOL PRODUCER MAGAZINE | MARCH 2019

mosomes in the cell nucleus. Then the work begins to determine whether the desired trait was successfully transferred and how the other parts of the yeast metabolism responded. Just as the cost and speed of sequencing have been dramatically reduced, the time and cost for genetic engineering is dropping. One of the new tools getting a lot of attention is CRISPR-CAS9. (CRISPR stands for clustered regularly interspaced short palindromic repeats). In researching the function of those unique CRISPR sequences in bacteria, scientists determined the CAS9 enzyme action was important in the microorganism’s immune system. When attacked, the bacteria produce enzymes to destroy a virus by cutting up its genetic material, storing some of the virus’ genetic code to be used to identify and enzymatically destroy similar viruses. By feeding manufactured code to the CRISPR sections, the researchers learned they could target a specific spot on a chromosome, knocking out the targeted gene and enabling a gene substitution, if desired. “CRISPER can do in one step what took multiple steps and multiple days with the old technique,” Teunissen says. The cost of the procedure has also dropped from around $20,000 to less than $50. Examining all the effects of genetic changes is important, Teunissen says. “We cannot always predict which genes are turned on or off, so we need to test that.” In adding a metabolic pathway to yeast using three enzymes to more efficiently convert glucose to ethanol, she says, the yeast began making different cellular byproducts. When changes like that are found, the researchers have to evaluate whether the impact

YEAST

FINAL FREEZE: Top yeast strains are preserved by freezing in small vials for future production and commercial-scale testing. PHOTO: MASCOMA LLC

is positive, negative or neutral. In the case of glycerol reduction strategies, it’s important to remember that glycerol contributes to yeast health, she says, and diverting too much into ethanol production might result in yeast cells that can’t handle the stresses later in the fermentation cycle. “Whenever you change something, you will need to test for the other effects,” Teunissen says. “What I notice when I talk to ethanol producers, particularly in the beginning when they began using GM yeast, was that they are hoping for something that fits into the process, so they don’t have to make changes,” Teunissen says. There needs to be a willingness to tweak process conditions to optimize for the new yeast, which might involve adjustments to pH, temperature staging or enzyme dosing. The success of a new yeast is dependent upon the interaction of the biotech team and technical sales and service team, Stonehouse says. “We have to understand what the real process is and how to scale our laboratory testing of new yeasts to more process-relevant testing.” The technical service team also works with plants to tailor the process to realize the value of the new yeast, she says. “It can be small tweaks, often it’s the timing of things.”

there were any impacts on the meat or milk. Getting GRAS status (generally recognized as safe) and a feed definition with the American Association of Feed Control Officials were important, though time-consuming, steps. The advances in genetic engineering are having an impact here as well, Stonehouse says. “The regulatory agency can ask us for the full genome sequence. It’s a powerful tool to make sure the targeted change

was done. Sequencing is a powerful tool to layer on top of genetic engineering to gain knowledge, but also to build trust in what you’ve done.” It’s important, she says, “that the industry understands the care that goes into the development of the new yeasts and the good science behind them.” Author: Susanne Retka Schill Freelance Journalist retkaschill@yahoo.com

Care and Good Science

“The competition among the companies is driving innovation,” Stonehouse says, and she notes that groups are already moving beyond glycerol reduction and glucoamylase expression to look at other enzymes and other metabolic enhancements. Engineering new yeasts goes beyond evaluating their performance in fermentation to ensuring the acceptability of the feed coproduct. In introducing TransFerm, Stonehouse says Lallemand went the full regulatory route of convening a panel of independent experts to evaluate their scientific work. Animal feeding trials involved not only feed performance, but whether

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EDUCATION

PLANTING

24 | ETHANOL PRODUCER MAGAZINE | MARCH 2019

THE SEEDS

Agricultural organizations in Kansas and Ohio have programs aimed at ethanol education, training teachers and even providing funding to ensure the knowledge is passed on to students. By Matt Thompson

ETHANOL IN ACTION: Teachers in Kansas who participate in the Seed to STEM program tour East Kansas Agri-Energy in Garnett. These teachers also got a chance to see the Renewable Fuels Associationâ&#x20AC;&#x2122;s custom E85 chopper. PHOTO: KANSAS CORN GROWERS ASSOCIATION

ETHANOLPRODUCER.COM | 25

EDUCATION

For at least one student in James Burkeâ&#x20AC;&#x2122;s engineering class at Trego County Community High School in WaKeeney, Kansas, ethanol is a passion. â&#x20AC;&#x153;I

have them test different factors and kind of design their own recipe for how they would best produce a fermentation for ethanol â&#x20AC;Ś. We havenâ&#x20AC;&#x2122;t got to it yet this year, that part of it. Weâ&#x20AC;&#x2122;re working on some wind energy stuff, and he keeps chomping at the bit wanting to get back to ethanol.â&#x20AC;? Burke says the student is so excited about ethanol, this is the second year heâ&#x20AC;&#x2122;s voluntarily enrolled in the engineering class. Burke, a science teacher, is able to use those ethanol production labs in his classes thanks, in part, to an education effort undertaken by Kansas Corn, a state corn advocacy group made up of the Kansas Corn Growers Association and the Kansas Corn Commission. The focus is on training Kansasâ&#x20AC;&#x2122; K-12 teachers to use ethanol, corn and biotechnology curriculum in the classroom, says Sharon Thielen, educational curriculum manager for Kansas Corn. â&#x20AC;&#x153;Our goal is to provide lessons and materials so that Kansas teachers can start teaching about these topics as early as kindergarten and then get

more advanced into those topics as they get closer to graduating high school.â&#x20AC;? Through the program, Kansas Corn hosts summer workshops for the stateâ&#x20AC;&#x2122;s teachers, and trains them on specific labs relating to corn and ethanol. Those efforts grew out of a request from the KCC, which is made up of Kansas farmers, and determines how to use the organizationâ&#x20AC;&#x2122;s checkoff funds. â&#x20AC;&#x153;About five years ago, they started having strategic planning meetings about where they were going to spend some of those commission dollars,â&#x20AC;? Thielen says. â&#x20AC;&#x153;They decided that they wanted to get more into the K-12 classroom and start making those linkages with science and agriculture, but specifically looking at biotechnology and ethanol, because we wanted our youth and our teachers to understand the importance of those two areas to our industry.â&#x20AC;? Burke is one of Kansas Cornâ&#x20AC;&#x2122;s lead teachers who writes some of the programâ&#x20AC;&#x2122;s labs and helps train other teachers during the workshops. He says the program is successful in helping students understand where and how the commodities grown in their backyards are used. â&#x20AC;&#x153;Thereâ&#x20AC;&#x2122;s a ton of science thatâ&#x20AC;&#x2122;s there. Thatâ&#x20AC;&#x2122;s just a really cool way to make it all relevant for students and tying it in with whatâ&#x20AC;&#x2122;s going on and whatâ&#x20AC;&#x2122;s

important in their environment and economy,â&#x20AC;? Burke says. Ohio Corn & Wheatâ&#x20AC;&#x2122;s education program, Feed the World, is also funded through the stateâ&#x20AC;&#x2122;s checkoff program. Unlike Kansasâ&#x20AC;&#x2122; program, Ohio focuses on middle school teachers, says Brad Reynolds, director of communications for Ohio Corn & Wheat. â&#x20AC;&#x153;One way that they really wanted to get in with the education part of it was ... reaching middle school science teachers who are more and more removed from the farms themselves,â&#x20AC;? Reynolds says. Like Kansasâ&#x20AC;&#x2122; program, Feed the World provides curriculum, supplies and training for teachers on topics related to corn and ethanol.

Ethanol Awareness

In Kansas, ethanol producers are happy with Kansas Cornâ&#x20AC;&#x2122;s education efforts. â&#x20AC;&#x153;The Renew Kansas board, which makes up basically all the CEOs of the ethanol plants in the state, are beyond supportive and very happy with how weâ&#x20AC;&#x2122;re doing,â&#x20AC;? Thielen says. â&#x20AC;&#x153;So just their continual support is evidence, too, that we must be doing a good job.â&#x20AC;? Derek Peine, CEO of Western Plains Energy and a member of the Renew Kansas board, says the success of the program is evident when he meets teachers at events and gatherings. â&#x20AC;&#x153;From my perspective,

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just random interactions with people have shown me that it is working and that there are teachers out there that are enjoying it and liking it,â&#x20AC;? he says. The benefits of the program, he adds, are providing teachers with supplies for educating students about ethanol, but also growing awareness about the fuel. â&#x20AC;&#x153;From my standpoint, I think itâ&#x20AC;&#x2122;s one of the best things we have going in the state as far as public awareness and education about ethanol,â&#x20AC;? he says. â&#x20AC;&#x153;Itâ&#x20AC;&#x2122;s actually, I think, just a huge success for the state of Kansas.â&#x20AC;? On a national level, Kansas Corn was recognized by Curriculum for Agricultural Science Education for partnering with Renew Kansas to provide teachers the supplies needed to use labs focused on emissions, energy in feed, renewable fuel and energy output in their classrooms. The effort to fund the supplies for CASEâ&#x20AC;&#x2122;s labs led to Kansas Corn partnering with CASE to train all the stateâ&#x20AC;&#x2122;s ag teachers. â&#x20AC;&#x153;The national CASE office has recognized the contribution that weâ&#x20AC;&#x2122;re making, and now theyâ&#x20AC;&#x2122;re allowing Kansas Corn, in partnership with them, to train all ag teachers in our state in that ethanol lab,â&#x20AC;? Thielen says. â&#x20AC;&#x153;Theyâ&#x20AC;&#x2122;ll at least be able to get trained in that one lab so that they can get the financial support for those supplies.â&#x20AC;?

EQUIPPED FOR SUCCESS: Two teachers begin the process of making ethanol during one of Ohio Corn & Wheatâ&#x20AC;&#x2122;s Feed the World workshops. The program provides teachers with curriculum and supplies to teach corn- and ethanol-specific labs in their classrooms. PHOTO: OHIO CORN & WHEAT

Beyond the Classroom

Thielen and Reynolds say their programs are exceedingly popular among teachers. â&#x20AC;&#x153;The teachers, they love it,â&#x20AC;? Thielen says. â&#x20AC;&#x153;They think our workshops are some of the best out there.â&#x20AC;? In 2018, Kansas Corn either trained or provided supplies

to 725 teachers during the school year, who, in turn, taught about 20,500 students. And, Thielen says, most of the budget for those programsâ&#x20AC;&#x201D;87 percentâ&#x20AC;&#x201D;went directly to Kansas teachers. â&#x20AC;&#x153;Basically, corn producers should feel really good that their dollars are going right back into education,â&#x20AC;? she says.

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ETHANOLPRODUCER.COM | 27

EDUCATION

CLEAN BURNING: A student burns kerosene for comparison with ethanol as part of a Curriculum for Agricultural Science Education clean smoke lab. PHOTO: KANSAS CORN GROWERS ASSOCIATION

“The ability to create curriculum and get that in front of students to learn about corn, and everything from corn dissections to making ethanol in a bag has really [had] great benefits for Ohio,” Reynolds says. According to Ohio Corn and Wheat, in 2018 the Feed the World program trained 82 teachers, and has trained a total of 320 since 2013. In addition, the lessons on ethanol are the most highly rated by the attending teachers. While there are differences between the programs, both offer supplies to teachers who attend the workshops, a benefit that does not go unnoticed by teachers and stakeholders in both states. “When they go through this program … [the teachers are] over the moon excited about taking this back to the classroom. How disappointing would that be if they go back and they can’t actually do it because they don’t have the tools? So we try to provide them all of the tools they need to do that. … It’s a critical component, I think, of what makes this a success,” Reynolds says. The same is true for Kansas, Thielen says. “What we have learned across all ethanol labs is they’re great, but the equipment is extremely expensive. So even though teachers are trained to teach them, they’re not teaching them because they don’t have the funds to buy the equipment.” Those supplies—and the continual support Kansas Corn offers teachers after they’ve attended 28 | ETHANOL PRODUCER MAGAZINE | MARCH 2019

the workshop—are what teachers appreciate the most about the program, she adds. “Basically, if they’re dedicated to teaching it, we want to keep giving them more resources.” Peine agrees. “One of the hardest things to do in public schools today, or any school, is funding. Especially funding for additional supplies. So it’s great to learn the material. It’s great to provide the teacher with the curriculum, but providing them with the supplies really makes a difference, and really allows them to take it to the next step. … The last thing we want to do is teach them something and then not give them the resources to teach it on to the students.” Several of Kansas’ larger school districts have encouraged all of their teachers to attend the workshops, Thielen says. “I think they see the vision and I think they know the importance of getting agriculture in the classroom, and so we’ve had a great response.” In fact, the program’s been so successful that many teachers have asked to attend the workshops more than once. But Thielen says that’s not ideal, as the focus is to allow as many teachers as possible to participate. But those requests led to a change in the upcoming summer’s workshops. “This summer is the first time we’re going to do a Seed to STEM 2.0, where we’re writing new labs, many about biodiesel and ethanol, where

those teachers are going to learn some more, and they’ll get more supplies to teach more labs. … If they are doing an exceptional job of teaching our labs already, let’s just keep them interested and keep providing more labs that they can teach, because that’s just going to help agriculture.” Seed to STEM is the part of the education program that focuses on curriculum for sixth through 12th graders. Kansas Corn’s workshops also feature tours to ethanol plants. “We work very closely with our ethanol plants and we now fund teachers to take their students to an ethanol plant after they do the distillation lab,” Thielen says. So does Ohio’s. “In Ohio, we have seven of them,” Reynolds says. “So, we try to get them into an ethanol facility where they can see it being made, see the process and how there’s not waste and it’s really environmentally friendly.” An ethanol plant visit for Burke’s engineering class is coming up this year, giving his ethanol-focused student a field trip to look forward to. “He’s really excited about that,” Burke says. Author: Matt Thompson Associate Editor, Ethanol Producer Magazine 701.738.4922 mthompson@bbiinternational.com

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BIOFILM

PREVENTATIVE PROTECTION: Chippewa Valley Ethanol Co.’s cooling towers are relatively new. Bob Jewell, CVEC’s energy systems chief, says the plant’s proactive approach to biofilm control helps protect the new cooling system. PHOTO: CHIPPEWA VALLEY ETHANOL CO.

32 | ETHANOL PRODUCER MAGAZINE | MARCH 2019

BIOFILM

the big Picture Chippewa Valley Ethanol Co.’s Bob Jewell shares what he’s learned about efficiency through biofilm control. By Matt Thompson

It takes a keen eye to notice, but it’s there, impacting systems and increasing inefficiencies. Because it’s hard to see, it likely won’t top every plant’s priority list, but controlling biofilm can have system-wide impacts.

Bob Jewell, energy systems chief at Chippewa Valley Ethanol Co. in Benson, Minnesota, says recognizing and controlling biofilm can pay off, not just by preventing fouling. “Being proactive can also result in numerous indirect benefits such as optimized usage and improved effectiveness of other treatment additives, reduced system corrosion rates, increased equipment longevity, heat exchangers

staying cleaner and for longer periods, and reduced maintenance requirements,” he says. Jewell says, historically, not all plants have paid much attention to biofilm control. He says that while there are plants with an effective biofilm control program, many plants’ cooling systems might not be operating as efficiently as they could be, because of biofilm. “I also think it is relatively common that the implications of biofilm are not fully appreciated or are undervalued.” But that’s changing, he says. With new technologies and more industry focus on the biofilm issue, more plants are starting to take a proactive look at detecting and controlling biofilm. “I don’t think that the industry’s response to the issue is related so much to attitudes, but rather to the problem just being

ETHANOLPRODUCER.COM | 33

BIOFILM

SPICK AND SPAN: Biofilm and solids have collected in cooling tower film fill on the left. The right shows new, clean cooling tower film fill. PHOTO: CHIPPEWA VALLEY ETHANOL CO.

brought to the forefront relatively recently and to education, awareness and perceptions catching up, which are all key in being able to identify, address and control it,” he says. Jim Lukanich, director of applied technology at U.S. Water Services, agrees. “That knowledge has been around for quite some time, but there’s just an increased focus on research because of what we understand in cooling water, and in other process water systems: that if we can control biofilm, it reduces the amount of problems we have with corrosion as well as mineral deposits,” he says. “However, due to the amount of research and observations in the field over the years, there is an increased understanding of bacteria, fungi and algae in general, … we understand biofilm can contribute to these other problems to a much larger degree than previously thought.” He adds that the recent focus on biofilm control isn’t unique to the ethanol industry. All industries that use cooling water in their operations have been more attentive to biofilm recently. It stems from an increased awareness about preventing Legionella, the bacteria

34 | ETHANOL PRODUCER MAGAZINE | MARCH 2019

that causes Legionnaires’ disease, Lukanich says. “By controlling biofilm and having an emphasis on biofilm, we not only minimize the potential for fouling, scale and corrosion, but also reduce the risks associated with the amplification of Legionella and associated disease.” According to Jewell, the American Society of Heating, Refrigerating, and Air Conditioning Engineers (ASHRAE) in 2015 developed a standard for preventing Legionella. While it isn’t enforceable unless it’s made a law by local or state governments, Jewell believes wider adoption is likely to happen in the future. “According to ASHRAE’s website, ASHRAE is asking its chapters to encourage municipal, state and federal governments and legislative bodies to adopt [the standard] into law,” he says. “The parts are all there and it was written with pathways for adoption in mind. Future cases or outbreaks are only likely to build more momentum toward its adoption.” Kevin Milici, vice president of marketing and technology for U.S. Water Systems, agrees. “In certain parts of the world … pathogen or risk management isn’t a

recommended best practice, it’s the law. … And there’s been a lot of discussion about, ‘Is that basic regulation coming to the United States?’ And I think the consensus is that, with time it will.” He adds that there are areas of the U.S.—like New York City—that have adopted the standard as law. But those laws apply to building water systems in places like hotels, healthcare facilities and apartment buildings, rather than industrial manufacturing facilities, like ethanol plants. Jewell says it’s natural for plants to begin focusing on controlling biofilm in their quest to optimize their operations. Many plants, he says, have undertaken improvements to debottleneck and increase efficiencies in other areas. With those visible issues resolved, plants are turning to less obvious ways to make their plants run more smoothly, and for many, that means evaluating their cooling systems and biofilm-control mechanisms. “As they’ve optimized and debottlenecked their processes and used up all that low-hanging fruit, it just leads them to looking other places that they hadn’t really entertained in the past,” Jewell says.

BIOFILM A Proactive Approach

For Jewell and CVEC, the interest in biofilm control began when the plant was evaluating debottlenecking opportunities. During that process, they discovered the plant’s cooling system was underperforming. “In trying to keep the plant efficient, and expand and debottleneck things, and optimize the plant, … we kept going after that cooling water system to try to optimize that as well, because that was one of the things that was holding us back. But in that whole exercise, it kind of revealed that we had some biofouling and biofilm issues, so we’ve been attuned to that for quite a few years.” A new cooling tower was eventually installed to replace the aging towers that had been in place since the plant’s construction in 1996, and Jewell says the plant’s current biofilm control measures grew, in part, out of a desire to protect the new components. “We wanted to make sure we protected those assets and didn’t end up reverting back to our old practices, which were not effective in preventing that biofilm,” he says. While Jewell says he doesn’t necessarily consider CVEC a model plant for fighting biofilm, he says they are proactive in their efforts. “I think that we are definitely one of the plants on the forefront regarding the utilization of some of the currently best available technology for measuring, monitoring, and control of biofilm. I do feel we are helping to bring awareness to the potential issues and implications of biofilm and to shape how that type of technology is utilized within the industry.” Part of the plant’s success comes from a continuous, real-time monitoring and control system for biofilm, which was installed by Solenis. Jewell says early detection is crucial in deploying biofilm countermeasures. “Recognizing biofilm formation at the onset is key, and a huge benefit in gaining control of it early before fouling occurs or other damage can be done,” he says.

‘A Holistic View’

That’s especially true since the conditions affecting biofilm growth are constantly changing. “Bacteria are moving targets, and they adapt quickly to change,” Lukanich says. As bacteria reproduce, they can become resistant to control methods that had worked in the past. “All of a sudden, they’ve turned on a mechanism by which they become more resistant to the level of chlorine being used, and so now

… you begin to get fouling, when for a year, everything was fine. … Nothing relating to an increase in organic carbon or anything else, it’s just an organism turned on a mechanism of adaptation. We have to pay attention to that activity.” But an increase in organic carbon— from a heat exchanger leak, for example— can be detrimental to biofilm control. “There’s nothing that causes the microbial activity in that tower to spike in an ethanol plant more than a heat exchanger leak,” Lu-

ETHANOLPRODUCER.COM | 35

BIOFILM

BIOFOULING: Biofilm led to fouling in this cooling tower fill. PHOTO: U.S. WATER

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kanich says. “You can go from being wellcontrolled to being out of control with respect to a fouling pretty much overnight.” He adds that small leaks can go unnoticed, so constant monitoring of the system is crucial to control biofilm. “If you go from 40 ppm of organic carbon in a tower to 100, there may not be anything in your daily testing that will tell you that, but it would change the activity of those organisms and therefore how you treat them on an ongoing basis,” he says. That constant monitoring can be difficult with some of the older methods, Jewell says. Prior to the plant’s current system, CVEC used coupons and other means to monitor biofilm and corrosion. The problem, he says, is the length of time it takes to get results. “You need to expose them to that for anywhere from seven to 90 days, depending on what you are trying to measure, so you end up responding to something that happened as much as three months previously. It’s hard to react to those types of delayed results.” Perhaps more important than continuous monitoring is understanding the cooling system and how it’s operating. “I think it’s one of the single most important things you can do, is just do an evaluation of all the equipment within the system to determine if you have deficiencies in cooling capacity or just performance of the equipment, and compare it to design or the as-built specifications,” Jewell says. “Because that’s the only way that you’re going to understand, number one, where your deficiencies lie, and that’s going to be how you determine if it’s just a deficiency in design or if it’s a deficiency due to some other reason such as biofilm.” Lukanich agrees. “You have to look at the facility from a holistic view. What are the things contributing to microbial growth and biofilm accumulation in your systems, what’s the history, what are you doing with respect to that? … I think it’s a starting point.” Author: Matt Thompson Associate Editor, Ethanol Producer Magazine 701.738.4922 mthompson@bbiinternational.com

36 | ETHANOL PRODUCER MAGAZINE | MARCH 2019

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CONSEQUENCES OF A LOWER PH Many plants decrease pH to treat bacterial contamination in fermentation, but this can cause numerous other issues. By Dennis Bayrock

38 | ETHANOL PRODUCER MAGAZINE | MARCH 2019

FERMENTATION

At an ethanol plant experiencing bacterial contamination, lowering the pH in the propagator/fermenter is typically the first step used to control bacterial growth. Phibro’s Diagnostic Kit services show that over 80 percent of the bacterial contamination in ethanol plants is from the lactic acid bacteria (LAB) family. The pH for optimal growth of most LAB is between 5.5 and 5.8. In contrast, S. cerevisiae yeast can tolerate and multiply better than most bacteria in acidic environments—from pH 4.0 to 6.5. Knowing this, it seems logical to lower the pH during contamination to inhibit the bacteria while allowing the yeast to grow. But should lowering the pH be the first option? Are there negative consequences to lowering pH for yeast and ethanol plants? Following is a list of issues to consider, along with a somewhat surprising solution: Raise the pH.

Sulfuric Acid

Lowering the pH in a fermenter requires an increased amount of sulfuric acid. With a liquefaction pH of 5.7 and a fermenter volume of 750,000 gallons, the amount of sulfuric acid typically needed to lower the fermenter pH to 5.2 is 4,286 pounds. But, to adjust the same fermenter to a pH of 4.5 requires 8,573 pounds of sulfuric acid. Sulfur is already a problem for many fuel ethanol plants to meet specifications for distilled ethanol. Excess sulfur in dried distillers grains with solubles (DDGS) also increases the risk of the fatal disease polioencephalomalacia (PEM) when fed to animals.

FLAWLESS FERMENTATION: Phibro Ethanol Performance Group’s Dennis Bayrock has a surprising suggestion to treat bacterial contamination in fermenters like these at Chippewa Valley Ethanol Co. in Benson, Minnesota. PHOTO: BBI INTERNATIONAL

CONTRIBUTION: The claims and statements made in this article belong exclusively to the author(s) and do not necessarily reflect the views of Ethanol Producer Magazine or its advertisers. All questions pertaining to this article should be directed to the author(s). ETHANOLPRODUCER.COM | 39

FERMENTATION

from the outside environment into the yeast require that a pH gradient is maintained across the membrane with a lower pH outside than inside. In addition, each yeast transporter has a specific and optimal pH. Moving the pH away from these optimal transport values decreases nutrient uptake.

Organic Acid Inhibition

The primary function of the yeast cell membrane is to physically house all of the various cellular organelles, structures, enzymes and biochemical reactions inside the cell. Another function of the yeast cell membrane is to keep materials that inhibit multiplication and metabolism—lactic and acetic acids, fatty acids and fusels—out of the cell.

GRAPHIC: PHIBRO ETHANOL PERFORMANCE GROUP

pKa and Inhibition

GRAPHIC: PHIBRO ETHANOL PERFORMANCE GROUP

Consequences for Yeast Growth

Yeast use pH as a signal for multiplication, fermentation and metabolism. In general, yeast multiply most efficiently at a pH higher than 5.0. In contrast, efficient fermentation is achieved at a pH lower than 5.0. Yeast does not grow below a pH of 2.8, although its metabolic activity continues, albeit at a lower rate than normal. Yeast multiplication rate increases nonlinearly as the pH increases, with optimal growth occurring at a pH of 5.5 to 6.0. During optimal propagation/fermentation, the pH is typically set to about 5.0. As the process continues, the pH naturally

40 | ETHANOL PRODUCER MAGAZINE | MARCH 2019

decreases, which also decreases the yeast growth rate. Complicating the picture somewhat is the fact that yeast cells have been evolutionarily “tuned” to multiply not only when the pH is about 5.5, but also when there is a natural pH difference of 1 to 2 units from the start to end of propagation/fermentation. If this pH difference is cut short (either by starting at a lower absolute pH, or preventing the pH from reaching this difference), yeast multiplication is significantly curtailed. Nutrition is part of the explanation. Many of the yeast cell transporters that move nutrients such as carbohydrates, ions and amino acids

Lactic and acetic acids are two of the more common organic acids found within contaminated fermenters at fuel ethanol plants. They are classified as weak acids with specific pKa values (lactic acid pKa is 3.86, acetic acid pKa is 4.74). By definition, weak acids do not fully dissociate in solution like strong acids, such as hydrochloric acid, do. If any solution of lactic acid is adjusted to a pH of 3.86, exactly one-half of the total lactic acid will be undissociated, and one half will be dissociated. Only the undissociated form of weak acids can physically cross the yeast cell membrane to cause inhibition. If the pH of the same lactic acid solution is decreased below pH 3.86, a larger proportion of the total lactic acid will be undissociated, and if the pH is increased above 3.86, a larger proportion of the lactic acid will be dissociated. In other words, when weak acids are present, decreasing the pH will increase the proportion of the undissociated form and thus increase the amount of yeast inhibition. So to fully understand how much yeast will be inhibited by weak ac-

FERMENTATION

ids, a plant must know both the pH and the amount of the acid using high performance liquid chromatography or gas chromatography. Fatty acids (FA) can also accumulate at an ethanol plant from hydrolysis of corn oil, bacterial contamination, methanator process upset, or as a result of yeast stress. FAs also have pKa values and so behave similarly to weak organic acids. Lastly, some fusel compounds (produced by yeast and contaminating bacteria) also have specific pKa values.

The Solution: Raise the pH

Bacteria inhibit yeast in two ways. They compete with yeast for nutrients (especially micronutrients), limiting yeast’s growth by starving them of micronutrients. And they can also produce toxic organic acids that inhibit yeast. The question becomes how to reduce one or both of these inhibition types on yeast. Antibiotics and antimicrobials can be added to yeast propagation and fermentation to limit the growth of bacteria. But what about the organic acids/FA already present even if 100 percent of the bacteria

are theoretically dead? Raising pH can limit the organic acid/FA inhibition on yeast. Phibro raised pH by 0.2 absolute at more than 43 fuel ethanol plants with contaminated fermenters that had been stalled for more than 32 hours. Yeast in these fermenters finished utilizing sugars and increased ethanol yield closer to levels of fermenters without bacterial contamination. This pH increase was achieved by adding into the fermenter either aqueous ammonia or caustic from clean-in-place (CIP) lines. But, would caustic raise the sodium level in the fermenter and further inhibit the yeast? Technically, the sodium concentration will increase. But, at a lower pH in a contaminated fermenter, it is likely that the degree of inhibition on yeast from organic acids is much greater than inhibition by sodium. Also, to raise the pH by 0.2 in a 750,000 gallon fermenter requires approximately 1,500 gallons of 5 percent CIP, which increases the concentration of sodium only by 3 percent in the fermenter. Would raising the pH allow contaminating bacteria to grow better and have a competitive advantage over the yeast? Technically, yes. But, in a contaminated,

stalled fermenter, most of the inhibition on the yeast at that time is caused by organic acid/FA inhibition and not nutrient competition. Also, a pH increase of only 0.2 absolute was needed to decrease organic acid/FA inhibition on the yeast without increasing bacterial numbers. Larger pH increases will certainly provide an additional boost to bacterial growth, negating the benefits of reduced inhibition by organic acid/FA. The routine practice of lowering pH to combat bacterial contamination does work. But it also increases the stress on the yeast by directly inhibiting the yeast, and it increases the chemical inhibition on the yeast from dissociated and undissociated forms of these acids. Raising the pH in more than 43 contaminated ethanol plants reduced sugars passed to distillation and partially recovered ethanol yield. Author: Dennis Bayrock Global Director Fermentation Research Lactrol Phibro Ethanol Performance Group 651.788.0602 dennis.bayrock@pahc.com

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