Final report for FNC19-1199
Project Information
The operation consists of 25 bee hives located on five farms and on top of one restaurant in Champaign Co. IL. Other participating apiaries are located in Georgia, Colorado, New Hampshire, Maine and Wisconsin. All beekeeping is conducted according to natural principles such as natural queen rearing, natural materials (wood, metal) and diverse farm environments that promote good nutrition. Chemicals used conform to USDA organic guidelines.
Yeast is fundamental to brewing. Most commercial yeast strains that are used for brewing are produced in laboratories and have standardized flavor profiles. Wild yeast, on the other hand, reflects the terroir (local climate, crops and soil).
Small farm breweries using wild yeast occupy a growing niche in the local food movement. However farmhouse breweries using wild yeast traditionally rely on open, spontaneous fermentation. This project was intended to help farmhouse breweries explore a new potential source of yeast for brewing. The purchase of commercial yeast is usually one one of the most expensive elements in brewing. When farmhouse brewers have local sources of wild yeast, it allows them to produce a product that is unique and economically attractive.
Our economic goal was to increase the commercial viability of farmhouse fermentation by expanding sources of local yeast. Our specific objective was to determine what wild yeast species could be isolated from raw honey. We were particularly interested in Saccharomyces yeasts that could have potential for use in making beer, cider or mead. Our educational goal was to demonstrate to farmers and small batch brewers how to exploit an important natural resource, wild yeast.
Twenty nine samples of honey were tested from six states. There were seven species of yeasts identified. Candida magnoliae was the most common species isolated. Other species of Candida were also identified. Several isolates could not be identified by the time this project concluded but are being investigated. In addition, yeast was also isolated from bees and wildflowers.
While our research did identify a number of yeasts existing in honey, Saccharomyces does not appear to be naturally found there. However, the diversity of yeasts that we isolated included several yeast isolates that have yet to be identified. A scientific outcome of this work was the potential for expanding an identification tool, MALDI-TOF (matrix assisted laser desorption ionization-time of flight mass spectrometry).
An unexpected outcome was the breadth of interest and potential for collaboration that the science of yeast inspires. Our project drew interest from small-batch commercial meaderies and breweries, home brewers, beekeepers, homemakers, chemistry students, university professors and yeast professionals. For example, thanks to this project, several undergraduate chemistry students acquired skills that aided yeast identification. We also corresponded with the Yeast Bay Company located in Portland Oregon about Metchnikowia reukaufii, a nectar yeast that we isolated from flowers. In addition, we corresponded with beekeepers throughout the United States in our quest for a suitable wild yeast. Their interest has encouraged us to plan an article for submission to a beekeeping and/or scientific journal.
- Evaluate the potential of wild yeast as a crop by identifying and testing wild yeast strains in particular terroirs (fruit farm, flower farm and brewery farm)
- Encourage farmers and beekeepers to exploit the wild yeast possibilities of their particular landscape and products (beverage, baking, cheese, pickling, etc.) by sharing findings through social media, educational presentations, conferences and written information
- Identify scientific methods that would be useful for capturing and propagating wild yeast with the help of a microbiologist
- Evaluate the impact of seasonal fluctuations and geographical constraints (such as monofloral environments) on the prevalence of wild yeast
- Encourage the development of innovative market bridges and income streams between local agriculture and local brewing
Cooperators
- - Producer
- - Producer
- - Producer
Research
Sources of honey: Honey was obtained from thirteen beekeepers from six states (Illinois, Wisconsin, Colorado, New Hampshire, Maine, and Georgia. Some beekeepers provided samples from multiple hives. The greatest number of hives came from central Illinois. One honey (Great Lakes Wild Hive brand) was purchased from a local supermarket. A sample of manuka honey was purchased online. A total of 29 honey samples were tested. Two samples were known to be at least a year old, and a third sample’s age could not be verified.
Yeast sources: Honey samples were diluted in sterile water to a density of 10-20 Brix (specific gravity 1.040-1.080) using a refractometer. Samples were incubated at ambient temperature (20-22°C) for three to five days, depending on how quickly they developed turbidity or observable mold growth. The honey-water mixtures were streaked with a sterile loop, or spread-plated onto yeast media, including Yeast-Mold Agar (YMA) and Sabouraud’s Dextrose Agar (SDA). In a few cases, Lin’s Wild Yeast Medium or Rose Bengal Agar was also used. Plates were incubated at 28-30°C until colonies developed, up to seven days. Individual colonies were streaked to obtain single colony isolates. Yeast isolates were stored on YMA or SDA at 4°C until identified.
Bee and flower isolations: As a side project, attempts were made to isolate yeast from bees and wildflowers. Bees were surface disinfected and dissected to remove the intestinal tract. The guts were ground in a sterile microcentrifuge tube containing sterile water. Plates of YMA or SDA were streaked from the suspension with a sterile inoculating loop. Seven species of Wisconsin wildflowers were also tested in June 2020. The flowers were columbine (Aquilegia canadensis), lily of the valley (Convallaria majalis), false solomon’s seal (Maianthemum racemosum), forget-me-not (Myosotis sylvatica), bunchberry (Cornus canadensis), Canada mayflower (Maianthemum canadense), and yellow clintonia (Clintonia borealis). Flowers were crushed in sterile water in a microcentrifuge and plated on either SDA or Rose Bengal Agar.
A previous collaborator, Dr. Jason Ridlon (University of Illinois Urbana-Champaign), also provided 34 samples from isolates cultured from honey provided by Maggie Wachter (Illinois). He has not shared information on how many samples he tested or what methods were used to isolate yeasts. Based on the isolate codes, it appears there were at least six different sources of honey for the isolates he shared.
Yeast identification: Isolates were analyzed using matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). MALDI-TOF analysis was conducted by Dr. James Lawrence in the Department of Chemistry (UWSP). Fresh plates (with colonies only two to three days old) were provided for analysis.
Of the 29 honey samples tested by Barta, yeast was recovered from 21 of them. There were seven different species of yeast identified by MALDI-TOF. The most common species recovered was Candida magnoliae (found in 11 samples). Candida magnoliae was also recovered from bees from Georgia. Other species isolated from honey were the following: Candida parapsilosis (2), Candida orthopsilosis (1), Candida guillermondii (2), Rhodotorula mucilaginosa (4), Zygosaccharomyces rouxii (4), and Aureobasidium pullulans (1). Eleven isolates could not be identified by MALDI-TOF. These isolates have been sent off to BCN Laboratories, Inc. for identification using PCR-DNA sequencing. Results are pending. No yeast was recovered from the manuka honey or the honey purchased from the supermarket. A summary of yeast identifications is shown in Table 1.
Of the isolates shared by Ridlon, Candida magnoliae was the main species identified. Candida parapsilosis was also identified from one source. Isolates from another source were identified as Saccharomyces cerevisiae.
Yeast was recovered from three of the seven wildflowers tested. Metchnikowia reukaufii was identified from Clintonia borealis and Convallaria majalis. A yeast isolate from Maianthemum canadense produced a distinct MALDI-TOF spectrum but from could not be identified by that technique.
Species of Candida were prominent in samples from Wisconsin and Illinois. Isolation of Candida from honey is not surprising based on what is reported in the scientific literature. These yeasts are not suitable yeasts for mead or beermaking, as these yeasts generally do not grow anaerobically. In addition, fermentation tests we performed in malt extract with Candida magnoliae and C. parapsilosis isolates confirmed poor fermentation ability. Candida magnoliae, the most common yeast identified, was identified in magnolia flowers and the bumblebee gut. Candida species are used to produce a variety of biotechnologically interesting compounds. Candida magnoliae is an industrially important yeast used to produce mannitol and the low calorie sweetener, erythitol. (2, 7) Zygosaccharomyces rouxii, which is capable of fermentation, is well known as a food spoilage yeast, and is tolerant to high levels of sugar, ethanol, and preservatives such as sorbic acid. Both Candida magnoliae and Zygosaccharomyces are common in honeybee-stored bee bread (1). Rhodotorula, a pink yeast, is in the phylum Basidomycota (as opposed to the phylum Ascomycota for the other yeasts). Rhodotorula yeasts are common in many environments including plants and soil. Rhodotorula occasionally inhabits nectar. These yeasts are being studied for their potential in bioremediation. A few species, including R. mucilaginosa, can cause infections in immunosuppressed people (6).
It is interesting that Saccharomyces cerevisiae was in the collection of yeasts isolated by Ridlon.. The hive from which it was isolated was located next to a small brewery. Bees are known to collect sugars from any source available, and we presume the bees were visiting the brewery and coming into contact with brewing yeast.
An interesting finding was the isolation of Metchnikowia reukaufii from Wisconsin wildflowers. This yeast is known as a nectar specialist (3), and it produces odors thought to attract pollinators. It has applications in beer brewing. An isolate from California is currently available from Yeast Bay (produced by White Labs). Because this yeast can ferment glucose but not maltose, it must be used in conjunction with another brewing yeast.
We did not find any Saccharomyces in honey, even in freshly-collected samples. But perhaps this is not surprising as the natural habitat for Saccharomyces is known to be the skin of fruits. No reports could be found in the literature of Saccharomyces residing in nectar. One hypothesis that could explain the lack of Saccharomyces in honey samples is that Saccharomyces has a lower osmotolerance than other yeasts. A study by Stratford et al. (4) showed that Candida and Zygosaccharomyces were more osmotolerant than Saccharomyces. However, Rhodotorula was less osmotolerant than Saccharomyces and yet was recovered in a few honey samples. The presence of Saccharomyces in the honey samples obtained from Dr. Ridlon argues against osmotolerance as a reason for its absence in honey.
The samples that did not produce any culturable yeasts are thought to have either been heated, or were too old, leading to a loss of cell viability. We did test year-old old honey from two sources and were not as successful at recovering yeast.
While small ribosomal RNA gene sequencing has been the standard in microbial identification, MALDI-TOF is an emerging tool for microbial identification (3). It is rapid, sensitive, and cost effective in terms of labor and materials. However, MALDI-TOF analysis is limited by the extent its species database. The data base is still growing.
Conclusion
The Sars CoV-2 pandemic created several challenges in carrying out this research. In particular, UW-Stevens Point was shut down for a period of time and access to campus was limited to essential personnel. In addition, an internal professional development grant that had been awarded to fund the molecular identification of the isolates was rescinded due to university budget pressures related to Covid. Despite these challenges, we were able to test 29 samples of yeast from six states and obtain yeasts from 21 of them. Since yeasts are widespread in nectar, we expected to find yeasts in honey. Although we did not find species of Saccharomyces, the most suitable type of yeast for beverage fermentation, we did isolate seven other species of yeast. This study adds to the body of knowledge about yeast ecology. In addition, the identification of the yet-identified isolates will expand the MALDI-TOF library.
Literature Cited
- Detry R, Simon-Delso N, Bruneau E, Daniel H-M. Specialisation of Yeast Genera in Different Phases of Bee Bread Maturation. Microorganisms. 2020; 8(11):1789. https://doi.org/10.3390/microorganisms8111789
- Lee JK, Koo BS, Kim SY, Hyun HH. Purification and characterization of a novel mannitol dehydrogenase from a newly isolated strain of Candida magnoliae. Appl Environ Microbiol. 2003; 69(8):4438-47.
- Pozo, MI, Herrera, CM, Alonso, C. 2014. Spatial and temporal distribution patterns of nectar-inhabiting yeasts: How different floral microenvironments arise in winter-blooming Helleborus foetidus. Fungal Ecol 11: 173-180
- Sibghal, N, Kumar, M, Kanauji, PK, and Virdi, JS. MALDI-TOF mass spectrometry: an emerging technology for microbial identification and diagnosis. Front. Microbiol. 2015; 6: 1-16. https://doi.org/10.3389/fmicb.2015.00791
- Stratford, M, Steel, H, Novodvorka, M, Archer, DB, Avery, SV. Extreme osmotolerance and halotolerance in food-relevant yeasts and the role of glycerol-dependent cell individuality. Front. Microbiol. 2019; 9: 1-14. https://doi.org/10.3389/fmicb.2018.03238
- Wirth, F, Goldani, LZ. Epidemiology of Rhodotorula: An Emerging Pathogen, Interdisciplinary Perspectives on Infectious Diseases, vol. 2012, Article ID 465717, 7 pages, 2012. https://doi.org/10.1155/2012/465717
- Yu JH, Lee DH, Oh YJ, Han KC, Ryu YW, Seo JH. Selective utilization of fructose to glucose by Candida magnoliae, an erythritol producer. Appl Biochem Biotechnol. 2006;131(1-3):870-879.
Educational & Outreach Activities
Participation Summary:
The SARE project was announced in the local newspaper, the News-Gazette. (Spring 2019)
Dr Barta and Maggie Wachter visited a yeast production company (Inland Island, Denver CO, winter 2020) and a wild fermentation brewery (Black Project Spontaneous & Wild Ales, winter 2020) where we consulted on mead-making.
An Instagram account called 'Fermentabee' was established to illustrate and inspire fermentation experimentation. (Winter 2021)
We announced the SARE project and issued a call to beekeepers in other parts of the country to contribute samples of raw honey for testing. We did this through letters to the editor published in the March 2021 issues of American Bee Journal and Bee Culture. We received 4 samples of honey from New Hampshire and Maine.
Maggie Wachter attended a yeast workshop with commercial mead-makers from a local micro-meadery. They discussed the SARE yeast project extensively. (winter 2021).
We recovered 8 yeasts that are not yet in the MALDI-TOF Yeast Library. The MADI-TOF Yeast Library is a comprehensive reference tool used by brewers and other food professionals to identify contaminants and spoilage agents. Our yeast samples will expand the capacity of the Library, contributing to food safety and brewing purity.
Dr. Barta collaborated with a chemistry professor in the training of at least 3 students in using the MALDI-TOF methodology. It is our hope that these students will continue to use and contribute to the MALDI Library.
In fall 2021, Steve Halfar and Maggie Wachter conducted 2 mead-making workshops in which yeast selection and use was discussed extensively. The workshops were advertised through the Parkland College Community Education brochure.
A product of this research is an online booklet on mead-making with a section on yeast techniques, written by Dr. Barta. An initial draft of the booklet, available on this website, will be updated in the future.
In 2020, the pandemic affected three planned educational and outreach events:
The project was announced to the local brew club, the BUZZ, in the fall of 2018 and again during the spring of 2019. The club's president and some members expressed interest in participating in the brewing portion of the project. One beekeeper/brewer club member who was starting a commercial meadery expressed particular interest in brew-testing the experimental yeast. Unfortunately the club suspended meetings in March 2020. Until the club resumes meetings, we plan to work with individual home brewers to help us test promising strains.
In August 2020, we planned to present our project at the annual meeting of the Eastern Apiculture Association. Unfortunately the conference was cancelled due to the pandemic.
In 2020, our consulting microbiologist, Dr. Barta, applied for and received an auxiliary grant from the University of Wisconsin for equipment to test air and surface quality around the hives. Such analyses might have helped us understand better how environmental factors surrounding bee hives might affect the presence of brewer's yeast in honey. Unfortunately, for economic reasons related to the pandemic, UW-Stevens Point withdrew faculty grant funding during 2020.
Learning Outcomes
We learned that while it seems reasonable to isolate strains of brewer's yeast in honey, our results suggest otherwise. We explored several yeast bioprospecting avenues and discussed the project with many yeast and food professionals. Along the way, the beekeepers involved acquired a better understanding of fermentation and how to improve the brewing process. We learned that some yeasts, such as the Metchnikowia reukaufii, are useful as flavor enhancers when used in combination with Saccharomyces during fermentation. The quest for novel yeast might benefit by turning instead to flowers and fruits.
Project Outcomes
While we have not yet isolated a yeast strain suitable for brewing, we have noticed that the hives located in isolated and forested areas, such as the Appalachian Mountains of Georgia or the wilderness of Southern Illinois contained better yeast diversity.