KBI/OSU Genomic Study
A Summary of Kombucha Microbiome Research

By Keisha Rose Harrison

The KBI/OSU SCOBY Genomic Study took place from November 2017 to September 2018 as a two-part study. Part 1 asked the questions “what does the Kombucha SCOBY consist of?” and “how diverse is the Kombucha SCOBY?” in regards to the collective microbiota (the bacterial and fungal populations). Participants were asked to submit representative samples from a SCOBY (solid starter culture) with information regarding current location and time of use.

Part 2 was designed as a follow-up study to ask more specific questions about Kombucha starter cultures and brewing practices. Participants were asked in the Part 2 study to submit either broth (liquid starter culture) or SCOBY samples and to answer a questionnaire about brewing practices (including tea type and brewing volume). Data collected from both studies were combined to strengthen the scope of the study and conclusions about microbiota patterns. A total of 107 SCOBY and 19 Broth samples were collected and evaluated from 7 countries and 26 US states.

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timeline of KBI OSU SCOBY genomics study

 

Before diving into the results, let’s review the study methods and approach. Both parts relied upon the same DNA sequencing technique to determine microbiota from sample submissions. DNA was extracted using a method modified from traditional column-based extraction protocols. Measures were used to ensure that samples were thoroughly mixed to best represent the sample. Amplicon sequencing was the method of DNA sequencing. During this process, conserved regions of bacterial rDNA and fungal DNA were amplified and sequenced using fluorescent markers.

What exactly does that mean? Each bacterial and fungal cell has a “fingerprint” in its DNA that is generally conserved among members of a genus and species. It is this “fingerprint” that is copied and read. A bioinformatics pipeline is subsequently used to assign species-level identification for yeast and genus-level identification for bacteria. Relative abundance is determined as the number of times a copy of the “fingerprint” is recognized in proportion to all of the fingerprints. Bacterial and fungal relative abundances are reported separately.

results of DNA sequencing of SCOBYs

 

How to interpret your report? Participants received a report that included the participant ID (number used to ensure confidentiality), a list of bacteria genera with relative abundance, and a list of yeast species with relative abundance. An example of a “bacterial profile” report is depicted below. From this report, we can observe the composition of the bacterial population from a submitted sample. Only bacteria that were present at >0.01% were included in the report.  A majority, 43.7%, of the bacterial population in the sample was identified as Lactobacillus genus. The method of interpretation can be used for the fungal or yeast reports.

Bacteria (Genus) K-XXX
Sporolactobacillus 0.0700
Lactobacillus 0.4367
Oenococcus 0.1133
Acetobacter 0.1033
Gluconacetobacter 0.2767

 

Now that the relative abundance of bacteria and yeast are known, the next question is “so what does this mean?” What does a percentage tell us about the fermentation, taste, and quality of kombucha? On an individual level, each sample can serve as a building block of knowledge. Any quality assurance and control plan begins with a detailed overview of the system. Troubleshooting and monitoring starter culture health are all contingent upon knowing the composition of a “normal” and “healthy” culture. The impact of this research is strengthened when we zoom out and look at the whole sample population.

What does the data from both Part 1 and Part 2 tell us? By looking at the microbiota of multiple samples from various geographic locations, we began to answer questions about starter culture diversity. With the information provided by study participants were able to identify the most abundant Kombucha microorganisms, determine the impact of location and culture conditions on the microbiota, and classify starter culture “types”. Samples were collected from both solid and liquid starter cultures have the same common bacteria and yeast populations. Regardless of source, the most common yeast species (based upon an average of relative abundance) are Brettanomyces bruxellensis, Brettanomyces anomala, and Issatchenkia orientalis. The most common bacterial genera are Lactobacillus, Komagtaebacter, and Acetobacter. The proportions of bacteria genera vary depending upon starter culture source so that lactic acid bacteria is more abundant on average in broth than the SCOBY pellicle. Respectively, more acetic acid bacteria is abundant on average in the SCOBY pellicle than the broth. This makes sense we consider that the bacterial species, Komagaetibacter xylnium, is often credited with the formation of the physical SCOBY structure.

Taking a step back, let’s consider what this means. More of the bacteria type that produces ethanol, lactic acid, and carbon dioxide are on average found in broth. Whereas, more of the bacteria type that oxidizes ethanol and produces acetic acid is found in SCOBY. This diversity may be in part because of how the floating physical structure creates an air-liquid interface to support an oxygen-rich environment. Furthermore, we compared physical and liquid samples from the same producer to better understand the dynamic.

Let’s breakdown the figure below. The outside ring represents the SCOBY microbiota and the inside represents the BROTH microbiota. The color blocks represent top 5 microbial groups (so the most abundant bacteria and yeast genera). Blocks of the same color depict the same microbial group. The size of the block correlates the amount of that microbial group present. From the figure, we are comparing 4 BROTH and 4 SCOBY samples each BROTH/SCOBY pair is from the same location. The figure illustrates that in some instances the microbiota is comparable between SCOBY and BROTH and in some instances they differ. What does this mean? We cannot assume that the BROTH and SCOBY are interchangeable.

biofilm vs broth comparison of microorganisms composition

How to best understand ALL of this data? SCOBY sequencing revealed that there are many, many, MANY different species of yeast and bacteria in the Kombucha starter culture population. Looking at just the average SCOBY or BROTH composition is not representative of most individual samples and underscores the importance of starter culture diversity. A better way to compare starter cultures of rich diversity is to group similar microbiota profiles. In other words, by clustering samples that have similar amounts of Bacteria A and Yeast B, we are able to determine “types” of starter cultures. We ultimately used a hierarchical clustering analysis approach to sort through individual data and identify 5 groups within the overall population. These groups represent patterns of yeast and bacteria that any individual samples may fall under. The figure below depicts how specific types of microorganism are likely to appear together in a starter culture. Starter cultures with a large amount of the yeast genera, Brettanomyces, are less likely to be present in a culture with Starmerella/Candida. The “types” of starter culture can be described as follows:

 DOMINANT BACTERIADOMINANT YEAST
IAcetobacter, high – moderate levels of secondary KomagataeibacterVariable
IIKomagaetibacterBrettanomyces
IIILactobacillusBrettanomyces
IVKomagataeibacterStarmerella (Starmerella/Candida clade)
VKomagaetibacter AND Mixed Komagaeteibacter/Acetobacter Komagaeteibacter/Lactobacillus Brettanomyces

To determine how each starter culture type influences fermentation parameters, a preliminary study was undertaken in Dr. Chris Curtin’s lab at Oregon State University. Those findings will be available later in the year in the form of an academic publication that will be shared on the KBI site.

5 types of scobys

READ PART TWO OF THE STUDY

webinar stills

KBI is delighted to report a wildly successful first webinar in our “Ask Us Anything” series – Fast Track to Distribution” (May 18, 2016). All of the attendees enjoyed the detailed and Kombucha-specific advice offered by our expert speakers. 

“Thank you so much for the webinar today! It was packed full of great information. The panel was so gracious with their time and experience. They gave very detailed answers to some really good questions. I really appreciated the real-life, real small business growing pain examples – especially mistakes made. I could easily see myself walking down that same path and making those same mistakes – thank you for saving me the expense and the agony!” -Elizabeth, BIP

Chris Montelius of Non-Prophet shared tips and advice from his former life experience as a beer distributor. Many members had written in with several questions and he graciously answered them all in a slide presentation.

Jared and Deanne of Kombucha on Tap covered distribution from their perspective as distributors of kegged kombucha in Southern California. They covered everything from equipment choices to costs involved in self-distribution vs. utilizing an outside distributor’s assistance, as well as keg-specific information regarding kegerators, keg washing, and more.

“Thanks for putting the Distribution webinar together, and thanks for injecting my questions into the conversation so late in the game. I found it tremendously informative, helpful, and timely as DZB begins to plan out the early phases of keg distribution. It’s also great to know that there are some tremendously knowledgeable and generous members of the Kombucha industry willing to shed some light on these topics. From our perspective, it has seemed a little isolating and unfriendly at times, but this webinar has proved contrary to that notion. Thanks again for facilitating this, and good luck with the future ones. I’ll be sure to tune in to the rest of the series.” -Joe, Dean’s Zesty Booch

Shane Dickman of High Country recounted the brand’s journey from tiny start-up in the late 2000s to its current size as a thriving national brand. From the tough early years of self-distribution, which sometimes involved drivers sleeping in the van on runs from Colorado to California, to the common mistakes many brands might make early on, with specific advice on how to avoid losing money on distribution deals, Shane’s experience provided a lot of insight into the lengths that sometimes must be traveled on the road to success.

“Loved the dist. webinar yesterday! Thanks for providing exceptional tools and resources to KBI community.” -Gayle, FedBrew

Did you miss this webinar? You’re in luck! The recorded version (as well as an audio-only version) is available in the member forums! Not a member? Join today!

 

On the heels of this successful first seminar, KBI is pleased to announce the second webinar in our “Ask Us Anything” series:

The Secrets of Successful Flavoring, June 21 from 1-3PM PST.

original

REGISTER HERE

only $20 for KBI members, $200 for non-members

Buy 3 webinars, receive the 4th one free!

Speakers

Darius Subatis of Health-Ade

Born and raised in Boston, Darius came out to California in 2006 to pursue a career in film. After spending some time working in production for NBC Universal he decided it was time for a change. Darius joined the Health-Ade team as employee hire #1 in 2013 and has used his skills from film production to run the brewery for Health-Ade. His passion for efficiency and doing things right has helped grow Health-Ade over 10x what it was just three years ago. If he’s not brewing kombucha for Health-Ade, Darius is enjoying life with his beautiful wife, Amanda, and their dog, Riley, in Santa Monica.

 

Alla Shapiro of NessAlla

Alla joined business partner Vanessa with the common vision of helping the community gain a deeper knowledge of health and well being. Both certified herbalists, we began teaching kombucha brewing classes in Madison, Wisconsin in 2007. We became well known for our kombucha knowledge and word of our expertise spread. Workshops sold out and we soon had a SCOBY farm growing! We decided to work together to create one of the nation’s first small, local kombucha breweries.

Hannah Crum the Kombucha Mamma of Kombucha Kamp

Hannah Crum of Kombucha Kamp

Hannah Crum is the Kombucha Mamma. Originator of the Kombucha Kamp workshop,she partnered with her husband Alex LaGory, aka Alex Kombucha, to create the top informational site for Kombucha in the world KombuchaKamp.com Their mission is to “change the world, one gut at a time.” Hannah is a popular speaker about Kombucha, fermentation and bacteriosapiens at corporate and health conferences, fermentation festivals and events around the world. As an outgrowth of their commercial consulting, industry reporting and marketing efforts, Alex & Hannah co-founded Kombucha Brewers International in 2014 to unite and advocate for the commercial Kombucha bottling industry around the world. Through Kombucha Kamp’s videos, blog posts and online support communities, they have mentored 100,000’s of homebrewers all over the world. Their newly released “Big Book of Kombucha” from Storey Publishing (featuring 260 “Flavor Inspirations”) has been met with critical acclaim and praise. With this impressive wealth of information, it is no surprise that they are the leading Kombucha experts in the world.

The Secrets to Successful Flavoring

Every brewer flavors in a different way- that’s one of the ways brands differentiate their products. Have you always wanted to try out some new ideas, but weren’t sure how to start? Looking for some new flavor inspirations? Had some frustrating setbacks and not sure how to proceed? This webinar is for you!

Webinar Includes:

When to Flavor

– Primary

– Secondary

– Reasons for flavoring in primary vs. secondary


Teas

How to Flavor with Tea

– Flavoring and potency of tea types

– Tea Blends

– Brand distinction with tea alone

– Preparing ingredients

fresh_fruit

How to Flavor with Fruit

– Preparing ingredients

– Whole or Sliced Fruit, Juice, Dried, Concentrate, Freeze Dried, Frozen Fruit

– Advantages and Disadvantages

How to Flavor with Herbs, Flowers and Spices

– How to prepare herbal infusions (dried vs. fresh)

essential oils and medical flowers herbs

How to Flavor with Essential Oils

How to Flavor with Different Types of Sugars

– How each type affects flavor

-White sugar, brown sugar, honey, Stevia, etc.

Unwanted flavors and solutions (ex. water quality)

– Herbal or fruit flavors that might compromise probiotic content

– Nutrition facts skewed by fruit inclusion (having each flavor tested and labeled)

– How to know when you’ve messed up. Can it be fixed?

Join us, and ask the speakers your questions in advance by emailing Morgan!

 

REGISTER HERE

only $20 for KBI members, $200 for non-members

Buy 3 webinars, receive the 4th one free!

Upcoming Webinars:

Control Your Brew- Reducing Alcohol Levels in Under-21 Kombucha

Alcohol Testing Methods- What Works, What Doesn’t, & How to Do It for Less

Under Pressure- Kegs, Cans, & Bottles