Kombucha Bacterial Cellulose - Revision history

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Introduction

Kombucha is a symbiotic living material made of a variable composition of different species of bacteria and yeast. Among the most common ones participating in the symbiosis we can find: Bacteria: Acetobacter xylinum, A. xylinoides, A. aceti, A. pasteurianus, Bacterium gluconicum. Yeasts: Schizosaccharomyces pombe, Kloeckera apiculata, Saccharomycodes ludwigii, Saccharomyces cerevisiae, Zygosaccharomyces bailii, Brettanomyces bruxellensis, B. lambicus, B. custersii and Pichia. Kombucha tea is a traditional health-promoting fermented beverage that exists since several thousand years. It is produced by the fermentation of sugared tea with a symbiotic colony of bacteria and yeast. Kombucha is a traditional beverage drunk for its wide range of health benefits. The cellulose matrix formed in the culture medium can be used as a bio-cellulose tissue to create clothes, or bio-paper. But it can also be used as a cosmetology for its anti-inflammatory, antioxidant and anticancer properties.

Content: What is a biofilm?

In fact, the kombucha sheet forming on top of the culture is a type of biofilm. Biofilms are the most common way in which microorganisms exist in nature. They are very resistant and have very interesting physical properties.

A biofilm is any group of microorganisms in which cells stick to each other and often these cells adhere to a surface. These adherent cells are frequently embedded within a self-produced matrix of extracellular polymeric substance (EPS). Biofilm extracellular polymeric substance, which is also referred to as slime (although not everything described as slime is a biofilm), is a polymeric conglomeration generally composed of extracellular DNA, proteins, and polysaccharides (like the bacterial cellulose we find in kombucha). Biofilms may form on living or non-living surfaces and can be prevalent in natural, industrial and hospital settings

Biofilms are usually found on solid substrates submerged in or exposed to an aqueous solution, although they can form as floating mats on liquid surfaces and also on the surface of leaves, particularly in high humidity climates. Given sufficient resources for growth, a biofilm will quickly grow to be macroscopic (visible to the naked eye). Biofilms can contain many different types of microorganism, e.g. bacteria, archaea, protozoa, fungi and algae; each group performs specialized metabolic functions. However, some organisms will form single-species films under certain conditions. The social structure (cooperation/competition) within a biofilm depends highly on the different species present.

Biofilms can be found in many different places and conditions: Biofilms can be found on rocks and pebbles at the bottom of most streams or rivers and often form on the surface of stagnant pools of water. In fact, biofilms are important components of food chains in rivers and streams and are grazed by the aquatic invertebrates upon which many fish feed. Biofilms can grow in the most extreme environments: from, for example, the extremely hot, briny waters of hot springs ranging from very acidic to very alkaline, to frozen glaciers. In the human environment, biofilms can grow in showers very easily since they provide a moist and warm environment for the biofilm to thrive. Biofilms can form inside water and sewage pipes and cause clogging and corrosion. Biofilms on floors and counters can make sanitation difficult in food preparation areas. Biofilms are present on the teeth of most animals as dental plaque, where they may cause tooth decay and gum disease. Biofilms are used in microbial fuel cells (MFCs) to generate electricity from a variety of starting materials, including complex organic waste and renewable biomass.

Picture: Electron microscopy of biofilm of Staphylococcus aureus biofilm on an indwelling catheter. Public Domain, https://commons.wikimedia.org/w/index.php?curid=2740748

Materials

Kombucha sheets (about one or two per person) Grean tea, vinegar, sugar Ink and organic colorants Dryers and ovens Press Knives Blender Bleach and hydrogen peroxide NaOH 1M Water and paper press Needles

Protocol: how start a culture of bacterial cellulose?

Instructions: Boil 1L of distilled water and add 20g tea in bags. Let infuse during 5 minutes and add 113 g of glucose. Let cool at room temperature and add 100mL of a previous batch (liquid medium and a piece of the cellulosic matrix). Add 100ml of cider vinegar. Seal the bottle and let ferment and grow the culture at room temperature, between 5 and 14 days.

The culture doesn’t need too much oxygen. Yeasts hydrolyze sucrose into glucose and fructose, producing ethanol and carbon dioxide, as metabolites. Acetic acid bacteria converts glucose into gluconic acid and fructose into acetic acid. The final pH reading should be between 2.5 and 3.2, to prevent the symbiotic culture from becoming contaminated by undesirable microorganisms.

Picture: Dr Peter Musk, a scientist catalyst at The Edge Southbank, with some of the vegan leather being produced from kombucha. Photo credits: Queensland University of Technology (Queensland, Australia).

Protocol: how to use and modify bacterial cellulose?

Before starting the protocol, we need to inoculate sugary tea with Kombucha for the week after. To do so, prepare the medium and the inoculum as described above and let it at room temperature in the soom, covered. After this, you can experiment with the Kombucha you got and treat the pre-made Kombucha sheets to modify their properties. Some options include: coloring / decoloring, blending, and desiccation. The information you’ll see below is mostly adapted from the iGEM Team Imperial College 2014 (you can check more on their website: http://http://2014.igem.org/Team:Imperial).

The team from iGEM Imperial 2014 did an intensive production and prototyping during some months and can serve as an example of what we would be able to do in a short term project around biomaterials. The goal of their iGEM project was to optimize the production of bacterial cellulose by engineering Gluconacetobacter xylinus. They also explored the processing of the biomaterial, producing and testing water filters, and functionalizing them with binding proteins to trap specific contaminants.

Bacterial cellulose (BC) exhibits a multitude of different properties depending on the processing, growth conditions, functionalization and strain used for production of the material. Acquiring large quantities of cellulose produced would allow testing of a broad variety of cellulose processing methods and functionalization steps. By mass producing cellulose this enables a better understanding of what material properties can be realistically produced during the short duration of iGEM. More importantly, it improves the likelihood of finding suitable processing candidates for the project’s aim of making a customisable ultrafiltration membrane, at the same time as allowing room for creativity and exploration of the remarkable properties of cellulose.

Kombucha processed sheets from iGEM Imperial College 2014. From left to right: thin kombucha, thick NaOH processed kombucha, and blue-dyed kombucha. Minimum requirements

Treatment of BC requires killing the cells, particularly if the cells are genetically engineered, which is the aim for putting the customisable in ultrafiltration membranes. Based on brainstorming with Central Saint Martins student Zuzana, removing the colour of BC is required as it looks displeasing to the eye otherwise, and seems counter-intuitive to filter clean water with cellulose coloured like turbid water. Removal of the smell of BC has also been raised as a requirement, particularly by producers who work in close contact with the processing facilities. Mass Production Methods

Setting up the mass production of cellulose was done according to the Kombucha media protocol , which involved setting up 61 trays with media and G. xylinus and yeast co-culture. The trays were left to grow up over 7 days, after which diminishing pellicle growth was detected. Upon harvesting, the pellicles were sorted according to granular pellicles and even pellicles (see pictures above). All pellicles were kept in distilled water in large plastic buckets or containers. Below shows the general workflow (adapted from IGEM Imperial 2014) employed to mass produce the cellulose and illustrates the process of manufacturing biomaterials with significantly different properties despite originating from the same BC source.

Inoculate sugary tea and incubate for 7 days at room temperature Harvest pellicules and assess quality of the BC Blend the bacterial cellulose to a paste Wash in water Treat chemically Dyson ocean blue stain in 900ml of distilled water and 100ml of olive oil Air drying without treatment Blend 200g with 250ml of water. Incubate at 0.1 M NaOH at 80°C for 3 hours Oven drying with press after NaOH treatment for 120 minutes Incubation in 1M NaHCO3 for 60 min at 120°C Put into a shape and dry Press dry Produces the most white cellulose if the film that covers the bottom of the pellicle is removed before treatment. BC capable of immersion into water, then it could be reshaped and re dried Produces poor results: brown cellulose Blue flexible cellulose, thin layer, not fragile Brownish cellulose with high moisture content left, material is flexible

Appendices: methods tested at iGEM Imperial 2014 (as stated in their wiki page)

0.1M NaOH for 60 min at 120C: Produces the most white cellulose but the process has been shown to produce some yellow/brownish cellulose if the film that covers the bottom of the pellicle was not removed before treatment. 1M NaHCO3 for 60 min at 120C: Produces quite poor results, 3 samples have been tested and even after 4 hours of treatment at 120C the samples were less white than similar samples treated in distilled water for the same duration. Heat treatment in distilled water at 120C: 3 samples still contained some

brownish tint after 4 hours of incubation, but the samples produced were considerably whiter than those treated with baking soda solution

Air drying without treatment first: Produces brownish cellulose with high moisture content left, material is flexible 120C Oven drying without press, without treatment for 180 min: Produces brittle paper like cellulose, it is fragile, brownish and prone to tears. Oven drying with press (1l Duran bottle on top of two tiles) after NaOH treatment for 120 min: Produces flexible more plastic cellulose capable of being shaped into a cone that filters water through. The cellulose was capable of immersion into water, which produced wet cellulose that could be reshaped and re dried. NaOH for 20 min at 120 C, followed by blending: produces cellulose that seems like it is much less ductile. Disadvantage: the functionalization will be blended just like the cellulose, so the proteins may be broken down mechanically. Distilled water treatment over 48 hours: Produced more white cellulose than what was harvested. The distilled water turned yellow giving evidence that the surface of cellulose actually did dissolve some of the medium’s colour 60 C incubation in tightly wrapped autoclave tape: New tape was applied every 3-8 hours during a 36 hour period. The pressure allowed water to escape and create a compact material of high hardness. Quite a promising result for hard cellulose.

Kombucha Textile : Cellulose Baterial Textile & “Vegetable” Leather

@ Suzanne Lee creation made from Kombucha

Here tuto super cool :) : Cuir végétale-Kombucha https://www.youtube.com/watch?v=vXrAxGx8dTY&feature=share

Protocol: Kombucha Textile

You Need : 200 cl water 20 cl cider vinegar 200 cl sugar 20 g of green tea (2 tea bag) 1 elastic 1piece of gaze fabric 1 strain of Kombucha SCOBY The “starter” : The liquid where the strain has growth (if you have not this liquid, don’t worry*) Glass Jar

Boil water (stop to heat the water) Add tea and let it infuse 15 min Put sugar in the pot and mix with a whisk Wait that the water cool down below 30°C Put vinegar + Water-Sugar-tea liquid + Starter into the Glass jar Put the Kombucha strain on the surface (white face in the top) Put the gaze fabric to close the jarr Leave the Jarr in a room without sun,

If you don’t have starter, put your strain of Kombucha on a plate. Put 5 tablespoons of white vinegar (pur, distilled, boiled : to avoid competition with other organisms)

Kombucha : What we can do with it & Future (To put on the presentation google slide):

“Now a team from Imperial College London have developed a set of DNA tools to control and engineer a strain of bacteria - normally found in a fermented green tea drink called kombucha tea - to produce modified bacterial cellulose on command. This technique also enables the team to “weave” proteins and other biomolecules into the fabric of the bacterial cellulose as it grows.” http://www3.imperial.ac.uk/newsandeventspggrp/imperialcollege/newssummary/news_27-5-2016-14-46-16

@@ Line 1: / Line 1: @@
 '''Kombucha''' tea is a traditional health-promoting fermented beverage that has been around for several thousand years. It is produced by fermenting sweetened tea with a symbiotic colony of bacteria and yeast. Kombucha is a traditional beverage consumed for its many health benefits. The cellulose matrix formed in the culture medium can be used as biocellulose fabric to create clothing or biopaper. But it can also be used in cosmetology for its anti-inflammatory, antioxidant and anti-cancer properties.
 <br>
-Bacteria: Acetobacter xylinum, A. xylinoides, A. aceti, A. pasteurianus, Bacterium gluconicum. Yeasts: Schizosaccharomyces pombe, Kloeckera apiculata, Saccharomycodes ludwigii, Saccharomyces cerevisiae, Zygosaccharomyces bailii, Brettanomyces bruxellensis, B. lambicus, B. custersii and Pichia.
+Bacteria: ''Acetobacter xylinum'', ''A. xylinoides'', ''A. aceti'', ''A. pasteurianus'', ''Bacterium gluconicum''. Yeasts: ''Schizosaccharomyces pombe'', ''Kloeckera apiculata'', ''Saccharomycodes ludwigii'', ''Saccharomyces cerevisiae'', ''Zygosaccharomyces bailii, Brettanomyces bruxellensis, B. lambicus, B. custersii'' and Pichia.
 <br>
 '''Protocol:''' how to start a bacterial cellulose culture?
@@ Line 24: / Line 25: @@
 With 10% glycerol, one can even get the 'dried' scoby to not crumble away, and have a more leather like texture.

← Older revision		Revision as of 20:48, 2 October 2023
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	'''LATEST''' '''EXPERIMENTS'''		'''LATEST''' '''EXPERIMENTS'''
		+
		+	We got some great 'jun' kombucha from Patrik d'Haeseleer and [[Jun scoby\|here is the current protocol]].
		+
		+	These make very clean and light-colored bacterial cellulose biofilms.
		+
		+	With 10% glycerol, one can even get the 'dried' scoby to not crumble away, and have a more leather like texture.
		+
		+
		+	Previous documentation here:

	aim: to grow big scobies in food trays.		aim: to grow big scobies in food trays.

@@ Line 16: / Line 16: @@
 '''EXPERIMENTS'''
 aim: to grow big scobies in food trays.
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 also had bottle that kept a good smell, but no scoby...
-expt2)
+expt2) 27jan2022 RA
 test Mg++ for aiding them grow?
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 about pH3...
-have 1/4 scoby for each and 1/2 to bottle with more fresh sweet tea to incubator...
+have 1/4 scoby for each and 1/2 to big jar with more fresh sweet tea to incubator...
 <br>

@@ Line 18: / Line 18: @@
 '''EXPERIMENTS'''
-aim: to grow big scobies in food trays.  test Mg++ for aiding?
+aim: to grow big scobies in food trays.
 Try 2 trays of tea, english breakfast. about 5L per tray + 500ml each kombucha tea and half of the fresh part of current scoby (from Javier's batch, 27jan2022)... check pH and maybe add vinegar!
-one try will get 2mM extra MgSO4 (make 2M MgSO4 with 12g/50ml - add 5ml to one of the trays.  let cool before adding scoby and have lids not sealed, so o2 can get in) <br>
+one try will get 2mM extra MgSO4 (make 2M MgSO4 with 12g/50ml - add 5ml to one of the trays.  let cool before adding scoby and have lids not sealed, so o2 can get in)

@@ Line 13: / Line 13: @@
 In fact, the kombucha sheet forming on top of the culture is a type of biofilm. Biofilms are the most common way in which microorganisms exist in nature. They are very resistant and have very interesting physical properties.
 <br>
-A biofilm is any group of microorganisms in which cells stick to each other and often these cells adhere to a surface. These adherent cells are frequently embedded within a self-produced matrix of extracellular polymeric substance (EPS). Biofilm extracellular polymeric substance, which is also referred to as slime (although not everything described as slime is a biofilm), is a polymeric conglomeration generally composed of extracellular DNA, proteins, and polysaccharides (like the bacterial cellulose we find in kombucha). Biofilms may form on living or non-living surfaces and can be prevalent in natural, industrial and hospital settings. <br>
+A biofilm is any group of microorganisms in which cells stick to each other and often these cells adhere to a surface. These adherent cells are frequently embedded within a self-produced matrix of extracellular polymeric substance (EPS). Biofilm extracellular polymeric substance, which is also referred to as slime (although not everything described as slime is a biofilm), is a polymeric conglomeration generally composed of extracellular DNA, proteins, and polysaccharides (like the bacterial cellulose we find in kombucha). Biofilms may form on living or non-living surfaces and can be prevalent in natural, industrial and hospital settings.

@@ Line 5: / Line 5: @@
 '''Protocol:''' how to start a bacterial cellulose culture?
 <br>
-Instructions: Boil 1.5L of distilled water and add 10-15g of tea bags (approximately 2gr of tea per bag). Allow to infuse for 5 minutes and add 1/2 cup sugar, glucose, fructose (1/2 cup agave, etc). Allow to cool to room temperature and add 100mL of a previous batch (liquid medium and a piece of the cellulosic matrix). (optional) Add 100mL of cider vinegar. Seal the bottle and let the culture ferment and grow at room temperature for 5-7 days. After this period you can restart and/or take out the fermented tea and start a second fermentation with citrus, hibiscus flower, ginger, etc. in another bottle for 2-3 days.
+Instructions: Boil 1.5L of distilled water and add 10-15g of tea bags (approximately 2gr of tea per bag). Allow to infuse for 5 minutes and add 1/2 cup sugar, glucose, fructose (1/2 cup agave, etc). Allow to cool to room temperature and add 100mL of a previous batch (liquid medium and a piece of the cellulosic matrix, the "mother" or baby kombucha). Seal the bottle and let the culture ferment and grow at room temperature for 5-7 days. After this period you can restart and/or take out the fermented tea and start a second fermentation with citrus, hibiscus flower, ginger, etc. in another bottle for 2-3 days. Optional: add 100mL of cider vinegar. The final pH reading should be between 2.5 and 3.2 (you can buy PH paper stripe in the pharmacy), to avoid contamination of the symbiotic culture with undesirable micro-organisms.
 <br>
-The culture does not need too much oxygen. Yeasts hydrolyse sucrose into glucose and fructose, producing ethanol and carbon dioxide as metabolites. Acetic acid bacteria convert glucose to gluconic acid and fructose to acetic acid. The final pH reading should be between 2.5 and 3.2, to avoid contamination of the symbiotic culture with undesirable micro-organisms.<be>
+The culture does not need too much oxygen. Yeasts hydrolyse sucrose into glucose and fructose, producing ethanol and carbon dioxide as metabolites. Acetic acid bacteria convert glucose to gluconic acid and fructose to acetic acid. <br>
 '''Content:''' What is a biofilm?