Cellulose & sustainability:

One of my favourite aspects of microbiology is the ability of simplistic, single cells to make things of value to us as a society. Somehow, it seems less exploitative than the use of animals and more sustainable than using crude oil-based resources. This is of course, true some of the time but not quite as simplistic as it first seems. Some of the more complex fermentations that make products like vaccines and antibodies will use tonnes of plastics, chemical resources and energy in their lifecycle. And in order to exploit fermentation products to replace the chemical building blocks within crude oil, we would need a consistent source of sugar without diverting it from the food industry. These aspects are not insurmountable, and often the sustainability of biological processes relies heavily on advances in other disciplines such as engineering (renewable energy) and chemistry (allowing the breakdown of complex waste into sugars). So, it is important that we persist in researching these processes so that we understand what is possible. 

One of my primary research interests is the production of bacterial cellulose via fermentation. Cellulose is a complex carbohydrate, made up of linked chains of glucose molecules, and is the most abundant biopolymer on Earth. The glucose bonds in a way that creates long overlapping polymer chains that have a high tensile strength and stability. One of the best-known uses of cellulose fibres in nature is the structural material in plant cell walls. Aside from plants, many bacterial species can produce cellulose which they secrete outside the cell to form biofilms. The cellulose fibres form a solid matrix that facilitates bacterial attachment to surfaces and can provide a protected microenvironment in which they can accumulate in higher densities. Cellulose is also a valuable commodity, being primarily used in the manufacture of paper & textiles. This article on Fortune Business Insights provides an excellent analysis of the current cellulose market. Looking at market trends is certainly not the job of a microbiologist. However, it is important to know the value of the research you are conducting. This value doesn’t need to be economic / financial – but there needs to be an end game for a scientist’s work, and this nowadays is referred to as impact. So how does this relate to my kitchen table?

Fermentation of bacterial cellulose is actually quite a simple process that can be carried out using household items. This process isn’t something that I designed – it is a well-known and established home brewing method that is widely documented and freely available in books and websites (here’s a good one from Make: magazine). I have had lots of success with home brewing in the past, and decided it was time to set-up a new batch of cellulose. Here’s one of my old completed pieces hanging out to dry on my washing line:

The fermentation broth:

In order to grow bacterial cellulose at home you need to provide an environment that is hospitable and allows the bacteria to divide and make your desired product. For the fermentation of cellulose, we need to provide the following:

• A food source for the bacteria. Depending on the bacterial strain, this can be quite prescriptive as some bacteria need specific molecules fed to them which they can’t make themselves. Luckily, the microbes we are growing aren’t that fussy, so we are giving them table sugar (sucrose – a disaccharide of glucose and fructose) at approx. 100 grams per litre of fermentation broth. The bacteria will break this down and use its components to make the building blocks required to divide and make new copies of themselves. 
• Extra nutrients. Alongside carbon and hydrogen (provided in the sugar) bacteria need smaller amounts of other elements such as nitrogen (part of a cells protein material), phosphates (forms DNA backbone), salts and trace elements. For this purpose, I added a teaspoon (about 5 g) of Marmite to the fermentation broth. 
• Acidity. The microbial community that ferments cellulose prefer to live in a slightly acidic environment. In order to provide this, I added 100 ml of white vinegar to the fermentation broth. 
• Water. These microbes will live in a planktonic culture (free floating in liquid) and need available water to survive. I used boiled tap water for this purpose.  

The table below shows the components for a bacterial growth media (Hestrin-Schramm Broth) that can be used to grow cellulose producing Acetobacter species in the microbiology lab. I have included the home-based substitutes in the table so you can see the comparison. 

Hestrin-Schramm Broth		Home-lab Broth Alternative Component
Ingredient (purpose)	Weight (per litre broth)	Ingredient	Weight (per litre broth)
D-Glucose (carbon and energy source)	20 g	Table sugar	100 g
Yeast Extract (nitrogen, salts, trace element source)	5 g	Marmite	Teaspoon (approx. 5 g)
Peptone (nitrogen, amino acid source)	5 g
Disodium Orthophosphate Dihydrate (buffer)	2.7 g	None	n/a
Citric Acid (acidity, carbon source)	1.15 g	Vinegar	100 ml
Type 2 water (solvent)	Make up to 1 litre	Tap water	1 litre boiled and cooled
Preparation notes:   Autoclave to sterilise		Preparation notes:   Sterilise glass vessel with boiling water

My fermentation broth is less defined and is certainly missing some specific ingredients. However, we are not using optimal conditions for the home brew – we just want to make some cellulose no matter the quality. Also, we are going to use a mixed microbial culture in our fermentation. I often use a commercially produced Kombucha scoby (symbiotic culture of bacteria and yeast) as a starting culture for my cellulose fermentation purchased from the Happy Kombucha Company.

IMPORTANT These are food grade starter cultures intended for making edible kombucha tea. I have varied the recipe so the product I make is not going to be kombucha tea and is not an edible food item. This is important – please do not follow my recipe above to make anything at home. 

The mixed culture will provide an undefined number of bacterial and yeast species that have been selected for their ability to ferment sugars and produce kombucha tea, and as a side product can also produce cellulose. Often in such cultures, the yeast will ferment the sugars and provide alcohol that certain bacterial strains can use as a carbon source. So having a mixed culture can be very advantageous. However, when I prepare cellulose in the microbiology lab, I use the model bacterial strain Komagataeibacter xylinus DSM 2325 purchased from the Leibniz Institute DSMZ German Collection of Microorganisms and Cell Cultures.

Setting things up:

Setting up the fermentation is quite easy – just maintain a clean area in order to avoid any contamination from bacteria on other surfaces / your skin etc. I used Pyrex casserole dishes with lids which I steam boil/sterilised by filling them with the correct volume (1 litre) of boiled water from a kettle prior to adding the rest of the ingredients. The dish has a large diameter which is important when making cellulose as the biofilm forms and floats on the surface of the vessel. the bigger the diameter of the vessel at the height of the liquid, the larger the piece of cellulose that you produce. Once the water had cooled, I added the sugar, vinegar and marmite and stirred using a spoon that had been blanched in boiling water. I then added the contents of the scoby to the broth and placed the dishes in a warmish area of the house (NB not accessible to children, pets or any other vulnerable individuals).

And finally:

Will it grow? This is a new set of ingredients for my home fermentation, so we will wait and see what happens. I will provide an update next week – hopefully with cellulose to show off!