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From 2014.igem.org

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Culturing G. hansenii

Introduction

G. hansenii requires a source of sugar (C source) and peptides (N source) in order to grow and produce bacterial cellulose (Kurosumi et al., 2009). There have been an extensive number of published investigations into how to the broad range of media in which it is possible to grow Gluconacetobacter however most focus on the varieties of carbon source (Masaoka et al., 1993; Ramana et al., 2000; Ha et al., 2008; Hong and Qiu, 2008; Kurosumi et al., 2009). The standard medium for growing Gluconacetobacter in a lab is HS medium.

Aims

One of the main aims of JuicyPrint is to create an easily accessible platform for 3D printing of bacterial cellulose (more info here). To help achieve this we needed to design high yielding media for the production of bacterial cellulose by G. hansenii which can be easily recreated by any individuals around the world. This meant limiting the ingredients of our media to those easily purchasable from supermarkets or grocers'.

Results

To find the best 'homebrew' media for producing bacterial cellulose we tested a number of shop bought products which could potentially be used as carbon sources or nitrogen sources for culturing G. hansenii (Figure 1).

Carbon sources: "Fruit Splash tropical juice drink" Orange juice from concentrate Apple juice from concentrate "Cloudy Apple Juice" (not from concentrate) Tomato Juice (not from concentrate) Orange Marmalade * Boiled potato peelings

Nitrogen sources: Dried skimmed milk powder Marmite Tea from fresh black teabag Tea from used black teabag

All ingredients were diluted to contain an equivalent mass of protein (for tested nitrogen source media) or sugar (for orange marmalade medium only) to that found in HS medium. The normal quantities of yeast extract and peptone for HS medium were added to tested carbon source media and the normal quantities of sugar for HS medium were added to tested nitrogen source media. Each medium was adjusted to approximately pH6.0 (that of HS medium) using lemon juice or bicarbonate of soda (CaCO3).

Figure 2 (error bars show SEM) shows a bar chart of tested media which produced any structurally coherent media after 110 hours of incubation at 30C.

Unfortunately none of the tested homebrew nitrogen sources resulted in growth of cellulose.

Of the carbon sources which resulted in growth of a cellulose pellicle, tomato juice medium appears to have given the highest quantity of cellulose (significantly larger mass than that produced in HS medium) however due to the thick particulate nature of tomato juice, this large mass is most likely to reflect the pieces of tomato juice which could not be washed out from the cellulose pellicle.

However more promisingly, medium containing orange juice from concentrate appears result in a substantial amount of cellulose pellicle which is not significantly worse than the lab standard HS medium. This also appears to be in line with published observations of orange juice being a favourable medium for cellulose production (Kurosumi et al., 2009). The exact composition of 1 litre of our orange juice medium is:

• 1L of Orange juice from concentrate (pictured, 75pence/L).
• 5g peptone
• 5g yeast extract
• 17g Sodium Bicarbonate powder to give a final pH of 5.95

Future directions

The lack of cellulose production from any of our tested nitrogen sources was disappointing. In order to achieve our goal of a 100% homebrew medium it is essential that we find easily obtainable replacements for lab grade peptone and yeast extracts. Testing a broader range of carbon sources and an analysis of cost per yield of cellulose are also experiments we plan to continue with.

Once both a best performing homebrew carbon and homebrew nitrogen source have been established the two can be combined and further tweaks to the formula through addition of ingredients containing acetic acid and ethanol can be evaluated.

References

Ha, J.H., Shehzad, O., Khan, S., Lee, S.Y., Park, J.W., Khan, T., Park, J.K., 2008. Production of bacterial cellulose by a static cultivation using the waste from beer culture broth. Korean Journal of Chemical Engineering 25, 812–815. doi:10.1007/s11814-008-0134-y

Hong, F., Qiu, K., 2008. An alternative carbon source from konjac powder for enhancing production of bacterial cellulose in static cultures by a model strain Acetobacter aceti subsp. xylinus ATCC 23770. Carbohydrate Polymers 72, 545–549. doi:10.1016/j.carbpol.2007.09.015

Kurosumi, A., Sasaki, C., Yamashita, Y., Nakamura, Y., 2009. Utilization of various fruit juices as carbon source for production of bacterial cellulose by Acetobacter xylinum NBRC 13693. Carbohydrate Polymers 76, 333–335. doi:10.1016/j.carbpol.2008.11.009

Masaoka, S., Ohe, T., Sakota, N., 1993. Production of cellulose from glucose by Acetobacter xylinum. Journal of fermentation and bioengineering 75, 18–22.

Ramana, K., Tomar, A., Singh, L., 2000. Effect of various carbon and nitrogen sources on cellulose synthesis by Acetobacter xylinum. World Journal of Microbiology and … 245–248.