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Team:Imperial/Mass Production and Processing
From 2014.igem.org
Mass Production and Processing
Introduction
Bacterial cellulose (BC) exhibits a multitude of different properties depending on the processing, growth conditions, functionalisation and strain used (Bismarck 2013) for production of the material. Acquiring large quantities of cellulose produced would allow testing of a broad variety of cellulose processing methods and functionalisation 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.
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-intuituve 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 as shown in figure 3. 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 (see figure 2 left) and even pellicles (figure 2 right). All pellicles were kept in distilled water in large plastic buckets or containers.
Below shows the general workflow we employed to mass produce our cellulose and illustrates the process of manufacturing biomaterials with significantly different properties despite originating from the same BC source.
Results
We have made bacterial cellulose of a quality high enough to be used for water filtration in an ultrafiltration membrane setting. From different trial and error, we tried making leather cellulose that can be used a potential fabric garment. We produced bacterial cellulose on a scale larger than any previous iGEM team has produced a biomaterial in the Manufacturing track before. This has provided us with opportunities to try treating cellulose with different low tech solutions that have been identified as invaluable to our collaborating Chemical Engineer Dr. Koonyang Lee. In his own words, we have produced more bacterial cellulose than he did during both his PhD and postdoc on BC, and come up with some simple solutions for practical issues. The ideas of ours that Dr. Lee has decided to carry on include using silicon coated baking paper in the protocol for measuring yield of BC to avoid BC sticking to the surface, and effectively using a non-bleach fabric stain remover to clean the BC thereby reducing the harmful impact of research in this area.
Appendices
- 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 residual 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 redried.
- NaOH for 20 min at 120 C, followed by blendering: produces cellulose that seems like it is much less ductile. Disadvantage: the functionalisation will be blended just like the cellulose, so the proteins may be broken down mechanically.
- NaOH for 20 min at 120 C, followed by blendering: produces cellulose that seems like it is much less ductile. Disadvantage: the functionalisation 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.
