Physiology of Escherichia coli at high osmolarity and its use in industrial ethanol production

Biofuels are becoming increasingly important in the light of climate change, increasing energy demands and higher fuel prices. Their production must be carefully balanced against the production of foods and use of fresh water, both of which are consumed by crop based biofuels such as corn ethanol. One proposed solution is to instead use waste materials such as plant matter including wood offcuts and plant trimmings. This waste can be turned into syngas (a mix of CO and H₂) and converted to ethanol using microorganisms. Production of ethanol using microorganisms however, is complicated as the ethanol produced by the cells becomes toxic at higher concentrations, inhibiting their growth and further production. The usual method of keeping the toxicity down to allow further production is to continuously distil ethanol off at low concentrations and consequently, a high cost. Since the mechanisms of ethanol damage to microbes are similar to those that occur during osmotic challenge: damage to the membrane, cytoplasmic dehydration, and protein unfolding, I hypothesized that we can use knowledge of osmoregulatory mechanisms to increase the resistance of cells to ethanol damage and decrease distillation costs. While working under this hypothesis I had to address some of the challenges one faces when understanding the physiology and growth of microbes, and for the purpose I have developed a number of useful techniques; a method for calibrating optical densities to cell number, a neural network for identifying cells and determining their concentrations via microscope imaging and a simple particle diffusion simulation for correcting errors due to confinement of particles within cells. In addition, I have produced a simplified model of industrial production to help evaluate economic impacts that changes to the growth of microbes and the plant process may have. To study any useful links between osmolarity and ethanol resistance, I chose to use Escherichia coli as the model organism due to the large amount of data available on its osmoregulatory mechanisms. It has been long known that when bacteria do grow at high but not lethal osmolarity, they grow at a reduced rate which, even if it increases the ethanol resistance, may have a detrimental effect on the desired production rates. So therefore, in addition to testing the ethanol tolerance of the bacteria under different osmotic conditions, and as a second focus of this project, I have tried to understand why the reduction in growth rates occurs, with the hope of mitigating this effect. This will offer a better understanding of osmotic growth and provide useful insights for industrial bio-production. To this end, I have tried to discern some of the possible reasons for this slower growth by measuring various cell physiological parameters such as batch-culture yield, cytoplasmic diffusion and proteome allocation using my newly developed techniques. I have found a reduction in the cell yield with increasing osmolarity of 50% with an increase of 1Osm of osmotic agent, a slight decrease in cytoplasmic diffusion and a slight decrease in RNA content at high osmolarity. I have also proposed a coarse-grained model of proteome partitioning to help integrate these results and explain growth at high osmolarity. It is still to be determined if, as a whole, the changes observed explain fully the reduction in growth. When it comes to ethanol resistance, and contrary to my hypothesis, I found that increasing the osmolarity of the medium with sucrose or NaCl reduced the ethanol resistance. However, I found that the proW gene provides significant ethanol resistance, indicating glycine betaine, or another substrate for this transporter, is highly useful as a protectant. And this transporter is a potential candidate for overexpression. A reduction in growth temperature also provides significant solvent tolerance at the expense of a reduction in growth rate and hence production.Restricted Acces