Isothermal microwave biology : catalysis and fermentation

This thesis looks directly into the controversial subject of the microwave field effect by the production of a versatile prototype isothermal microwave reactor for the investigation of enzymatic and microbiological reactions. The observed results from the prototype reactor and experiments conducted conclude that there is a nonthermal, nonlinear response between the exposure microwave power and rate and yield of cellulose saccharification. The nature of the nonthermal response is controversial and may be dependent on the definition of "nonthermal,' leading to ambiguity of exact mechanism. Enzymatic and microbial conversion of cellulosic material to ethanol is a highly desirable industrial process. Whether the demand is for the mitigation of climate change, political obligations or energy independence, the use of arable land for energy crops limits the available glucose carbon sources for conversion to bioproducts. To prevent this limitation, cellulose (~-l,4-linked glucose polymers) are touted as the "silver bullet" to prevent carbon exhaustion or impinging on food crops. The technical constraint for the industrialization of cellulose based processing is the rate limitation in the cellulase enzymatic action on cellulose. The enzyme rate is limited by feedback cycles and limited mechanical freedom, therefore a relatively high enzyme concentration is required to speed up the process. To date, the associated enzyme production costs and infrastructure prevents bulk volume exploitation. Biomolecular advances (amino acid substitutions, recombination of expression vectors etc) have gone some way to increase either enzymatic rate or enzyme concentration. The work presented in this thesis differs by increasing the rate of the enzyme without molecular modification. Using a microwave field, the work presented shows that by separating the system into its base units, irradiation of the enzyme/substrate complex in an aqueous environment can increase both the initial enzyme rate and the saccharification yield without alteration of the temperature set point. This study shows that the rate increase is not proportional to the microwave field power. An optimal power in each study is either found or suggested. The results cited show that in the three systems (Endoglucanase and cellobiohydrolase with cellulose, endoglucanase and cellobiohydrolase and ~- glucosidase with cellulose, and ~-glucosidase with cellobiose) the initial rates can be increased by 201 %, 65.5% and 69% respectively. In the total hydrolytic process (endoglucanase and cellobiohydrolase and ~-glucosidase on a cellulose substrate) the final glucose yield was increased by 43% in comparison to the conventional thermal control reaction. This is shown in Figure 1. 10.000 1 9.000 1 8.000 j 7.000 6.000 o 20 40 60 80 100 120 140 160 180 I I 1 I U 5.000 r:: o u 4.000 3.000 2.000 j i t t , f 1.000 0.000 Time (hours) =->=OOOW Glucose' ?012W Glucose ?p025W Glucose ~050W Glucose ?075W Glucose Figure 1. Microwave irradiated "cellulase" enzymes with cellulose substrate I For development into an industrial system and looking towards simultaneous saccharification and fermentation (SSF), the yeast Saccharomyces cerevisiae was subjected to irradiated microwave fermentations on a glucose substrate. Although inconclusive in terms of rate increase, cell density 1 was comparable across the power range showing that the irradiation does not have a derogatory effect. ! The natural evolution of the conclusions drawn would be development of the system into a SSF or SSCF configuration for bio-product formation is possible with irradiation up to SOW. ii The novelty of the experiments conducted is twofold. Firstly, the reactor has been designed to ensure that the microwave irradiation is independent of the bulk temperature therefore allowing the exploration of the microwave field effect independently to the thermal effect. Secondly, the microwave source is a continuous microwave irradiation (none pulse irradiation) ensuring that the reaction is subjected to the microwave field for the entire reaction