The evaluation of the fungi using tower fermenters

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The development of filamentous fungi in submerged culture

During the 24 hour period following inoculation, aggregation of spores and sporelings can have an important effect on the subsequent growth of filamentous fungi in submerged culture. This early phase of growth does not appear to have received much attention, and it was for this reason that the author's research was started. The aggregation, germination and early growth of the filamentous fungus Aspergillus niger have been followed in aerated tower fermenters, by microscopic examination. By studying many individual sporelings it has been possible to estimate the specific growth rate and germination times, and then to assess the branching characteristics of the fungus over a period of from 1 to 10 hours after germination. The results have been incorporated into computer models to simulate the development of the physical structure of individual and aggregated sporelings. Following germination, and an initial rapid growth phase, fungi were found to grow exponentially: in the case of A.niger the mean germination time was about 5 hours and the doubling time was as short as 1.5 hours. Branching also followed an exponential pattern and appeared to be related to hyphal length. Using a simple hypothesis for growth along with empirical parameters, typical fungal structures were generated using the computer models : these compared well with actual sporelings observed under the microscope. Preliminary work suggested that the techniques used in this research could be successfully applied to a range of filamentous fungi.

Fermentation methods for dextransucrase production

Several fermentation methods for the production of the enzyme dextransucrase have been employed. The theoretical aspects of these fermentation techniques have been given in the early chapters of this thesis together with a brief overview of enzyme biotechnology. A literature survey on cell recycle fermentation has been carried out followed by a survey report on dextransucrase production, purification and the reaction mechanism of dextran biosynthesis. The various experimental apparatus as employed in this research are described in detail. In particular, emphasis has been given to the development of continuous cell recycle fermenters. On the laboratory scale, fed-batch fermentations under anaerobic low agitation conditions resulted in dextransucrase activities of about 450 DSU/cm3 which are much higher than the yields reported in the literature and obtained under aerobic conditions. In conventional continuous culture the dilution rate was varied in the range between 0.375 h-1 to 0.55 h-1. The general pattern observed from the data obtained was that the enzyme activity decreased with increase in dilution rate. In these experiments the maximum value of enzyme activity was ∼74 DSU/cm3. Sparging the fermentation broth with CO2 in continuous culture appears to result in a decrease in enzyme activity. In continuous total cell recycle fermentations high steady state biomass levels were achieved but the enzyme activity was low, in the range 4 - 27 DSU/cm3. This fermentation environment affected the physiology of the microorganism. The behaviour of the cell recycle system employed in this work together with its performance and the factors that affected it are discussed in the relevant chapters. By retaining the whole broth leaving a continuous fermenter for between 1.5 - 4 h under controlled conditions, the enzyme activity was enhanced with a certain treatment from 86 DSU/cm3 to 180 DSU/cm3 which represents a 106% increase over the enzyme activity achieved by a steady-state conventional chemostat. A novel process for dextran production has been proposed based on the findings of this latter part of the experimental work.

Batch and continuous fermentation methods for the production of dextransucrase

The available literature concerning dextransucrase and dextran production and purification has been reviewed along with the reaction mechanisms of the enzyme. A discussion of basic fermentation theory is included, together with a brief description of bioreactor hydrodynamics and general biotechnology. The various fermenters used in this research work are described in detail, along with the various experimental techniques employed. The micro-organism Leuconostoc mesenteroides NRRL B512 (F) secretes dextransucrase in the presence of an inducer, sucrose, this being the only known inducer of the enzyme. Dextransucrase is a growth related product and a series of fed-batch fermentations have been carried out to extend the exponential growth phase of the organism. These experiments were carried out in a number of different sized vessels, ranging in size from 2.5 to 1,000 litres. Using a 16 litre vessel, dextransucrase activities in excess of 450 DSU/cm3 (21.67 U/cm3) have been obtained under non-aerated conditions. It has also been possible to achieve 442 DSU/cm3 (21.28 U/cm3) using the 1,000 litre vessel, although this has not been done consistently. A 1 litre and a 2.5 litre vessel were used for the continuous fermentations of dextransucrase. The 2.5 litre vessel was a very sophisticated MBR MiniBioreactor and was used for the majority of continuous fermentations carried out. An enzyme activity of approximately 108 DSU/cm3 (5.20 U/cm3) was achieved at a dilution rate of 0.50 h-1, which corresponds to the maximum growth rate of the cells under the process conditions. A number of continuous fermentations were operated for prolonged periods of time, with experimental run-times of up to 389 h being recorded without any incidence of contamination. The phenomenon of enzyme enhancement on hold-up of up to 100% was also noted during these fermentations, with dextransucrase of activity 89.7 DSU/cm3 (4.32 U/cm3) being boosted to 155.7 DSU/cm3 (7.50 U/cm3) following 24 hours of hold-up. These findings support the recommendation of a second reactor being placed in series with the existing vessel.