Inoculum regime
Growth of P. putida ML2 in benzene culture had been slow and the final cell yield low. It was believed that P. putida ML2 was capable of utilising fluorobenzene as a carbon and energy source. The use of benzene in the first set of shake flasks was substituted for fluorobenzene. However fluorobenzene gave rise to no detectable cell growth. Later correspondence (Lynch, 1994) revealed that P. putida ML2's industrial importance is an enzyme defect that prevents it metabolising fluorocatechol, the product fluobenzene oxidation, hence leading to its accumulation.
In order to grow cells on entirely defined media the peptone was excluded from the first set of shake flasks. When benzene was used as a sole carbon source it was found that concentrations in the region of 2000 pL L'^ inhibited growth and did not increase the cell yield. Sampling meant that the vaporised benzene was lost when the Subaseal was removed and so it was not possible to construct growth curves in this first shake flask stage.
The use of fructose-grown cells was resumed because inoculum cultures could be grown to greater densities. It was easier to grow larger volumes of cell suspension with fructose than benzene because of faster growth and equipment limitations o f the flask culture system. Due to the volatility of benzene the shake flasks had to be sealed. Scaling up the stoppered benzene flasks was difficult, as two litre flasks could not be stoppered effectively. The volume of SFl used to inoculate the SF2's was increased and this shortened the overall incubation time. The only other variation in the inoculum regime was the use of a larger volume of SF2 flask. (IPP6) Finally the inoculum was used at high enough densities at mid growth phase to allow very rapid growth.
Cells could only be grown to 0.4 g dry wt L’^ in benzene shake flasks. The inhibitory nature of high benzene concentrations and oxygen starvation in the stoppered flasks limited growth. This was not the case with the fructose-grown cells which could be cultured to concentrations upto 400% higher. The use of two phase system of flask culture proved to be effective. The first allowed cells to be screened for Toi plasmid activity (only cells with the plasmid can utilise benzene) and the second increased the cell concentration. This reproducible approach provided stable populations of cells in mid growth phase for use as an inoculum.
Fermenter medium composition
The initial media formulation and method suggested by Shell (PPFl) adequately produced cells. In order to allow collaborative work within the department it was decided to work with a researcher investigating on-line monitoring of biomass during fermentations. The method of testing for biomass changes was the measurement of capacitance using a device, known as a Biomass Probe. The presence of an excess of ammonium sulphate increased the electrical conductivity of the fermenter medium and interfered with the operation of the Biomass Monitor.
The majority of problems with fermentations appear to have arisen from variable inocula. A lack of standardisation in the inoculum regime can lead to low cell concentrations and cells possibly not being in the chosen growth phase. The fermenter media composition was not significantly changed during this work but the preparation of the components was altered. The main changes were in the nitrogen source. Ammonium sulphate at a concentration of 1 g appears to be adequate provided an strong inoculum is introduced into the vessel and extra nitrogen is fed in the form of ammonia solution for pH control. To prevent any slowing of growth due to nitrogen limitation an excess was eventually used. The initial concentration of ammonium sulphate of 8.4 g proved satisfactory.
Fermenter operating parameters
It was considered important to standardise the fermenter operating conditions as well as the inoculum regime. P. putida ML2 is an aerobic organism and has a large oxygen requirement. In order to avoid oxygen limitation effects it was decided to prevent the DOT dropping below 20% and fix the operating parameters from the start of the run. Aeration rates were in excess, in the region of 1 vessel volume minute" ^ were used with agitation speeds of upto 1000 rpm. This gave rise to two problems. One of the reasons for the slow growth of benzene grown cells was believed to be because of the high stirrer speeds damaging the solvent weakened cell membranes. During scale up to 25 L fermentations the use of an aeration rate of 25 L m in'l lead to foaming problems when the culture reached stationary phase. It was reduced and compensated for by increased stirrer speed.
Chapter 2 - Fermentation developm ent D iscussion
Another advantage of the final regime was the convenience of being able to divide the work into weekly units:
Friday revive stock cultures in the benzene shake flasks (SFl) Monday inoculate fructose shake flasks (SF2)
prepare the fermenter and allow the probes to settle over night Tuesday inoculate the vessel
Wednesday harvest cells
downstream processing or assay testing Thursday cell damage testing
Friday results analysis
revive stock cultures in the benzene shake flasks
The fermentation protocol PPF6 successfully produced cells for assay and downstream processing testing. There was no reported loss of biotransformation activity and the method proved to be inexpensive in terms of media costs and preparation time.
3 Analytical development
3.1 Introduction
3.1.1 Methods for quantifying cell viability and damage
As was mentioned in Chapter 1, quantifying the damage suffered by a cell population is not a straightforward task. Simply measuring loss of viability can provide insufficient information about the damage process. The term viability is not rigidly defined and has a number of different meanings.
The following chapter sets out to show how a reproducible assay test system was developed to quantify cell damage. The introductory section provides a review of current 'viability' and 'cell damage' tests and is divided into the following sections:
• Measurement of cellular viability • Measurement of cellular disruption • Measurement of the ATP content of cells • Methods of cell disruption at laboratory scale
3.1.1.1 Growth analysis
It is often necessary to report on the size of the bacterial population in a sample. A Viable Cell Count assumes that a visible colony will develop from each organism. Bacteria are however, rarely separated from each other and are often clumped together in large numbers particularly if they are actively reproducing (Collins et a l, 1989). A single colony may therefore develop from one organism or from hundreds or even thousands of organisms. Each colony develops from one viable unit, known as a Colony Forming Unit (CPU). As any agitation, eg in the preparation of dilutions, can break up or induce the formation of clumps, it is obviously difficult to obtain reproducible results. Bacteria are seldomly evenly distributed throughout the sample and as only small samples are usually examined very large errors can be introduced. Many of the bacteria present in a sample may not grow on the medium used at the pH or incubation temperatures employed or in the time allowed.
In Viable Cell Count methods, it is recognised that large errors are inevitable, even if numbers of replicate plates are used. Some of these errors are, as indicated above