Data Obtained By Direct Measurement

Direct measurement by the methods described in chapter 4, (Materials And Methods) produced the data presented in the previous chapter. All the data presented here is from fermentations, or portions of fermentations which were running to plan, and in which no fault had as yet occurred.

In this chapter graphs are presented showing mean, 2o and 3o confidence limits for each of the data reported. The choice of 2 and 3 standard deviations reflects typical industrial statistical process control practice. If the data is normally distributed, 2 standard deviations would encompass 95% of the data, and 3 standard deviations 99.5% of the data. The actual data are shown as fainter broken lines.

In a fault detection scheme, defective fermentations should show up by having broth solids concentrations in samples outside the confidence limits at the point in the fermentation at which the sample was drawn.

Fig 6.1 Expected Broth solids Trajectory Limits, showing mean, 2 a and 3 a confidence limits.

50 45 40 35 25 20 15 10 5 0 0 20 40 60 80 100 120 140 160 Time h

Note: In this chapter bold lines are used to denote mean, 2a, and 3a trajectories. The central bold line denotes mean, the next bold lines out represents the 2 a trajectory, and the outer bold lines represent the 3a trajectory. Raw data from all fermentations yielding useful information is shown as points on the graph.

Fig 6.2 Expected Penicillin Production Limits

Time h

140

This graph and the previous broth solids data (fig 6.1) illustrate the level of scatter from fermentations run to plan using different starting and feed conditions. This data is virtually unusable for fault detection purposes because the limits of

detection which it yields are so broad.___________________________________ Directly measured fermentation parameters can be expected to vary widely especially in fermentations conducted for R&D purposes, such as the

fermentations conducted in the small scale fermenter suites in pharmaceutical companies' research and development laboratories, which is the subject of this thesis. This is because these fermentations are used to investigate the impact of changes in operating conditions, medium components and new organism strains. A useful extension to this work could be to group fermentation data from experiments with similar objectives or characteristics.

6.2 Rate Data

In addition to the production of broth solids and penicillin basic rate data was derived. This is of interest in that, in a correctly functioning fermentation, the rate of broth solids production and carbon dioxide evolution is a function of the rate of nutrient addition and the maintenance of suitable growing conditions in the

parameter data. A rapid change in rate data is indicative of a number of possible faults as will be seen in chapter 8.

Fig 6.3 Carbon Dioxide Evolution Rate Data : Mean,

2c

and 3 a confidence limits.

0.8 ■ •

W 0 .6 - -

Time h

160

Carbon dioxide evolution per litre of fermentation, based on data from all batches, unless obvious or planned faults were in progress. As with biomass and penicillin data, the raw CER data is not useful in establishing that a fault has ocurred.______

6.2.1 Specific Growth Rate

Specific growth rate is defined as

6.1

Specific Growth Rate g/g/hr Broth solids g

Time h

The following equation was used to estimate specific growth rate:

The time increment in the above equation is taken as the time interval over which splines were calculated (10 hours).

The differential nature of the calculation and the sparse nature of the available dry weight concentration measurements resulted in rather disturbed time courses of specific growth rates. However, two phases of growth can be distinguished in all time courses, an initial period of rapid growth followed by a decline in growth rate to a low value for the remainder of the fermentation, which corresponds to the production phase. The disturbed nature of the specific growth rates makes it difficult to determine the values of maximum specific growth rate (P-max)» the specific growth rate during the production phase, accurately. However, the values of and the mean values of p during the production phase, identified for each successful fermentation are presented below.

Table 6.1 Observed Specific Growth Rate Fermentation _Mmax (h-1 ) t Mean p during the production phase (h-i) FERM04 0.068 0.016 FERM08 0.086 0.015 FERM09 0.065 0.014 FERMI 1 0.063 0.013 FERM12 0.070 0.012 FERMI 3 0.086 0.015 FERM2C 0.051 0.013 FERM3B 0.061 0.014 FERM3C 0.053 0.015 FERM5B 0.057 0.012 FERM5C 0.067 0.014 Overall Mean 0.065 0.014

t p estimated using equation 6.2

The calculated values of Pmaxare lower than, for example, those of Mou and Cooney (1983a) or Nestaas and Wang (1981) who reported values of 0.11 h’ ^ and 0.17 h"l respectively for Pénicillium chrysogenum P2 grown under similar conditions to those used in this work. This could be because of the presence of non-biomass solids in the medium, which made the calculation of specific growth rate inaccurate during the growth phase in this work. This is unlikely however, since such solids are unlikely to significantly change in concentration fi"om sample to sample (although they will decline over the course of the fermentation as they are diluted by input streams of glucose, PAA and ammonium hydroxide streams)

The mean specific growth rates during the production phase are qualitatively consistent with the glucose feed rates. In practice, the rate of production of broth solids varied widely due to the differing feed rates used.

Fig 6.4 Specific Growth Rate (p ,) Trajectory Limits 0.7 0.6 0.5

I

I

I

1 0.4 0.3 0.2 -0.2 -0.3 0 20 40 60 80 100 120 140 160 180 Time h

Specific growth rate data. This is based on broth solids accumulation / litre of broth per hour. The presence of solids from com steep liquor does potentially impact specific growth rate figures because the concentration of these solids may be decreasing over the first 30-40 hours, for a discussion of this issue, see section 5 .5.1. The data is somewhat depressed by the fact that the broth is continuously diluted. This is because the rate of material addition due to feeds and pH control exceeds the rate of material lost into the exit gas stream and sampling.

The relatively larger errors seen in the first 30 hours on this chart is largely due to differences in timing of peak growth rates, as some fermentations went from lag to exponential growth phase earlier than others, and some depending on glucose feed regimes continued to grow exponentially for a few hours more than others.

A better comparison of fermentations can be obtained by dividing specific growth rate by carbon fed. This reduces the impact of dififering feed profiles as can be seen in fig 6.5.

Fig 6.5 Normalised specific growth rate (p, /S) trajectory limits showing mean, 2 a and 3 a confidence limits.

0.08 M 0.06 S

I

0.04 o W 0.02 ■0.02 60 140 160 0 20 40 80 100 120 180 Time h

This figure illustrates the usefulness of normalising fermentation data for batch to batch comparison. In effect, the basis for comparison is now between the relative yields of broth solids on glucose Y^g, rather than specific growth rates. The parameter Y^g, is likely to be significantly more stable than in fermentations where glucose addition rates are varied.

6.2.2 Penicillin Production Rate (nip)

Penicillin production rate can be defined as

P

wt) /

/ Xp =

Y 6.3

= Penicillin concentration g/1

= Specific penicillin production rate g penicillin / g broth solids (dry hr

The specific penicillin production rates (m?) were calculated for all fermentations using Equation 6.4

(P *-P ,.,)/A t

=

The specific production rate for all fermentations are presented below

Fig 6.6 Specific Penicillin Production Rate, showing 2a and 3 a confidence limits. 0.15 X 0.1 ■■ _ 0.05 --

I

S 0 I c -0-05 + a w ^.1 . . -0.15 -- -0.2 20 40 60 80 Time h 100 120 140 160

This graph neatly illustrates why Gaussian distributions cannot be applied, the distribution is distorted by outliers. This is the reason that 2 and 3 standard deviation confidence limits are shown rather than 95% and 99% confidence limits.

This data also shows the difficulties of working with limited data sets. More linear confidence limits can be expected given a larger data base

The mp data is of limited value for fault detection purposes as it is highly sensitive to measurement noise. Each data point is obtained from a combination of four measurements, two of broth solids and two of penicillin concentration.. The maximum values specific penicillin production rate, excluding obvious outliers was 0.03-0.04 h'^. These values are comparable with those reported by Cooney (1979) of 0.04 h'^ (reported as 6000 units.(g.cell)'^h*^ where 10^ units = 0.667 g

penicillin G)

The conclusion fi-om this section is that it is necessary to process the data with a view to compensating for batch to batch process variations if an effective fault detection scheme is to be constructed.

In document On-line fault detection in fermentation development facilities (Page 129-138)