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The alanine and glucose fermentation was carried out in a 20 L fermenter with

Streptom yces lividans TK24. The media was M SMM4 (C) with alanine as described in chapter 5.

According to HPLC measurements at the start o f the fermentation, the C:N ratio was 9.3 and the alanine to glucose molar ratio was 0.95. The total carbon available in the medium was 314 mM. The average specific growth rate was 0.04 hr' 1 and the biomass reached its peak at 2.53 g/L after 47 hours. At this point the glucose had ran out, but som e alanine was still left in the culture broth. The biomass, glucose and valine concentrations are plotted in Figure 6-7. The organic acid concentrations are plotted in Figure 6 - 8 and the carbon evolution and the oxygen utilisation rates are plotted in Figure 6-9.

35 T 3.0 30 25 20 15 10 5 0 10 20 30 0 40 50 Glucose Tim e (hrs) ■ A - - Alanine 2.5 2.0 60 1.5 § c o o 1.0 w_TO E o m 0.5 0.0 - ♦ Biomass

Figure 6-7, Biomass, glucose and alanine concentrations in the alanine and glucose fermentation. 9 8 7 6 5 4 3 2 1 0 20 30 40 50 60 10 0 Tim e (hrs)

-A— Pyruvate ■ a-Ketoglutarate

Figure 6-8, Organic acid concentrations in the alanine and glucose fermentation.

* *

40 50 60 70 80

Tim e (hrs)

X Oxygen Utilisation Rate - Carbon Evolution Rate

Figure 6-9, Carbon evolution rate and oxygen utilisation rate in the alanine and glucose fermentation.

The m ax im u m pyruvate concentration present in the culture broth was 8.36 m M at 31 hours and the m ax im u m a -k e to g lu ta ra te concentration was 1.22 m M at 47 hours (the

point were the biom ass reached its m ax im u m concentration). a -K e to g lu ta rate was produced at a steady rate throughout the active growth phase. Pyruvate seem ed to be secreted at an exponential rate up until 31 hour before stagnating. Only at 47 hours did it start to be reassim ilated.

Out o f the 314 m M carbon available at the start o f the culture, about 105 m M went into biomass, about 180 m M was lost in respiration and about 30 m M (~ 10 %) was lost in acid production. Hence, the overall carbon balance was correct. T he overall nitrogen balance was also checked and this was also found to be satisfactory.

Some o f the carbon m ass balances for intermediate time intervals did not balance. The high assim ilation o f glucose betw een 0 and 22.75 hours was for exam ple not reflected in the accum ulating biomass. T he secreted acids and the carbon evolution rate could not account for the extra carbon apparently being m etabolised either. As the nitrogen balance seem ed to be correct, both the am ino acid and biom ass m easurem ents were assum ed to be fine. According to the assays, the glucose was also used up before the am ino acid, which is unusual. The discrepancy may therefore have been due to

M athem atical M odelling o f B iochem ical Pathways Fermentations

inaccurate glucose assays, or an undetected carbon metabolite secreted early in the culture and reassimilated later.

One unknown substance was seen to accumulate throughout the fermentation. The compound was detected on the Aminex HPLC column using the RI detector and had an approximate retention time, of 8.5 minutes. (This was comparable to one o f the unknown metabolites seen accumulating during valine and glucose fermentation.) The secretion pattern o f the detected metabolite did not match the secretion pattern expected for a metabolite that would have accounted for the mismatch in the carbon balance. The metabolite accounting for the missing carbon would have had to have been secreted early in the culture and then reassimilated later on. The detected compound was secreted all the way throughout the fermentation.

It was suspected that the unidentified HPLC peak might be citrate, as observed in other

Streptom yces cultures (Dekleva and Strohl, 1987). However, comparing standard relative retention times and UV/RI ratios for citrate (Bio-Rad) with the respective values for the unidentified HPLC peak, it soon became evident that the peak could not be citrate. In fact, none o f the about 50 common carbon metabolites described by Bio-Rad fitted the HPLC peak observed. The overall carbon balance for the fermentation was correct. Hence, it was assumed that this unidentified metabolite was probably not a major carbon compound and would therefore not be o f importance in metabolic carbon flux studies.

The alanine and glucose fermentation reached the highest acid concentration of the three fermentations. Pyruvate was secreted at an exponential rate up until 31 hours. Only after 47 hours did the reassimilation at start. This was unexpected behaviour, and it is suspected that pyruvate secretion continued after 31 hours, resulting in a maximum pyruvate concentration somewhere between 31 and 47 hours before it was reassimilated. Hence, at 47 hours the pyruvate level might just have happened to be at a comparable level to that at 31 hours.

M athem atical M o d e llin g o f B iochem ical Pathways A Sim ple Structured M odel

7.

A SIMPLE STRUCTURED MODEL FOR THE GROWTH OF