unid de 1 lote + 1 und

S. lividans TK24 grew well in starch-nitrate medium and produced biomass levels of up to 2.8g-L‘^ DCW at 96h (Figure 3.50), which was greater than in glucose- nitrate medium (2.1g-L'^). DNA analysis indicated that replicatory growth would have continued beyond 120h and the end of biomass accumulation probably reflects the exhaustion of starch at 72h (Figure 3.51). Nitrate was present throughout. Both pyruvate and a-ketoglutarate appeared in the medium at around 30h but neither increased to the levels recorded for glucose-nitrate medium. Pyruvate reached a maximum concentration of 23.8mg-L'^ at 54h and a-ketoglutarate a maximum of 57.0mg-L'^ at 72h. Acid production was accompanied by a slight fall in pH from 6.5 to 6.0, which was not as significant as in earlier cultures, although after 96h the pH rose sharply to 7.5 (Figure 3.52). The lowest pH corresponded to the highest acid levels. Had the culture continued beyond 120h, this high pH may have affected the organism. Biomass levels fell by 20% in the period of greatest pH increase, although the rise in pH accompanied the reuse of acids from the medium. Pyruvate was completely reused by 96h and may have been utilised to supply carbon during biomass accumulation up to 96h. a-Ketoglutarate was virtually completely reused by the end of the culture. Since a-ketoglutarate was not exhausted despite being excreted at only low levels, this suggests that this compound is not readily utilised by S. lividans as a potential carbon source in the way that pyruvate appears to be in defined media.

There were three significant differences between acid production in starch- nitrate and glucose-nitrate cultures. A comparable amount of carbon was consumed by both cultures (around 2g L'^). First, acid production was lower. Maximum pyruvate and a-ketoglutarate levels represented just 0.5% and 1.0% respectively of the consumed carbon in starch-nitrate medium (Figure 3.53), compared to proportions of 8.1% and 2.6% in glucose-nitrate medium. Second, the acid maxima occurred at different times, whereas all glucose media containing nitrate exhibited simultaneous acid maxima (Sections 3.2.2.1 and 3.2.2.3). Third, by weight, almost twice as much carbon was excreted as a-ketoglutarate, perhaps reflecting the extended period of production of this acid relative to pyruvate in starch-nitrate compared to glucose-nitrate medium.

g 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0 24 48 0 72 96 120 9 ^ TimeOi)

Figure 3.50: Graph showing biomass ( - • —j and deoxyribose f—T~j changes during a culture grown in SMM containing 0.5% w/v starch and 30mM nitrate.

u a) 6 500 5 400 4 300 w 3 2 1 0 0 1 0 =^0 120 24 48 0 72 96 Time ^i)

Figure 3.51: Graph showing changes in carbon source, starch (-m-), and organic acids,

pyruvate ( ) and a-ketoglutarate ( - A - ) during a culture grown in SM M containing

% 6

24 48 72

TimeOî)

96 120

Figure 3.52: Changes in p H during a culture grown in SMM containing 0.5% w/v starch and 30mM nitrate.

100 1 I = = A o 120 48 72 96 24 0 Time(h)

Figure 3.53: Graph showing decrease in total carbon ( - 0 - ) and proportion o f carbon present in organic acids, pyruvate (~0~) and a-ketoglutarate f-A-j, expressed as a % o f the total carbon consumed during a culture grown in SMM containing 0.5% w/v starch and 30mM nitrate.

s. lividans grew moderately well but slowly in maltose-nitrate medium (Figure 3.54). The maximum DCW recorded was 2.7g*L’^ at 120h, which was also an improvement on glucose-nitrate medium. DNA analysis indicated that replicatory growth continued beyond 120h (data not shown). Very little maltose was used in the initial stages of culture, levels falling by only around 6% in the first 72h (Figure 3.55). After 72h the carbon source was consumed more rapidly until around 75% had been used in total. This period of rapid maltose utilisation was accompanied by the appearance of a very small amount of pyruvate in the medium (10.8mg-L‘^) at 96h (not shown), corresponding to 0.07% of the total consumed carbon. No pyruvate was detected at other times during the culture, and no a-ketoglutarate was detected at all. The pH fell to 6.0 by 96h and rose rapidly to 7.0 by 120h (data not shown). Nitrate remained present throughout.

The biomass yields of S. lividans TK24 in glycerol-nitrate and fructose-nitrate media (Figure 3.54) did not exceed those of glucose-nitrate cultures, although glycerol was completely consumed (Figure 3.55). Both grew slowly and DNA analysis showed replicatory growth to continue beyond 120h (data not shown). Fructose-containing cultures grew steadily to a maximum DCW of 1.6g L'^ at 120h, but little fructose was utilised during the culture (Figures 3.54 and 3.55). Glycerol cultures grew slowly until 72h with the consumption of around 20% of the glycerol. Between 72 and 120h, the biomass doubled to a maximum dry weight of 2.0g-L‘^ and the remainder of the glycerol was utilised. Nitrate was present throughout cultures in both media. No acids were detected and the pH did not change from 6.5 (data not shown). The biomass in all cultures described in this section was in the form of finely dispersed mycelia.

The results demonstrated that organic acid excretion was not as copious when S, lividans TK24 was grown in defined media containing nitrate and an alternative carbon source to glucose. Those cultures excreting detectable amounts of acids were also those containing carbon sources which are metabolised directly to glucose (starch and maltose). The starch used was found to contain 0.2% reducing sugars in the form of glucose (manufacturer’s notes), corresponding to O.Olg-L'* in the media described in this section. It is possible that this small amount of glucose induced acid production, causing higher acid production in starch-based media compared to maltose media. Maltose as supplied did not appear to contain glucose.

35 3.0 3.0 25 25 Ü a) 2.0 _2.0 ■1) f 15

S

Figure 3.54: Graph showing biomass changes during three cultures in SMM containing maltose and nitrate ( - * - ); glycerol and nitrate f - # - ); or fructose and nitrate (—h —). Details are given in the text.

g c

-#0 120 Time(h)

Figure 3.55: Graph showing changes in carbon sources during three cultures in SMM containing maltose (-m~) and nitrate (not shown); glycerol (-#-) and nitrate (not shown); fructose ( -àr- ) and nitrate (not shown). Details are in the text. Nitrate was present