Phosphate Limitation

Phosphate Limitation

A mutant defective in the synthesis of a given biomolecule, which must thus be supplied for its normal growth is known as an auxotrophic mutant (Auxotroph). The utility of auxotrophs in fermentation processes is not surprising, as blockage of a known metabolic pathway, such as by limitation, represents a simple and direct method of shunting metabolism in predictable patterns.

Homoserine auxotrophs were isolated from N-methyl'-N'-nitro-Nnitrosoguanidine mutagenized cells of the thermophylic methylotroph, strain NOA2. Auxotrophy was confirmed in liquid cultures by demonstrating that either homoserine or threonine and methionine were required for growth.

Growth of the homoserine auxotroph NOA2 L20#1 in a 20-liter reactor required the feeding of both threonine and methionine. This was accomplished by adding 17.6 g/l L-threonine and 8.8 g/l of L-methionine to the phosphate-magnesium-calcium feed solution. When the production of lysine in the shake flask was found to be non-growth associated, the media were designed to limit growth by either phosphate or sulfate depletion. The pump feeding the amino acid-magnesium-calcium solution was stopped after growth of the cells had ceased, and the methanol feed was changed from the methanol-trace metals mix to pure methanol. This prevented the toxic effects caused by high concentrations of several of the trace metals. Fed-batch fermentations of NOA2 L20#1 HNV#3, using either phosphate or sulfate limitation to obtain 5 g of CDW per liter, resulted in the formation of 8.2 and 7.8 g L-lysine per liter, respectively. Figure 3 shows the non-growth-associated formation of lysine under phosphate-limited conditions in prior experiments performed in a 7-liter reactor (98, 112, 190, 203, 216, 230).


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Fed-Batch Cultivation of NOA2 L20#1

Fed-Batch Cultivation of NOA2 L20#1


Analysis of the culture supernatants from this fed-batch fermentation by high performance liquid chromatography (HPLC) revealed that aspartic acid also accumulate in the reactor (Fig. 3). The aspartic acid appeared only after lysine accumulation had begun and reached final levels that were between 30 and 60% of the final lysine concentration. No differences in the levels of aspartic acid were found when either phosphate or sulfate limitation was used. Production of lysine at cell densities of 20 g/liter, using phosphate limitation, resulted in L-lysine concentrations of 19 g/liter and aspartate concentrations of 7 g/liter after 32 h of cultivation (Fig. 4) (98, 112, 141, 199, 203, 216, 228, 230).


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