Submerged citric acid fermentation of whey permeate by Aspergillus niger : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Massey University

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Massey University
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The feasibility of using lactic casein whey permeate as an alternative source of raw material for the production of citric acid by Aspergillus niger was studied. A. niger (10 strains) and A. carbonarius (1 strain) were screened for their ability to produce citric acid from lactic casein whey permeate in shake-flask culture. Of the organisms tested, A. niger IMI 41874 produced the highest citric acid concentration of 5.0 g/l, representing a yield of 13.5% (w/w) based on lactose utilized. When the permeate was supplemented with additional lactose (final concentration 140 g/l), a concentration of 8.2 g/l was obtained, representing a yield of 15.5% (w/w). This organism was selected for further study including strain improvement work by induced mutation using UV light. A mutant strain (MH 15-15) was isolated which produced a citric acid concentration of 10.2 g/l in lactose-supplemented whey permeate. Using a sucrose-based synthetic medium a concentration of 52.8 g/l (yield 48% (w/w)) was observed, compared with 34.0 g/l (yield 33% (w/w)) produced by the parent strain. This mutant was used throughout subsequent experiments. In fermenter culture experiments using lactose-supplemented whey permeate a citric acid concentration of 14.8 g/l was obtained. When extra nitrogen was fed to the culture after the onset of citric acid production, a concentration of 19.5 g/l was observed. Experiments with decationized whey permeate, supplemented with various amounts of different trace elements, proved unsuccessful in respect of improved citric acid production when compared with untreated whey permeate. Experiments with different sugar sources using a synthetic medium demonstrated a marked effect of the sugar source on citric acid production. Thus, concentrations of 52.8 g/l, 31.0 g/l, 23.0 g/l, 5.0 g/l and 0 g/l were obtained from sucrose, glucose, fructose, lactose and galactose respectively. Good mycelial growth was observed with all the sugars. Similar experiments in fermenter culture showed the same trend of results, but in contrast to the experiments using whey permeate, citric acid production was lower than in shake-flask culture. The activities of some TCA-cycle enzymes in mycelial cell-free extracts were investigated during fermenter culture experiments using the different sugar sources in synthetic medium and whey permeate. The initial activities of aconitase and both NAD- and NADP-linked isocitric dehydrogenase showed a strong relationship with subsequent citric acid accumulation. During citric acid accumulation the activities of these enzymes decreased significantly compared with those found during growth phase, but did not completely disappear. 2-oxoglutarate dehydrogenase disappeared completely when citric acid production was high but activity was maintained when production was low. The activity of pyruvate carboxylase increase considerably during citric acid production but little activity was detected when citric acid was not produced. It was concluded that accumulation of citric acid is not a consequence of the complete disappearance of the activity of aconitase or isocitric dehydrogenase (both NAD- and NADP-linked), but rather the accumulation is caused by the repression of 2-oxoglutarate dehydrogenase causing a block in the TCA-cycle, and the concomitant increase in pyruvate carboxylase activity. It was hypothesized that glucose and fructose cause repression but galactose does not. Experiments using various combinations of glucose and galactose as sugar source demonstrated that galactose caused competitive inhibition of citric acid production from glucose. The inhibition showed a strong relationship with the levels of activity of 2-oxoglutarate dehydrogenase and pyruvate carboxylase. The effect of methanol on citric acid production from lactose, glucose, galactose and whey permeate was investigated. In shake-flask culture, 1% (v/v) methanol caused increased production and yields of citric acid from both glucose and lactose. Citric acid production from galactose was also observed (12.5 g/l). In fermenter culture, using whey permeate, the presence of 3% (v/v) methanol gave a 69% increase in citric acid production (25.0 g/l compared with 14.8 g/l in the absence of methanol). The presence of methanol showed a general inhibitory effect on the various TCA-cycle enzymes studied, in particular 2-oxoglutarate dehydrogenase. Overall, it was concluded that the main obstacle to the improved production of citric acid from whey permeate is the nature of the sugar source rather than the other components of the substrate. In particular, the galactose moiety of lactose is not a favourable sugar source.
Casein whey, Citric acid, Aspergillus niger, Fermentation