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    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
    (Massey University, 1983) Hossain, Moazzem
    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.
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    Citric acid production using Aspergillus niger by solid substrate fermentation : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Process and Environmental Technology at Massey University
    (Massey University, 1995) Lu, Minyuan
    The aim of this work was to investigate solid substrate fermentation for citric acid production using Aspergillus niger, in an attempt to provide systematic information and an understanding of the process. Initial experiments were performed to select an appropriate substrate and organism. Thus, kumara and a strain of Aspergillus niger, Yang No.2 were found favourable for citric acid production, while potato was a poor substrate due to its excessive nitrogen content. The fermentations were earned out under various conditions, i.e. inoculum size, moisture content and particle size of the substrate to optimize these parameters. Inoculum sizes between 10 4 and 10 6 spores/40 g kumara, moisture contents between 65 and 71% and particle sizes between 4 and 6 mm were optimal for citric acid production. It was found that the organism takes up nutrient by penetrating into the substrate, thus the fermentation had a direct relationship with the available surface area. The solid substrate was found to have the potential ability to overcome the adverse effect of high concentrations of metal ions. Addition of 150 mg/kg substrate of Fe2+, 25 mg/kg substrate of Cu2+, 75 mg/kg substrate of Zn2+ and 150 mg/kg substrate of Mn2+ had slightly stimulatory effects on citric acid production rather than inhibitory effects. Based on the optimized conditions, the kinetics of the solid substrate fermentation in flasks were studied. The maximum observed gravimetric rate, maximum observed specific rate and overall productivity of citric acid production were 1.5 g/kg.h, 122 mg/g.biomass.h and 0.48 g/kg.h, respectively. To develop the solid substrate fermentation process, experiments were performed in different types of reactors, including a gas-solid fluidized bed, a gas-liquid-solid fluidized bed, a rotating drum and a packed bed. Except for the packed bed reactor, these systems were found to be unsuitable for the fermentation, due to harsh conditions of abrasion, friction, low moisture supply, or combinations of these factors. The fermentation in the packed bed reactor was optimized with respect to air flow rate, bed loading and particle size. Based on these optimized conditions, the kinetics were studied, and it was determined that the fermentation allowed much higher rates of citric acid production than were observed in flasks, i.e. a maximum observed gravimetric rate of 1.9 g/kg.h and an overall productivity of 0.82 g/kg.h. In an attempt to understand mass and heat transfer in the solid substrate fermentation, experiments were conducted in a multi-layer packed bed reactor. However, because of the complexity of mass transfer in solid substrate fermentation, the understanding of this aspect in this process was rather limited. Nevertheless, the multi-layer packed bed reactor improved the mass transfer considerably compared with the single layer packed bed with the same bed loading, and allowed precise measurement of the gradients for gases, citric acid, biomass and starch. The results suggest that the multi-layer packed bed reactor is a suitable reactor for further investigations, and has the possibility of being used for large scale production of citric acid in solid substrate fermentation. This systematic investigation of solid substrate fermentation for citric acid production, which is the first reported, provides detailed information and understanding of this fermentation technology.
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    Mechanism of citric acid accumulation by Aspergillus niger in solid state fermentation : a thesis presented in partial fulfilment of the requirement for the degree of Doctor of Philosophy in Process and Environmental Technology at Massey University
    (Massey University, 1996) Leangon, Sirichom
    The main purpose of this work was to study the mechanism of citric acid accumulation in solid state fermentation of Aspergillus niger. Two strains, Yang No.2 and MH 15-15, represented the high-accumulating organisms from which the low-accumulating mutants, SL-1 and SL-2, were generated by ultraviolet treatment. Comparative solid state fermentations, with a starch-containing material as the substrate, were conducted in petri-dishes, a technique which conferred a major advantage in allowing recovery of metabolically active mycelia for biochemical assays. Apart from the decreased citric acid accumulation, the selected mutants displayed lower starch consumption and enhanced production of oxalic acid, while their growth were generally equal to that of their respective parents. Evidence on elevated levels of free glucose in the cultures of the mutants, despite there being no alteration of α- amylase and glucoamylase from their parents, has prompted a hypothesis that the mutants were defective in the rates of glucose uptake. The biochemical work started with the primary steps of carbon assimilation, viz measurement of glucose uptake and activity assay of hexokinase. The results confirmed the reduced glucose uptake rates by the mutants and a hypothesis that this is caused by some defects in certain components of the glucose transport mechanism, but not at membrane ATPase, has been proposed. In addition, hexokinase showed higher in vitro activities in the parents and, presumably, their glycolytic fluxes were greater than those of their mutants. Investigation of activities of some selected TCA cycle enzymes and other metabolic steps in vitro strongly indicated the decreased activity of 2-oxoglutarate dehydrogenase and, possibly, NAD-and NADP-specific isocitrate dehydrogenases in the parents. Although most other enzymes decreased their activities during the later phase of cultivation, there was no definite difference between each parent and its mutant. However, oxaloacetate hydrolase, for oxalate formation, was at higher activity in the mutants than in the parents. Measurements of intracellular concentrations of products of certain enzymes and adenine nucleotides were conducted in order to assess the in vivo catalytic function of the enzymes of interest. It was concluded that internal accumulation of citrate or oxalate is an immediate cause of its excretion. Supplemented by evidence from the ratio of ATP/AMP in the cells, a complete hypothesis describing citrate accumulation in A.niger Yang No.2 and MH 15-15 is proposed. Hence, the rate by which glucose is taken up into the cells is the primary trigger determining the capacity of glycolytic metabolism and it is proposed that the primary cause of citric acid accumulation in the high-accumulating strains is the deregulation of glucose uptake. When the glucose supply exceeds the requirement of the cells, i.e. when growth is slow, the TCA cycle is balanced by allosteric deactivation of isocitrate dehydrogenases by ATP which is excessively generated via the active glycolysis. The observed low level of activity of 2-oxoglutarate dehydrogenase is, therefore, a result of this metabolic block, rather than a cause. Because of the equilibria of the reactions, citrate is accumulated and then excreted out of the cells. In contrast, when the glucose supply is below such level, this enzyme regulation does not occur and oxalate, instead of citrate, acts as the drain of excess carbon going around the fully operative TCA cycle. In conclusion, the current hypothesis for citric acid accumulation is basically similar to that proposed for submerged fermentation conditions. However, the rationale for carbon sink via oxalate is novel. Finally, it has been shown that two A.niger strains of different origins displayed a similar mechanism, although the fine control may be different.