Regulation of citric acid production by oxygen: Effect of dissolved oxygen tension on adenylate levels and respiration in Aspergillus niger

Summary The mechanism of the control of citric acid accumulation by oxygen was investigated by means of pilot plant fermentation using Aspergillus niger. The critical dissolved oxygen tension (DOT) for oxygen uptake of this fungus was about 18–21 and 23–26 mbar for trophophase and idiophase, respectively. Minimal DOT for citric acid production was about 25 mbar. Citric acid production increased steadily between 40–150 mbar. Short time changes in the DOT produced immediate, irreversible changes in the rate of product formation. Adenine nucleotides paralleled growth but showed no evidence for control function in the oxygen effect on citric acid fermentation. A branched respiratory system was identified by experiments using specific inhibitors (antimycin, cyanide, azide, rotenone, amytal and salicylhydroxamic acid). Growth was sensitive towards inhibitors of the standard respiratory chain, but only slightly sensitive towards salicylhydroxamic acid (SHAM). Citric acid synthesis was highly sensitive towards SHAM during trophophase, but sensitive towards antimycine during idiophase. Interruptions in aeration cause an impairment of the SHAM sensitive oxidase during trophophase, and of the antimycin sensitive oxidase during idiophase.Dedicated to emeritus Professor Dr. Richard Brunner on the occasion of his 80th birthday     

Sudden substrate dilution induces a higher rate of citric acid production by Aspergillus niger.

On the basis of the present knowledge of Aspergillus niger metabolism during citric acid fermentation, an idea on how to improve the process was formed. Initially, a higher sucrose concentration was used for the germination of spores, which caused a higher intracellular level of the osmoregulator, glycerol, to be present. When citric acid started to be excreted into the medium, the substrate was suddenly diluted. Optimization of this procedure resulted in a nearly tripled volumetric rate (grams per liter per hour) of acid production, while the overall fermentation time was halved compared with the usual batch process. Yet, a characteristic delay was observed at the start of the acid excretion after the dilution. Hypo-osmotic shock caused a prominent elevation of intracellular cyclic AMP levels. Simultaneously, the specific activity of 6-phosphofructo-1-kinase increased significantly, probably due to phosphorylation of the protein molecule by cyclic AMP-dependent protein kinase. Specific 6-phosphofructo-1-kinase activity was much higher in the treated than in the normally growing mycelium. The metabolic flow through glycolysis was expected to be higher, which should contribute to a higher volumetric rate of acid production.     

Application of carbon balance to submerged citric acid production by Aspergillus niger

Summary In an air-lift fermenter, the following production was obtained from 125 g sucrose in mineral medium at pH 2.5 : 15.76 g mycelium dry wt, 107.2 g citric acid anhydrous and 0.594 mol CO₂ within 138 h (run I) and 13.72 g mycelium dry wt, 114.28 g citric acid and 0.516 mol CO₂ within 144 h (run II). Initially, the carbon content of consumed sugar and products did not balance. At the end of fermentation, the carbon content of the products was 0.9%–5.5% higher than that of the consumed sugar. For the purpose of the calculations the carbon content in mycelium was accepted as 0.462.The work was a part of Project No 04.11 CPBP, topic No 2.24     

Parasexual hybridisation and citric acid production by Aspergillus niger

Summary In this investigation hybridisation experiments were performed with two auxotrophic mutants of Aspergillus niger. A heterozygous diploid was derived from them and it produced segregants including parental haploids and a recombinant. Their yield characters were studied.     

Application of kaolin to improve citric acid production by a thermophilic Aspergillus niger

Citric acid production by a thermophilic strain of the filamentous fungus Aspergillus niger IIB-6 in a medium containing blackstrap cane molasses was improved by the addition of kaolin to the fermentation medium. The fermentation was run in a 7.5-l stirred bioreactor (60% working volume). The optimal sugar concentration was found to be 150 g/l. Kaolin (1.0 ml) was added to the fermentation medium to enhance volumetric production. The best results in terms of product formation were observed when 15 parts per million (ppm) kaolin was added 24 h after inoculation. With added kaolin, citric acid production was enhanced 2.34-fold, compared to a control fermentation without added kaolin. The length of incubation to attain this product yield was shortened from 168 to 96 h. The comparison of kinetic parameters showed improved citrate synthase activity of the culture (Y (p/x)=7.046 g/g). When the culture grown at various kaolin concentrations was monitored for Q (p), Q (s), and q (p), there was significant improvement in these variables over the control. Specific production by the culture (q (p)=0.073 g/g cells/h) was improved several fold. The addition of kaolin substantially improved the enthalpy (DeltaH (D)=74.5 kJ/mol) and entropy of activation (DeltaS=-174 J/mol/K) for citric acid production, free energies for transition state formation, and substrate binding for sucrose hydrolysis. The performance of fuzzy logic control of the bioreactor was found to be very promising for an improvement ( approximately 4.2-fold) in the production of citric acid (96.88 g/l), which is of value in commercial applications.     

Metabolism of citric acid production by Aspergillus niger: model definition, steady-state analysis and constrained optimization of citric acid production rate

In an attempt to provide a rational basis for the optimization of citric acid production by A. niger, we developed a mathematical model of the metabolism of this filamentous fungus when in conditions of citric acid accumulation. The present model is based in a previous one, but extended with the inclusion of new metabolic processes and updated with currently available kinetic data. Among the different alternatives to represent the system behavior we have chosen the S-system representation within power-law formalism. This type of representation allows us to verify not only the ability of the model to exhibit a stable steady state of the integrated system but also the robustness and quality of the representation. The model analysis is shown to be self-consistent, with a stable steady state, and in good agreement with experimental evidence. Moreover, the model representation is sufficiently robust, as indicated by sensitivity and steady-state and dynamic analyses. From the steady-state results we concluded that the range of accuracy of the S-system representation is wide enough to model realistic deviations from the nominal steady state. The dynamic analysis indicated a reasonable response time, which provided further indication that the model is adequate. The extensive assessment of the reliability and quality of the model put us in a position to address questions of optimization of the system with respect to increased citrate production. We carried out the constrained optimization of A. niger metabolism with the goal of predicting an enzyme activity profile yielding the maximum rate of citrate production, while, at the same time, keeping all enzyme activities within predetermined, physiologically acceptable ranges. The optimization is based on a method described and tested elsewhere that utilizes the fact that the S-system representation of a metabolic system becomes linear at steady state, which allows application of linear programming techniques. Our results show that: (i) while the present profile of enzyme activities in A. niger at idiophase steady state yields high rates of citric acid production, it still leaves room for changes and suggests possible optimization of the activity profile to over five times the basal rate synthesis; (ii) when the total enzyme concentration is allowed to double its basal value, the citric acid production rate can be increased by more than 12-fold, and even larger values can be attained if the total enzyme concentration is allowed to increase even more (up to 50-fold when the total enzyme concentration may rise up to 10-fold the basal value); and (iii) the systematic search of the best combination of subsets of enzymes shows that, under all conditions assayed, a minimum of 13 enzymes need be modified if significant increases in citric acid are to be obtained. This implies that improvements by single enzyme modulation are unlikely, which is in agreement with the findings of some investigators in this and other fields.     

Optimization of nonlinear biotechnological processes with linear programming: Application to citric acid production by Aspergillus niger

The metabolic pathway and the properties of many of the enzymes involved in the citric acid biosynthesis in the mold Aspergillus niger are well known. This fact, together with the availability of new theoretical frameworks aimed at quantitative analyses of control and dynamics in metabolic systems, has allowed us to construct a mathematical model of the carbohydrate metabolism in Aspergillus niger under conditions of citric acid accumulation. The model makes use of the S-system representation of biochemical systems, which renders it possible to use linear programming to optimize the process. It was found that maintaining the metabolite pools within narrow physiological limits (20% around the basal steady-state level) and allowing the enzyme concentrations to vary within a range of 0.1 to 50 times their basal values it is possible to triple the glycolytic flux while maintaining 100% yield of substrate transformation. To achieve these improvements it is necessary to modulate seven or more enzymes simultaneously. Although this seems difficult to implement at present, the results are useful because they indicate what the theoretical limits are and because they suggest several alternative strategies. (c) 1996 John Wiley & Sons, Inc.     

Galactose inhibition of citric acid production from glucose by Aspergillus niger

Summary Previous work in this laboratory has demonstrated that although Aspergillus niger can readily utilize galactose, no citric acid is produced from this carbon source (Hossain et al. 1984). Experiments were now conducted where galactose was added at various concentrations to synthetic growth medium containing glucose as carbon source, so that the effect of galactose on citric acid production from glucose could be observed. The results showed that the presence of galactose or a product of galactose metabolism caused inhibition of citric acid production, and also reduced the rate of glucose utilization. Enzyme analyses using mycelial cell-free extracts indicated that galactose interfered with the glucose-repression of the key enzyme 2-oxoglutarate dehydrogenase.     

Improved reproducibility of citric acid production in Aspergillus niger

Summary We report the derivation of Aspergillus niger strains having a lower standard deviation in citric acid production by the use of ⁶⁰Co gamma radiation and by the parasexual cycle. On the basis of the results obtained by isolating diploids and segregants, diploidization seems to be sufficient for reducing variability. The implications of these results in terms of improvement of industrial strains are discussed.     

Morphology and citric acid production of Aspergillus niger PM 1

Summary Aspergillus niger PM 1 was grown in a tubular loop and a stirred tank bioreactor. Batch fermentations were performed under various agitation conditions and pH. Citric acid, oxalic acid, extracellular polysaccharides and proteins were assayed. The following morphological parameters were measured: mean perimeter of clumps, mean perimeter of the central core of clumps, mean length of filaments and mean diameter of filaments. Citric acid production and morphology in both reactors were dependent on agitation intensity and pH. The length of the filaments was shown to be the only parameter that could be related to citric acid production in both reactors: the shorter the filaments the more citric acid was produced. However, for the same amount of citric acid produced the morphology of the organism grown in the stirred tank differed considerably from that grown in the loop reactor.     

The effect of methanol on citric acid production from galactose by Aspergillus niger

Summary The effect of methanol on the ability of several strains of Aspergillus to produce citric acid from galactose has been investigated. In the absence of methanol, very little production (less than 1 g/l) was observed. In the presence of methanol (final concentration 1% v/v), however, citric acid production and yeilds were increased considerably. Strong relationships were observed between citric acid production and the activities of the enzymes 2-oxoglutarate dehydrogenase and pyruvate carboxylase in cell-free extracts. During citric acid production, in the presence of methanol, the activity of 2-oxoglutarate dehydrogenase was low and that of pyruvate carboxylase high. In the absence of methanol, where little citric acid was produced, the reverse was true. It is suggested that the presence of methanol may increase the permeability of the cell to citrate, and the cell responds to the diminished intracellular level by increasing production via repression of 2-oxoglutarate dehydrogenase.     

The effect of the sugar source on citric acid production by Aspergillus niger

Summary Under otherwise identical fermentation conditions, the sugar source has been shown to have a marked effect on citric acid production by Aspergillus niger. Sucrose was the most favourable source, followed by glucose and fructose and then lactose. No citric acid was produced from galactose. Strong relationships were observed between citric acid production and the activities of certain enzymes in myccelial cell-free extracts prepared from fermentation samples. When sucrose, glucose, or fructose was the sugar source pyruvate carboxylase activity was high, but 2-oxoglutarate dehydrogenase activity was not detected. When galactose was the sugar source pyruvate carboxylase activity was low, but 2-oxoglutarate dehydrogenase activity was high. It is suggested that whereas glucose and fructose repress 2-oxoglutarate dehydrogenase, thereby causing accumulation of citric acid, galactose does not. The activity of aconitase showed a direct relationship to the citric acid production rate. Thus, the activity was highest when sucrose was the sugar source, and lowest when galactose was the source. It is suggested that when large amounts of citric acid are lost from the cell the activity of aconitase increases as a response to the diminished intracellular supply of its substrate.     

Biochemical studies of citric acid production and accumulation by Aspergillus niger mutants

The biochemical rationale for the inhibition of citric acid fermentation by Aspergillus niger in the presence of Mn²⁺ ions has been investigated using high citric acid-yielding, Mn²⁺ ion-sensitive as well as Mn²⁺ ion-tolerant mutant strains of A. niger. In the presence of Mn²⁺ (1.5 mg/l), citric acid production by the Mn²⁺ ion-sensitive strain (KCU 520) was reduced by about 75% with no apparent effect on citric acid yield by the Mn²⁺ ion-tolerant mutant strain (GS-III) of A. niger. The significantly increased level of the Mn²⁺ ion-requiring NADP⁺-isocitrate dehydrogenase activity in KCU 520 cells and the lack of effect on the activity level of the enzyme in GS-III mutant cells by Mn²⁺ ions during fermentation seem to be responsible for the Mn²⁺ ion inhibition of citric acid production by the KCU 520 strain and the high citric acid yield by the mutant strain GS-III of A. niger even in the presence of Mn²⁺.     

Stimulation of citric acid production in Aspergillus niger by addition of viscous substances in shake culture

When glucose (120mg/ml) was used as a carbon source, Aspergillus niger Yang no. 2. showed a markedly low citric acid productivity in shake culture (15.4 mg/ml) but a high productivity in semi-solid and surface cultures (72.3 mg/ml and 67.6 mg/ml, respectively). Since the viscosity of the medium was assumed to be one of the important factors for citric acid productivity in shake culture, the effects of the addition of viscous substances on citric acid productivity of strain Yang no. 2 were examined. The addition of 2.0–6.0 mg gelatin/ml as a viscous additive to the medium containing glucose as a carbon source increased slightly the medium viscosity but substantially increased the citric acid productivity in shake culture to levels of 52.0–53.3 mg/ml, about 3.4 times as much as that without gelatin. However, no influence of gelatin addition was observed in semi-solid and surface cultures, i.e. under static cultivation conditions. Different mycelial morphologies of the strain were observed when cultivations were done in shake culture with or without the addition of gelatin. Addition of 5.0 mg agar/ml, 5.0 mg carageenan/ml, 2.5 mg carboxymethylcellulose/ml and 2.5 mg polyethylene glycol 6000/ml, to the medium containing glucose as a carbon source also increased the citric acid productivity in shake culture to levels of 39.2–54.7 mg/ml. Since Yang no. 2 does not utilize these viscous substances, these results suggested that the viscous substances functioned as protectants for the mycelium from physiological stresses due to shaking and as a consequence resulted in a remarkably increased citric acid productivity in shake culture.     

Aconitase and citric acid fermentation by Aspergillus niger

In view of the often-cited theory that citric acid accumulation is caused by an inhibition of aconitase activity, the equilibrium of the reaction of aconitase was investigated by comparing in vivo steady-state concentrations of citrate and isocitrate in Aspergillus niger grown under various citric acid-producing conditions. With the equilibrium catalyzed by the A. niger enzyme in vitro, similar values were obtained. The validity of our in vivo measurements was verified by the addition of the aconitase inhibitor fluorocitrate, which appreciably elevated the citrate:isocitrate ratio. The results strongly argue against an inhibition of aconitase during citric acid fermentation.     

Improved production of citric acid by a diploid strain of Aspergillus niger

Aspergillus niger strain CGU 87 was treated with UV radiation and some auxotrophic mutants were obtained. These mutants were less productive than CGU 87, which produced an average of 7.4% citric acid. All possible crosses in pair wise combinations were carried out between these auxotrophs, and three heterokaryons were synthesised. Finally, one heterozygous diploid was isolated from each of them. These heterokaryons and diploids showed improved productivity when compared with their component parents, but except in one diploid D5, all others produced less citric acid than CGU 87. The yield of D5 exceeded that of CGU 87 by 1.2 times and it produced 9% citric acid. This is a significant improvement and the increased productivity seems to be the result of successful adaptation of D5 to its fermentation environment.