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⁺.     

Continuous production of citric acid from sugarcane molasses using a combination of submerged immobilized and surface stabilized cultures of Aspergillus niger KCU 520

Summary A high performance fermentation process for the continuous production of citric acid from sugarcane molasses by using the combination of submerged calcium alginate-immobilized and surface-stabilized cultures of Aspergillus niger KCU 520 in a continuous flow horizontal bioreactor is described. The citric acid productivity was dependent on the dilution rate with an optimum value of 0.015/h. Presaturation of fermentation medium with sterile air, in addition to surface aeration, before feeding into the bioreactor enhanced the citric acid productivity. The highest productivity, citric acid product concentration and yield obtained were 1.7 kg M^–3h^–1, 110kg M^–3 and 91% respectively. The cultures were continuously used for 30 days without any apparent loss in citric acid productivity.     

Repeated batch production of citric acid from sugarcane molasses using recycled solid-state surface culture ofAspergillus niger

Summary The recycled solid-state surface fermentation (SSF) culture ofAspergillus niger KCU520 was used for repeated batch production of citric acid from sugarcane molasses. The rate of citric acid production was doubled, reducing the fermentation time to half, compared to the normal single cycle batch submerged or surface fermentation process. About 80% sugar was converted to citric acid in five-day batch fermentation and three batches were carried out with the same fungal mat without any significant loss of productivity.     

Metabolic effects of manganese deficiency in Aspergillus niger: evidence for increased protein degradation

The effect of manganese deficiency on macromolecule synthesis has been studied in a citric acid producing strain of Aspergillus niger: pulse labelling experiments showed that the synthesis of both protein and RNA was not influenced by the presence of manganese; however, increased protein degradation occurred under manganese deficiency. This was also reflected by the increased activity of an intracellular proteinase activity under these conditions. In replacement cultures addition of inhibitors of RNA, DNA or protein synthesis revealed that only emetine and cycloheximide (which both act at the ribosome) successfully antagonized the adverse effect of manganese ions on citric acid accumulation. Manganese deficiency was also characterized by a decreased portion of polysomes and 80 S ribosomes.     

Concomitant loss of respiratory chain NADH: ubiquinone reductase (complex I) and citric acid accumulation in Aspergillus niger

Summary Growth, citric acid production and enzymatic activity of the mitochondrial respiratory enzymes of a wild-type and a citric-acid-producing mutant of Aspergillus niger have been compared during fermentation under citric-acid-accumulating and non-accumulating conditions. Under non-accumulating conditions, both strains showed standard growth and no citric acid production. The mutant strain was characterized by delayed onset of growth and lowered cell yield. Under citric-acid-accumulating conditions the wild-type strain exhibited decelerated growth and a maximal citric acid concentration of 12 g l^–1. Reduced, but continuing growth and citric acid production of 32 g l^–1 was observed for the mutant strain. In general, the mutant strain exhibited reduced activity for the proton-pumping respiratory complexes and enhanced activity for the alternative respiratory enzymes. In contrast to the stable activity of complex I in the wild-type strain, this complex was selectively lost in the mutant strain at the onset of citric acid production, while the alternative NADH dehydrogenases were kept at enhanced and constant activity. A possible causal connection between the loss of complex I and citric acid accumulation is discussed. Offsprint requests to: J. Wallrath     

Accumulation of 2-oxo acids in mutants ofAspergillus niger requiring lysine

Abstbact Mutants ofAspergillus niger 194A and 178 requiring lysine differ from the original prototrophic strain K 10 and from each other on the course of accumulation of organic acids. In both mutants less citric acid accumulates during the first phase of cultivation but considerably more 2-oxoglutarate and 2-oxoadipate accumulate than in the original strain. Whereas in the 194A mutant this state remains unchanged also during the second phase of cultivation, in the 178 mutant oxo acids are degraded and citric acid is synthesized intensively. The accumulation of 2-oxoglutarate and 2-oxoadipate in the fermentation medium indicates that inA. niger lysine is synthesizedvia the homocitrate pathway.     

Citric acid production by 2-deoxyglucose-resistant mutant strains of Aspergillus niger

Summary Many mutant strains showing resistance to 2-deoxy-d-glucose (DG) on minimal medium containing glycerol as a carbon source were induced from Aspergillus niger WU-2223L, a citric acid-producing strain. The mutant strains were classifiable into two types according to their growth characteristics. On the agar plates containing glucose as a sole carbon source, mutant strains of the first type showed good growth irrespective of the presence or absence of DG. When cultivated in shake cultures, some strains of the first type, such as DGR1–2, showed faster glucose consumption and growth than strain WU-2223L. The period for citric acid production shortened from 9 days for strain WU-2223L to 6–7 days for these mutant strains. The levels and yields of citric acid production of the mutant strains were almost the same as those of strain WU-2223L. The mutant strains of the second type, however, showed very slow or no growth on both the agar plates containing glucose and fructose as sole carbon sources. In shake cultures, mutant strains such as DGR2-8 showed decreased glucose consumption rates, resulting in very low production of citric acid.     

Semi-pilot scale studies on citric acid fermentation by a gamma-ray induced mutant of Aspergillus niger

Summary Semi-pilot scale production of citric acid was investigated with a gamma-ray induced mutant (HB3) of Aspergillus niger using 500, 1000 and 1500 ml medium in 51 fermentation jars. Yield of citric acid was found to be seven-fold higher compared to the parent in 1000 ml medium and the corresponding increase was two-fold in the 500 ml medium. With 1500 ml/fermentation jar the yield was low with both the parent and the mutant strain though the mutant gave higher yield compared to the parent.     

Correlation between manganese-deficiency,loss of respiratory chain complex I activity and citric acid production in Aspergillus niger

The correlation between manganese deficiency, loss of mitochondrial respiratory chain NADH: ubiquinone oxidoreductase (complex I) activity and citric acid overproduction in the Aspergillus niger strain B 60 was analysed. With increasing manganese-supplementation of the production medium the loss of complex I activity and the production of citric acid was reduced. Addition of manganese during growth stopped further loss of complex I activity and further increase of citric acid production. A possible causality between complex I deficiency and citric acid overproduction is discussed.     

Citric acid production from sugar cane molasses by 2-deoxyglucose-resistant mutant strain ofAspergillus niger

Citric acid production from sugar cane molasses byAspergillus niger NIAB 280 was studied in a batch cultivation process. A maximum of 90 g/L total sugar was utilized in citric acid production medium. From the parental strainA. niger, mutant strains showing resistance to 2-deoxyglucose in Vogal's medium containing molasses as a carbon source were induced by γ-irradiation. Among the new series of mutant strains, strain RP7 produced 120 g/L while the parental strain produced 80 g/L citric acid (1.5-fold improvement) from 150 g/L of molasses sugars. The period of citric acid production was shortened from 10 d for the wild-type strain to 6–7 d for the mutant strain. The efficiency of substrate uptake rate with respect to total volume substrate consumption rate,Q _s (g per L per h) and specific substrate consumption rate,q _s (g substrate per g cells per h) revealed that the mutant grew faster than its parent. This indicated that the selected mutant is insensitive to catabolite repression by higher concentrations of sugars for citric acid production. With respect to the product yield coefficient (Y _p/x), volume productivity (Q _p) and specific product yields (q _p), the mutant strain is significantly (p≤0.05) improved over the parental strain.     

N+ ion beam implantation of tannase-producing Aspergillus niger and optimization of its process parameters under submerged fermentation

Low-energy ion implantation as a novel mutagen has been increasingly applied in the microbial mutagenesis for its higher mutation frequency and wider mutation spectra. In this work, N⁺ ion beam implantation was used to enhance Aspergillus niger TA9701 in tannase yield. The optimization of process parameters under submerged fermentation was carried out to further improve the tannase yield of the mutant, Aspergillus niger J-T18. The results indicate that an excellent mutant J-T18 with a yield of 38.5 IU/mL, that is five times that of the original strain, was achieved by nine successive implantations under the conditions of 10 keV and 30–40 (×2.6?×?10¹³) ions/cm². This optimization further increased the yield of the mutant by 42 %, i.e. 53.6 U/mL which occurred in the mutant cultivated in the optimal fermentation culture medium composed of: rice flour 5 %; ammonium sulfate 1 %; tannic acid 2 %; calcium carbonate 0.5 %; manganese sulfate 0.1 %; and dipotassium phosphate 0.3 %; incubated at 30°C and 180 rpm for 72 h.     

Impairment of DNA formation is an early event in Aspergillus niger under manganese starvation

Summary The effect of managanese on Aspergillus niger ATCC 11414 was studied during the trophophase in a citric acid-accumulating sucrosemineral medium. The absence of manganese ions restricted growth and caused characteristic morphological aberrations. Labelling with (8¹⁴-C) adenine in-vivo revealed a reversible inhibition of DNA synthesis, but not of RNA synthesis in mycelia starved of manganese. A strong inhibition of DNA synthesis in the presence of 0.2 M Mn²⁺ was observed upon addition of 200 mM hydroxyurea (HU) which is known as specific inhibitor of the ribonucleotide reductase.The hypothesis is proposed that inhibition of DNA formation in A. niger may be traced back to an impairment of manganese dependent ribonucleotide reduction which provides the monomeric precursors for DNA replication.     

Influence of a pulsed electric field on the spores and oxygen consumption of Aspergillus niger and its citric acid production

Spores of Aspergillus niger were exposed to a pulsed electric field. After treatment by the electric field, the viability of the conidia of A. niger varied depending on the field strength, pulse width and frequency. In all cases, these parameters reduced the viability rate of the conidia from 2.0 × 10⁷ to a range from 6.2 × 10⁶ to 8.5 × 10⁶ spores/ml (3.1 to 42.6%). After pulse treatment, the conidia were used as the inoculum for citric acid fermentation in shake flasks. The highest increase in citric acid yield (about 1.4-fold) was reached at a field strength of 2.85 kV/cm, a frequency of 1 Hz and a pulse width of 1 ms. When the parameters of the electric field increased there were important changes in the respiration rate of the Aspergillus niger mycelium (48-h-old) after electric shock treatment. The highest consumption of dissolved oxygen (22.9%) in the medium by Aspergillus niger mycelium was observed at an electric field strength of 2.85 kV/cm, a 1 Hz frequency, a pulse width of 1 ms and a 1-min exposure period. It seems that an electric-field stimulation of the conidia prior to inoculation may offer an important method of improving the efficiency of citric acid. The treatment of the conidia is both simple from the technical point of view and extremely rapid.     

Influence of cultivation conditions on citrate production by Aspergillus niger in a semi-pilot-scale plant

The influence of some fermentation parameters on the semi-pilot scale (alteration of growth conditions,e.g., sugar concentration, incubation temperature and initial pH) on citrate production was demonstrated in parent and mutant strains ofAspergillus niger. Raw material from sugar industry (cane molasses) was examined as basal fermentation medium in a stirred stainless-steel 15-L fermentor. After growth on medium with 150 g/L sugar, the parent strain produced 51.2 g/L citric acid; the mutant strain achieved production maximum of 96.2 g/L. Comparing the growth, kinetic (volumetric substrate uptake rate, rate of substrate consumption and volumetric productivity rate) and production parameters it was found that the mutant strain grows more rapidly, with slightly changed morphology (intermediate, shiny round pellets with diameter 0.6–0.7 mm), and exhibits a higher citrate production and higher efficiency of sugar utilization.     

Characterization of metabolically active developmental stage of Aspergillus niger cells immobilized in polyacrylamide gel

Summary The in-situ development of Aspergillus niger entrapped in polyacrylamide gel from spores and the gel surface characteristics were studied during the repeated shake flask batch citric acid fermentation. A marked increase in the rate of citric acid production was observed with the periodic replacement of culture with fresh media at an interval of 6 days reducing the fermentation time nearly to half. The metabolically active A. niger cells for citric acid production were characterized by the appearance of thick and bulbous hyphae scattered in and on the gel surface.     

Influence of nitrilotriacetic acid and 8-hydroxyquinoline on the production of citric acid from molasses using Aspergillus niger

The effect of nitriloacetic acid (NTA) and 8-hydroxyquinoline on the production of citric acid by Aspergillus niger was investigated. The complexing agents showed an effect only during inoculation of the microorganism. Subsequent addition after inoculation did not produce any significant increase in citric acid yield. When 200 ppm of NTA was added during inoculation, an increase of 10.9 g·dm⁻³ citric acid over that produced by the control culture was observed. 8-Hydroxyquinoline, on the other hand, produced a higher concentration of citric acid which was 34 g·dm⁻³ over that of the control culture. The use of 8-hydroxyquinoline is therefore suggested for the production of citric acid from molasses using Aspergillus niger.     

Effect of manganese and other heavy metals on submerged citric acid fermentation of molasses

The addition of as little as 2 ppb of manganese to ferrocyanide-treated beet molasses during citric acid fermentation by Aspergillus niger NRC A-1-233 caused a 10% reduction in acid yield and undesirable change in the morphology of the organism from the normal pelletlike form to the filamentous from. Still smaller additions (0.4-2ppb) caused undesirable pellet clumping, while greater additions (2-100 ppb) gave further decreases in yield. The yield obtained at 100 ppb was less than 25% of that obtained at 1 ppb or less. None of the other metals tested (Al³⁺, Ca²⁺, Co²⁺, Cu²⁺, Fe²⁺, Mg²⁺, Ni²⁺, Zn²⁺) visibly changed pellet morphology, and only Al³⁺, Fe²⁺, and Zn²⁺ at relatively higher concentrations (5–25ppm) reduced acid yield. The adverse effect of manganese on growth and acid production was not affected by addition of the other metals.     

The kinetic basis of the role of Ca++ ions for higher yield of citric acid in a repeated-batch cultivation system

The present investigation deals with role of Ca⁺⁺ ions in increasing the yield of citric acid in a repeated-batch cultivation system (working volume 9-1) and its kinetic basis. Five different hyper-producing strains of Aspergillus niger were evaluated for citric acid production using clarified cane-molasses as basal substrate. Among the cultures, NG^GCB101 (developed by u.v./chemical mutation in our labs) gave maximum production of citric acid i.e., 87.98 g/1, 6 days after mycelial inoculation. The addition of CaCl₂ to the culture medium promoted the formation of small rounded fluffy pellets (1.55 mm, diameter), which were desirable for citric acid productivity. CaCl₂ at a level of 2.0 M, added during inoculation time, was optimized for commercial exploitation of molasses. During repeated-batch culturing, a yield of citric acid monohydrate of 128.68 g/1 was obtained when the sampling vs. substrate feeding was maintained at 4-1 (44.50% working volume). The incubation period was reduced from 6 to only 2 days. The values of kinetic parameters such as substrate consumption and product formation rates revealed the hyperproducibility of citric acid by the selected Aspergillus niger NG^GCB101 (LSD = 0.456a, HS). Case studies are highly economical because of higher yield of product, lower energy consumption and the use of raw substrate without any additional supplementation.     

Citric Acid Fermentation by Aspergillus niger on Low Sugar Concentrations and Cotton Waste

The possible use of cotton waste as a carbohydrate source of citric acid production by Aspergillus niger was examined. No citric acid was produced when A. niger was grown on cotton waste as a sole carbon source. In two-stage fermentations, however, mycelium obtained from surface cultures in cotton waste medium yielded more citric acid when transferred to sucrose-containing media than when directly inoculated to sucrose-containing media. It is concluded that cotton waste can be used for saving sucrose and for increasing yields of citric acid fermentation by A. niger.     

Redox potential in submerged citric acid fermentation

Summary The role and importance of the redox potential phenomena in submerged citric acid production are discussed. The redox potential of the fermentation broth is the result of oxydo-reduction processes where the metabolic activity of the microorganism Aspergillus niger plays the most significant role. The course of the redox curve for a good yielding citric acid production is presented and interpreted. The experiments of submerged citric acid production were carried out on beet molasses treated with potassium hexacyanoferrate and inoculated with A. niger spores.