Coffee husk: an inexpensive substrate for production of citric acid by Aspergillus niger in a solid-state fermentation system

Aspergillus niger CFTRI 30 produced 1.3 g citric acid/10 g dry coffee husk in 72 h solid-state fermentation when the substrate was moistened with 0.075 M NaOH solution. Production was increased by 17% by adding a mixture of iron, copper and zinc to the medium but enrichment of the moist solid medium with (NH₄)₂SO₄, sucrose or any of four enzymes did not improve production. The production of about 1.5 g citric acid/10 g dry coffee husk at a conversion of 82% (based on sugar consumed) under standardized conditions demonstrates the commercial potential of using the husk in this way.The authors are with the Department of Microbiology and Bioengineering, Central Food Technological Research Institute, Mysore-570 013, India;     

Sugarcane-pressmud as a novel substrate for production of citric acid by solid-state fermentation

Sugarcane-pressmud, a by-product of cane-sugar manufacture, was used as a substrate for production of citric acid by Aspergillus niger CFTRI 30, in a solid-state fermentation system. Of the 170 g of sugar supplied, 131 g were consumed, with a 79% yield of citric acid over 120 h. Potassium ferrocyanide improved the conversion to about 88% and lowered the fermentation time by 24 h. Enrichment with sugar and NH₄NO₃ was essential to improve productivity. About 174 g citric acid/kg dry sugarcane-pressmud were produced after 120 h in ferrocyanid-treated medium which initially contained 12.5% (w/w) effective sugar and 0.1% (w/w) NH₄NO₃. About 3% (w/w) of the original sugar present in the sugarcane-pressmud was non-utilizable. This is the first report on the potential of sugarcane-pressmud for citric acid production.V.S. Shankaranand and B.K. Lonsane are with the Fermentation Technology and Bioengineering Discipline, Central Food Technological Research Institute, Mysore-570 013, India     

Comparison of citric acid production byAspergillus niger immobilized in gels and cryogels of polyacrylamide

Aspergillus niger was immobilized in cryogels and in conventional gels of polyacrylamide. The growth of cells entrapped in two kinds of gels and the production of citric acid by the immobilized cells were investigated and compared. Cells immobilized in cryogels were more suitable for citric acid production.     

Short communication: Influence of inoculum preparation on citric acid production by Aspergillus niger

The type of sporulation medium and time of incubation had an effect on spore viability and citric acid production by mycelia grown from Aspergillus niger spores. Shu & Johnson agar (SJA) and potato dextrose agar gave higher citric acid titres than malt-extract agar. SJA also gave better germinability than the other media. Viability increased with time of incubation, but higher production of citric acid was achieved with spores incubated for less than 7 days.     

Mutation of Aspergillus niger for hyperproduction of citric acid from black strap molasses

Spore suspensions of Aspergillus niger GCB 75, which produced 31.1 g/l citric acid from 15% sugars in molasses, were subjected to u.v.-induced mutagenesis. Among three variants, GCM 45 was found to be the best citric acid producer and was further improved by chemical mutagenesis using NTG. Out of 3 deoxy-D-glucose-resistant variants, GCM 7 was selected as the best mutant which produced 86.1 ± 1.5 g/l citric acid after 168 h of fermentation of potassium ferricyanide + H₂SO₄-pretreated black strap molasses (containing 150 g sugars/l) in Vogel's medium. On the basis of comparison of kinetic parameters, namely the volumetric substrate uptake rate (Q _s), and specific substrate uptake rate (q _s), the volumetric productivity, theoretical yield and specific product formation rate, it was observed that the mutants were faster growing organisms and had the ability to overproduce citric acid.     

A proposed biochemical mechanism for citric acid accumulation by Aspergillus niger Yang No. 2 growing in solid state fermentation

Aspergillus niger Yang No. 2 and its mutant strain SL1 were grown in solid state fermentation. Samples were taken after 2, 4 and 6 days of incubation and the mycelia were analysed for their intracellular concentrations of some organic acids and adenylates and the activities of selected enzymes. Strain Yang No. 2 contained high concentrations of citrate with very little oxalate, while strain SL1 contained lower concentrations of citrate but considerably higher concentrations of oxalate. As the fermentation proceeded, strain Yang No. 2 showed a much higher ratio of ATP:AMP than did strain SL1. In addition, the enzyme ATP:citrate lyase became undetectable during citrate accumulation in strain Yang No. 2, while its activity remained high during oxalate accumulation in strain SL1. It is proposed that citrate accumulation by strain Yang No. 2 during solid state fermentation is due to blockage of its metabolism in the mitochondrion via inhibition of isocitrate dehydrogenase by the high ATP:AMP ratio, and in the cytosol by repression of ATP:citrate lyase activity.     

Continuous production of citric acid from dairy wastewater using immobilized<Emphasis Type="Italic">Aspergillus niger</Emphasis> ATCC 9142

The continuous production of citric acid from dairy wastewater was investigated using calcium-alginate immobilizedAspergillus niger ATCC 9142. The citric acid productivity and yield were strongly affected by the culture conditions. The optimal pH, temperature, and dilution rate were 3.0, 30°C, and 0.025 h⁻¹, respectively. Under optimal culture conditions, the maximum productivity, concentration, and yield of citric acid produced by the calcium-alginate immobilizedAspergillus niger were 160 mg L⁻¹ h⁻¹, 4.5 g/L, and 70.3% respectively. The culture was continuously perfored for 20 days without any apparent loss in citric acid productivity. Conversely, under the same conditions with a batch shake-flask culture, the maximum productivity, citric acid concentration, and yield were only 63.3 mg L⁻¹ h⁻¹, 4.7 g/L and 51.4%, respectively. Therefore, the results suggest that the bioreactor used in this study could be potentially used for continuous citric acid production from dairy wastewater by applying calcium-alginate immobilizedAspergillus niger.     

Influence of impeller speed on citric acid production and selected enzyme activities of the TCA cycle

Summary The effect of impeller speed on citric acid production and selected enzyme activities of the TCA cycle was studied. The highest yield of citric acid (28 g/l) was obtained in culture agitated at lower speed (300 rpm). The activity of citrate synthase decreased with the increase of speed of agitation, while the activity of aconitase and isocitrate dehydrogenase increased with the increase in agitation speed.     

Influence of dissolved oxygen concentration on intracellular pH for regulation of Aspergillus niger growth rate during citric acid fermentation in a stirred tank bioreactor

In this paper we report the regulation of Aspergillus niger growth rate during citric acid fermentation in a stirred tank bioreactor. For this, the influence of dissolved oxygen concentration in a medium on intracellular pH values and consequently on overall microbial metabolism was emphasized. Intracellular pH of mycelium grown under different concentrations of dissolved oxygen in the medium was determined. Sensitivity of proteins toward proton concentration is well recognized, therefore pH influences on the activities of key regulatory enzymes of Aspergillus niger were determined at pH values similar to those detected in the cells grown under lower dissolved oxygen concentrations. The results have shown significantly reduced specific activities of hexokinase, 6-phosphofructokinase and glucose-6-phosphate dehydrogenase in more acidic environment, while pyruvate kinase was found to be relatively insensitive towards higher proton concentration. As expected, due to the reduced specific activities of regulatory enzymes under more acidic conditions, overall metabolism should be hindered in the medium with lower dissolved oxygen concentration which was confirmed by detecting the reduced specific growth rates. From the studies, we conclude that dissolved oxygen concentration affects the intracellular pH and thus growth rate of Aspergillus niger during the fermentation process.     

Optimizing medium constituents and fermentation conditions for citric acid production from palmyra jaggery using response surface method

The quantitative effects of pH, temperature, time of fermentation, sugar concentration, nitrogen concentration and potassium ferrocyanide on citric acid production were investigated using a statistical experimental design. It was found that palmyra jaggery (sugar syrup from the palmyra palm) is a suitable substrate for increasing the yield of citric acid using Aspergillus niger MTCC 281 by submerged fermentation. Regression equations were used to model the fermentation in order to determine optimum fermentation conditions. Higher yields were obtained after optimizing media components and conditions of fermentation. Maximum citric acid production was obtained at pH 5.35, 29.76 °C, 5.7 days of fermentation with 221.66 g of substrate/l, 0.479 g of ammonium nitrate/l and 2.33 g of potassium ferrocyanide/l.     

Manganese deficiency leads to elevated amino acid pools in citric acid accumulating Aspergillus niger

Free amino acid pools have been investigated in a citric acid accumulating strain of Aspergillus niger during batch growth under manganese sufficient and deficient conditions by means of an improved chromatographic method. Studies on the mycelial content of several nitrogenous compounds under manganese sufficient and deficient conditions showed that manganese deficiency resulted in lower amino acid pool sizes during trophophase and considerable accumulation during idiophase, and in a reduction of the protein and nucleic acid contents. Addition of cycloheximide to mycelia grown with sufficient manganese also caused an elevation of free amino acid pool sizes, thus indicating that impairment of protein synthesis by manganese deficiency is responsible for the observed rise in amino acid concentration. Furthermore it was observed that the manganese deficient mycelia excreted high amounts of all amino acids suggesting that manganese deficiency may also affect membrane permeability.     

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⁺.     

Enzymes of the reductive citric acid cycle in the autotrophic eubacterium Aquifex pyrophilus and in the archaebacterium Thermoproteus neutrophilus

The autotrophic carbon fixation pathway was studied in the thermophilic hydrogen oxidizing eubacterium Aquifex pyrophilus and in the thermophilic sulfur reducing archaebacterium Thermoproteus neutrophilus. Neither organism contained ribulose-1,5-bisphosphate carboxylase activity suggesting that the Calvin cycle is not operating. Rather, all enzymes of the reductive citric acid cycle were found in A. pyrophilus. In T. neutrophilus ATP citrate lyase activity was detected which has not been achieved so far; this finding corroborates earlier work suggesting the presence of the reductive citric acid cycle in this archaebacterium. The reductive citric acid cycle for autotrophic CO₂ fixation now has been documented in the eubacterial branches of the proteobacteria, in green sulfur bacteria, and in the thermophilic Knallgas bacteria as well as in the branch of the sulfur dependent archaebacteria.     

Citric acid production and morphology of Aspergillus niger as functions of the mixing intensity in a stirred tank and a tubular loop bioreactor

The relationship between Aspergillus niger morphology and citric acid production was investigated in two reactor systems with different configurations, a tubular loop and a stirred tank bioreactor, with operating volumes of 6 and 8 dm³, respectively. Morphology was quantified by image analysis. In each system, morphology, characterized by the parameter P (mean convex perimeter of clumps), and citric acid production, were agitation-dependent and closely linked. Increased agitation caused a reduction of clump sizes and results when both reactors demonstrate that the parameter P should not exceed a threshold value in order to achieve increased productivities. The results obtained from the two reactors were in agreement, both qualitatively and quantitatively. Reducing the fundamentally different mixing conditions of the two bioreactors to the order of the dimensionless mixing parameter relative mixing time (τ_m), results showed that the loop simulated the stirred tank. Also, relationships valid for one system accurately described the results obtained from the other system, demonstrating the validity of the relationship between morphology and productivity for the particular fermentation, regardless of the reactor type. Previous attempts to evaluate the use of loop configurations as scale-up tools and their performance as bioreactors, neglected the morphology of the producer micro-organisms. This study demonstrated the close link between morphology and productivity for citric acid production by A. niger, and identified a morphology parameter that was used successfully to characterize the process performance.     

Presence and regulation of ATP:citrate lyase from the citric acid producing fungus Aspergillus niger

ATP:citrate lyase (EC 4.1.3.8) has been identified in cell-free extracts from the filamentous fungus Aspergillus niger. The enzyme was located in the cytosol. It exhibits an activity at least ten times that of acetate-CoA-kinase (EC 6.2.1.1) during growth on carbohydrates as carbon sources, and is thus considered responsible for acetyl-CoA formation under these conditions. It is formed constitutively and its biosynthesis does not appear to be controlled by changes in the nitrogen or carbon source or type. ATP:citrate-lyase appears to be very labile during conventional purification procedures; a method involving fast protein liquid anion exchange chromatography was thus developed in order to obtain enzyme preparations sufficiently free of enzymes which could interfere with kinetic investigations. This preparation displays commonly known characteristics of ATP:citrate lyase with respect to substrate affinities and cofactor requirements, with the exception that the affinity for citrate is rather low (2.5 mM). No activator was found. The enzyme is inhibited by nucleoside diphosphates, nucleoside monophosphates and palmitoyl-CoA. Regulation of ATP:citrate lyase be the energy charge of the cytosol in relation to lipid or citric acid accumulation is discussed in view of these findings. Present address: Institut für Allgemeine Biochemie, Universität Wien, Währingerstrasse 38, A-1090 Wien, Austria     

Leaching of iron from kaolins by a spent fermentation liquor: Influence of temperature,pH, agitation and citric acid concentration

Summary The efficiency of a biological bleaching process on a highly contaminated iron oxides kaolin was analyzed. The bio-bleaching method consists of two steps: first, the fermentation of a chemically-defined medium byAspergillus niger leads to a spent liquor which, in a second step, is employed as leaching agent for kaolin. In the leaching process 43% of iron oxides was removed when the kaolin was treated at 60°C for 5 h and the whiteness index was increased to 67%. Extracellular enzymes were not responsible for leaching, which is basically due to organic acids produced during the fermentation.     

Citric acid fermentation from starch and dextrose syrups by a trace metal resistant mutant of Aspergillus niger

Potato starch and both untreated and decationized dextrose syrups were used as substrates for submerged citric acid biosynthesis using a mutant of Aspergillus niger. The same yield of product (80%) was achieved with both syrups and the starch despite having different trace metals content. The obtained mutant was more sensitive than the parent to Cd²⁺, Mo²⁺, and As³⁺, with decreasing yields of citric acid at 10 mg of ions l^–1. Fe²⁺, Mn²⁺, V²⁺ below 50 mg l^–1 and Cr³⁺, Ni²⁺, Cu²⁺ up to 100 mg l^–1, did not significantly inhibit citric acid production.     

Influence of manganese on morphology and cell wall composition of Aspergillus niger during citric acid fermentation

Morphology and cell wall composition of Aspergillus niger were studied under conditions of manganese sufficient or deficient cultivation in an otherwise citric acid producing medium. Omission of Mn²⁺ (less than 10^-7 M) from the nutrient medium of Aspergillus niger results in abnormal morphological development which is characterized by increased spore swelling, and squat, bulbeous hyphae. Fractionation and analysis of manganese deficient cell walls revealed increased chitin and reduced -glucan contents as well as reduction of galactose containing polymers, as compared to cell walls from manganese sufficient grown hyphae. Addition of copper induced the same effect as manganese deficiency, both on morphology and cell wall composition. Addition of cycloheximide also produced a very similar type of morphology with increased chitin and reduced -glucan contents of the cell wall but its effect on galactose was less pronounced.Dedicated to emer. Prof. Dr. J. Kisser on the occasion of his 80th birthday     

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.     

Mathematical modelling and assessment of the pH homeostasis mechanisms in Aspergillus niger while in citric acid producing conditions

In this work we introduce an extended model of the Aspergillus niger metabolism while in citrate production conditions. The model includes many recent findings related to various transport processes. It now considers a new information about the fructose uptake system and the proton and amino acids carriers between cytoplasm and the external medium. It also accounts for recent information about both the malate-citrate antiport between mitochondria and cytoplasm and the dihydrogen citrate ion excretion symport with protons. Finally, the model also accounts for new information about the glycerol-3-phosphate shuttle and pH buffering systems. Provided with this updated representation and after having assessed its quality and dynamic behaviour, we were able to explain the observed pH homoeostasis found in A. niger while in citrate producing conditions. The model also serves to enhance our comprehension of the molecular mechanisms operating in order to keep homoeostasis of pH in A. niger and other fungi, bacteria and yeast of biotechnological relevance.