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.     

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.     

A High Performance Fermentation Process for Citric Acid Production by Aspergillus niger NGGCB-101, Using Vermiculite as an Additive in a Stirred Bioreactor

The present study describes the use of vermiculite for enhanced citric acid productivity by a mutant strain of Aspergillus niger NG^GCB-101 in a stirred bioreactor of 15.0 l capacity. The maximum amount of citric acid (96.10 g/l) was obtained with the control 144 h after mycelial inoculation. To enhance citric acid production, varying levels of vermiculite were added as an additive into the fermentation medium. The best results were observed when 0.20 g/l vermiculite was added into the medium 24 h after inoculation resulting in the production of 146.88 g citric acid monohydrate/l. The dry cell mass and residual sugar were 11.75 and 55.90 g/l, respectively. Mixed mycelial pellets (1.08–1.28 mm, dia) were observed in the fermented culture broth. When the culture grown at different vermiculite levels was monitored for Q _p , Q _s and q _p , there was a significant enhancement (P 0.05) in these variables over the control (vermiculite-free). Based on these results, it is concluded that vermiculite might affect mycelial morphology and subsequent TCA cycle performance to improve carbon source utilization by the mould, basic parameters for high performance citric acid fermentation.     

Citric acid fermentation by the mutant strain of theAspergillus niger resistant to manganese ions inhibition

Summary A new mutant strain,Aspergillus niger GS-III, showing resistance to manganese ions inhibition of citric acid fermentation on a sugarcane molasses containing medium was induced fromAspergillus niger KCU 520, a high citric acid-yielding strain. In submerged, surface or continuous cultures in the presence of manganese ions concentration upto 1.5 ppm the mutant strain yielded citric acid about 90 KgM^–3 . The citric acid yield was comparable to that obtained with the parental strain KCU 520 in the absence of manganese ions, but it was atleast 3-fold higher than that obtained by the latter in the presence of manganese ions. The mutant strain immobilized in calcium alginate beads was used in combination with surface-stabilized cultures for about 36-days in a continuous flow horizontal fermenter without any apparent loss in citric acid productivity. These results indicate that the manganese-resistant mutant is stable and may be used in the presence of sufficient manganese ions concentration (1.5 ppm) in the fermentation medium. This capability of the mutant strainA. niger GS-III has been correlated with greatly reduced levels (about one-thirds) of the NADP⁺ -isocitric dehydrogenase, one of the control points for citric acid accumulation.     

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.     

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⁺.     

Ram horn peptone as a source of citric acid production by <Emphasis Type="Italic">Aspergillus niger</Emphasis>, with a process

The present study deals with the production of citric acid from a ram horn peptone (RHP) by Aspergillus niger NRRL 330. A medium from RHP and a control medium (CM) were compared for citric acid production using A. niger in a batch culture. For this purpose, first, RHP was produced. Ram horns were hydrolyzed by treatment with acids (6 N H₂SO₄, 6 N HCl) and neutralizing solutions. The amounts of protein, nitrogen, ash, some minerals, total sugars, total lipids and amino acids of the RHP were determined. RHP was compared with peptones with a bacto-tryptone from casein and other peptones. The results from RHP were similar to those of standard peptones. The optimal concentration of RHP for the production of citric acid was found to be 4% (w/w). A medium prepared from 4% RHP was termed ram horn peptone medium (RHPM). In comparison with CM, the content of citric acid in RHPM broth (84 g/l) over 6 days was 35% higher than that in CM broth (62 g/l). These results show that citric acid can be produced efficiently by A. niger from ram horn.     

Comparative studies on citric acid production by Aspergillus niger and Candida lipolytica using molasses and glucose

Citric acid production by Aspergillus niger NCIM 548 and Candida lipolytica NCIM 3472 has been studied in shake culture using glucose and molasses as carbon sources. Methanol addition (3% v/v) at 40 h of fermentation enhanced the production of citric acid by Aspergillus niger whereas a reduction in citric acid production by Candida lipolytica was observed with addition of methanol. Maximum citric acid concentration of 12 kg/m³ was obtained with Aspergillus niger using molasses in the presence of methanol, while maximum citric acid concentration of 8.4 kg/m³ was obtained with Candida lipolytica using glucose without methanol. It appears that product formation by Aspergillus niger is either non-growth associated or partially growth associated depending on the substrate. Methanol addition changes the nature of product formation in case of Candida lipolytica.     

Phenotypes of gene disruptants in relation to a putative mitochondrial malate–citrate shuttle protein in citric acid-producing Aspergillus niger

The mitochondrial citrate transport protein (CTP) functions as a malate–citrate shuttle catalyzing the exchange of citrate plus a proton for malate between mitochondria and cytosol across the inner mitochondrial membrane in higher eukaryotic organisms. In this study, for functional analysis, we cloned the gene encoding putative CTP (ctpA) of citric acid-producing Aspergillus niger WU-2223L. The gene ctpA encodes a polypeptide consisting 296 amino acids conserved active residues required for citrate transport function. Only in early-log phase, the ctpA disruptant DCTPA-1 showed growth delay, and the amount of citric acid produced by strain DCTPA-1 was smaller than that by parental strain WU-2223L. These results indicate that the CTPA affects growth and thereby citric acid metabolism of A. niger changes, especially in early-log phase, but not citric acid-producing period. This is the first report showing that disruption of ctpA causes changes of phenotypes in relation to citric acid production in A. niger.     

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     

Citric acid production by selected mutants of Aspergillus niger from cane molasses

The present investigation deals with citric acid production by some selected mutant strains of Aspergillus niger from cane molasses in 250 ml Erlenmeyer flasks. For this purpose, a conidial suspension of A. niger GCB-75, which produced 31.1 g/l citric acid from 15% (w/v) molasses sugar, was subjected to UV-induced mutagenesis. Among the 3 variants, GCM-45 was found to be a better producer of citric acid (50.0 +/- 2a) and it was further improved by chemical mutagenesis using N-methyl, N-nitro-N-nitroso-guanidine (MNNG). Out of 3,2-deoxy-D-glucose resistant variants, GCMC-7 was selected as the best mutant, which produced 96.1 +/- 1.5 g/l citric acid 168 h after fermentation of potassium ferrocyanide and H2SO4 pre-treated blackstrap molasses in Vogel's medium. On the basis of kinetic parameters such as volumetric substrate uptake rate (Qs), and specific substrate uptake rate (qs), the volumetric productivity, theoretical yield and specific product formation rate, it was observed that the mutants were faster growing organisms and produced more citric acid. The mutant GCMC-7 has greater commercial potential than the parental strain with regard to citrate synthase activity. The addition of 2.0 x 10(-5) M MgSO4 x 5H2O into the fermentation medium reduced the Fe2+ ion concentration by counter-acting its deleterious effect on mycelial growth. The magnesium ions also induced a loose-pelleted form of growth (0.6 mm, diameter), reduced the biomass concentration (12.5 g/l) and increased the volumetric productivity of citric acid monohydrate (113.6 +/- 5 g/l).     

Selection of mutants of Aspergillus niger showing enhanced productivity of citric acid from starch in shaking culture

Mutants with enhanced citric acid production from soluble starch were induced from Aspergillus niger WU-2223L. After UV-irradiation of a conidial suspension of strain WU-2223L, mutants were selected on modified starch-methyl red agar plates on the basis of higher amylolytic activity and acid productivity. The 8 mutants selected showed enhanced citric acid production from soluble starch in shaking culture. Among them, a representative mutant strain, 2M-43, produced 48.0gg/l of citric acid from 120 g/l of soluble starch in 9 d of cultivation in shaking culture, whereas strain WU-2223L produced 35.1 g/l. Glucoamylase activities in the culture filtrates of strains 2M-43 and WU-2223L reached maximum levels of 3.62 U/ml and 2.11 U/ml, respectively, both at 3 d of cultivation, and thereafter decreased.     

Gene Identification and Functional Analysis of Methylcitrate Synthase in Citric Acid-Producing Aspergillus niger WU-2223L

Methylcitrate synthase (EC 2.3.3.5; MCS) is a key enzyme of the methylcitric acid cycle localized in the mitochondria of eukaryotic cells and related to propionic acid metabolism. In this study, cloning of the gene mcsA encoding MCS and heterologous expression of it in Escherichia coli were performed for functional analysis of the MCS of citric acid-producing Aspergillus niger WU-2223L. Only one copy of mcsA (1,495 bp) exists in the A. niger WU-2223L chromosome. It encodes a 51-kDa polypeptide consisting of 465 amino acids containing mitochondrial targeting signal peptides. Purified recombinant MCS showed not only MCS activity (27.6 U/mg) but also citrate synthase (EC 2.3.3.1; CS) activity (26.8 U/mg). For functional analysis of MCS, mcsA disruptant strain DMCS-1, derived from A. niger WU-2223L, was constructed. Although A. niger WU-2223L showed growth on propionate as sole carbon source, DMCS-1 showed no growth. These results suggest that MCS is an essential enzyme in propionic acid metabolism, and that the methylcitric acid cycle operates functionally in A. niger WU-2223L. To determine whether MCS makes a contribution to citric acid production, citric acid production tests on DMCS-1 were performed. The amount of citric acid produced from glucose consumed by DMCS-1 in citric acid production medium over 12 d of cultivation was on the same level to that by WU-2223L. Thus it was found that MCS made no contribution to citric acid production from glucose in A. niger WU-2223L, although MCS showed CS activity.     

Citric acid production from cellobiose by 2-deoxyglucose-resistant mutant strains of Aspergillus niger in semi-solid culture

Citric acid production from cellobiose by Aspergillus niger was studied by a semi-solid culture method using bagasse as a carrier. From the parental strain Yang no. 2, mutant strains showing resistance to 2-deoxy-d-glucose (DG) on minimal medium containing glucose as a carbon source were induced. The representative mutant strain M155 was selected and subjected to further mutation. The new series of mutant strains showing resistance to DG on minimal medium containing cellobiose as a carbon source was induced, and among them the best mutant strain C192 showed higher citric acid productivity than Yang no. 2 in semi-solid culture when glucose was used as a carbon source. Moreover, in semi-solid culture, the strain C192 produced 49.6 g/l of citric acid, 1.6 times as much citric acid as Yang no. 2 produced, from 100 g cellobiose/l and showed enhanced -glucosidase production. In shake culture, the extracellular -glucosidase activity of C192 was higher than that of Yang no. 2 when not only cellobiose but also glucose and glycerol, catabolite repressors, were used as a carbon source. These results indicate that mutant strains such as C192 are insensitive to catabolite repression. Correspondence to: S. Usami     

Submerged citric acid fermentation: rheological properties ofAspergillus niger broth in a stirred tank reactor

Summary A large number of submerged citric acid fermentations in a beet molasses substrate was studied. The development of Aspergillus niger from conidia to pellets was followed. Rheological characteristics of the fermentation broth including the pellets were determined. The results obtained confirm the fact that the non-Newtonian pseudoplastic behaviour of the fermentation broth was due to the presence of mycelial pellets. The most significant changes in rheological properties occurred during the period of maximal citric acid production and increase in biomass. Offprint requests to: M. Berovi     

The oxygen requirements of yeasts for the fermentation of d-xylose and d-glucose to ethanol

Summary The effect of oxygen availability on d-xylose and D-glucose metabolism by Pichia stipitis, Candida shehatae and Pachysolen tannophilus was investigated. Oxygen was not required for fermentation of d-xylose or d-glucose, but stimulated the ethanol production rate from both sugars. Under oxygen-limited conditions, the highest ethanol yield coefficient (Y_e/s) of 0.47 was obtained on d-xylose with. P. stipitis, while under similar conditions C. shehatae fermented d-xylose most rapidly with a specific productivity (q_pmax) of 0.32 h^-1. Both of these yeasts fermented d-xylose better and produced less xylitol than. P. tannophilus. Synthesis of polyols such as xylitol, arabitol, glycerol and ribitol reduced the ethanol yield in some instances and was related to the yeast strain, carbon source and oxygen availability. In general, these yeasts fermented d-glucose more rapidly than d-xylose. By contrast Saccharomyces cerevisiae fermented d-glucose at least three-fold faster under similar conditions.Nomenclature q_pmax maximum specific rate of ethanol production (g ethanol per g dry biomass per hour) - Y_e/s ethanol yield (g ethanol per g substrate utilized) - Y_p/s polyol yield (g polyol per g substrate utilized) - Y_x/s biomass yield (g dry biomass per g substrate utilized) - _max maximum specific growth rate (per hour)     

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.     

Formation of autodiploid strains in Aspergillus niger and their application to citric acid production from starch

Autodiploid strains were induced by colchicine treatment of Aspergillus niger WU-2223L, a citric acid-producing strain. In shaking culture, a representative autodiploid strain, L-d1, yielded higher citric acid than the parental strain, WU-2223L. When glucose was used as a carbon source, L-d1 and WU-2223L produced 67.2 g/l and 62.0 g/l of citric acid, respectively, from 120 g/l of glucose in 9 d-cultivation. Furthermore, the autodiploid strain L-d1 produced 49.6 g/l of citric acid, 1.4 times as much as that produced by WU-2223L from 120 g/l of soluble starch. During the whole period of cultivation with starch, the extracellular glucoamylase activity of L-d1 was on the same level as that of WU-2223L, but the extracellular acid-protease activity of L-d1 was much higher. The addition of pepstatin, an inhibitor of acid protease, to the culture broth at 2 d greatly increased the extracellular glucoamylase activity, and citric acid production by L-d1 reached a level of 59.0 g/l. During several subcultivations on both minimal and complete agar media, the autodiploid strains were genetically stable since they formed diploid conidia in their uniform colonies without producing sectors, and maintained citric acid productivity. However, when cultivated on minimal and complete agar media containing benomyl as a haploidizing reagent, the autodiploid strains readily formed sectors of haploid segregants. The properties of the haploid strains obtained by the benomyl treatment of the autodiploid strains were similar in morphology and citric acid productivity to those of the parental strain, WU-2223L. These results indicated that the enhanced production of citric acid from soluble starch by the autodiploid strains was due to autodiploid formation but not to gene mutation caused by the colchicine treatment.     

Microbial production of citric acid in fixed-bed column bioreactors

Summary Aspergillus niger NRRL 567 was cultured on the solid substrate, fruit pomace, in fixed-bed column bioreactors to produce citric acid. The rates of substrate consumption and citric acid production were strongly influenced by (1) the rate of aeration, (2) the fermentation temperature, (3) the initial moisture content of the substrate, and (4) the size of the inoculum. This culture method yielded approximately 130 g of citric acid per kg of apple pomace fermented under optimum conditions.