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.     

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.     

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     

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⁺.     

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.     

Overexpression of the NADP+-specific isocitrate dehydrogenase gene (icdA) in citric acid-producing Aspergillus niger WU-2223L

In the tricarboxylic acid (TCA) cycle, NADP⁺-specific isocitrate dehydrogenase (NADP⁺-ICDH) catalyzes oxidative decarboxylation of isocitric acid to form α-ketoglutaric acid with NADP⁺ as a cofactor. We constructed an NADP⁺-ICDH gene (icdA)-overexpressing strain (OPI-1) using Aspergillus niger WU-2223L as a host and examined the effects of increase in NADP⁺-ICDH activity on citric acid production. Under citric acid-producing conditions with glucose as the carbon source, the amounts of citric acid produced and glucose consumed by OPI-1 for the 12-d cultivation period decreased by 18.7 and 10.5%, respectively, compared with those by WU-2223L. These results indicate that the amount of citric acid produced by A. niger can be altered with the NADP⁺-ICDH activity. Therefore, NADP⁺-ICDH is an important regulator of citric acid production in the TCA cycle of A. niger. Thus, we propose that the icdA gene is a potentially valuable tool for modulating citric acid production by metabolic engineering.     

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     

Assimilation of galacturonate byAspergillus carbonarius

A citric acid accumulating strain ofAspergillus carbonarius could assimilate galacturonate as the sole carbon source as efficiently as glucose.A. carbonarius lost the citric acid productivity when grown on galacturonate medium, and partially restored it when galacturonate was utilized by the preformed mats. Galacturonate degradation by this mold appeared to follow two different pathways, one phosphorylated, the other nonphosphorylated.     

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.     

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.     

Regulation of hdc expression and HDC activity by enological factors in lactic acid bacteria

Aims: The aim of this work was to study the influence of enological factors on the histidine decarboxylase gene (hdc) expression and on histidine decarboxylase enzyme (HDC) activity in Lactobacillus hilgardii, Pediococcus parvulus and Oenococcus oeni. Methods and Results: Cell extracts and whole cells were used. Glucose, fructose, malic acid and citric acid diminished the hdc expression. Ethanol did not increase hdc expression or activity in cells, but increased HDC activity. Temperature and pH had effect on the activity of HDC but not on hdc expression. Tartaric acid and l -lactic acid, and sulphur dioxide (SO₂) had no effect on enzyme synthesis and activity. Bacterial species differ in the relative enzymatic activity but all the factors affected similarly to L. hilgardii, P. parvulus and O. oeni. Conclusions: The hdc gene expression was lowered by glucose, fructose, malic acid, and citric acid, whereas ethanol enhanced the HDC enzyme activity. The conditions that normally occur during malolactic fermentation and later on, could favour histamine production. SO₂ could prevent bacterial growth, but does not diminish the HDC enzyme activity. Significance and Impact of the Study: Information on hdc expression and HDC activity can contribute to the prevention of histamine formation during wine production and storage.     

Production of citric acid from methanol by a fluoroacetate-resistant mutant of Candida sp. Y-1

Summary Fermentative production of citric acid from methanol by an isolated yeast, Candida sp. Y-1, was investigated using a medium containing fluoroacetate, a potential inhibitor of aconitase. Culture conditions were optimized, and the results showed that efficient production of citric acid required several factors; (1) the optimum concentration of fluoroacetate, (2) an addition of yeast extract with corn steep liquor, (3) a low nitrogen source concentration, and (4) strictly aerobic conditions. We then isolated a fluoroacetate-resistant mutant strain MA92 with threefold higher citric acid productivity than the wild strain. This mutant strain had lower aconitase activity than the wild strain and produced 4.6 g/l citric acid from methanol after 4 days of culture. Offprint requests to: Y. Tani     

Citric acid production from sucrose by recombinant Yarrowia lipolytica using semicontinuous fermentation

The genetically modified yeast strain Yarrowia lipolytica H222‐S4(p67ICL1)T5 is able to utilize sucrose as a carbon source and to produce citric and isocitric acids in a more advantageous ratio as compared to its wild‐type equivalent. In this study, the effect of pH of the fermentation broth (pH 6.0 and 7.0) and proteose‐peptone addition on citric acid production by the recombinant yeast strain were investigated. It was found that the highest citric acid production occurred at pH 7.0 without any addition of proteose‐peptone. Furthermore, two process strategies (fed‐batch and repeated fed‐batch) were tested for their applicability for use in citric acid production from sucrose by Y. lipolytica. Repeated fed‐batch cultivation was found to be the most effective process strategy: in 3 days of cycle duration, approximately 80 g/L citric acid was produced, the yield was at least 0.57 g/g and the productivity was as much as 1.1 g/Lh. The selectivity of the bioprocess for citric acid was always higher than 90% from the very beginning of the fermentation due to the genetic modification, reaching values of up to 96.4% after 5 days of cycle duration.     

The global regulator LaeA controls production of citric acid and endoglucanases in <Emphasis Type="Italic">Aspergillus carbonarius</Emphasis>

The global regulatory protein LaeA is known for regulating the production of many kinds of secondary metabolites in Aspergillus species, as well as sexual and asexual reproduction, and morphology. In Aspergillus carbonarius, it has been shown that LaeA regulates production of ochratoxin. We have investigated the regulatory effect of LaeA on production of citric acid and cellulolytic enzymes in A. carbonarius. Two types of A. carbonarius strains, having laeA knocked out or overexpressed, were constructed and tested in fermentation. The knockout of laeA significantly decreased the production of citric acid and endoglucanases, but did not reduce the production of beta-glucosidases or xylanases. The citric acid accumulation was reduced with 74–96 % compared to the wild type. The endoglucanase activity was reduced with 51–78 %. Overexpression of LaeA seemed not to have an effect on citric acid production or on cellulose or xylanase activity.     

Transcriptomic Analysis of Temperature Responses of Aspergillus kawachii during Barley Koji Production

The koji mold Aspergillus kawachii is used for making the Japanese distilled spirit shochu. During shochu production, A. kawachii is grown in solid-state culture (koji) on steamed grains, such as rice or barley, to convert the grain starch to glucose and produce citric acid. During this process, the cultivation temperature of A. kawachii is gradually increased to 40°C and is then lowered to 30°C. This temperature modulation is important for stimulating amylase activity and the accumulation of citric acid. However, the effects of temperature on A. kawachii at the gene expression level have not been elucidated. In this study, we investigated the effect of solid-state cultivation temperature on gene expression for A. kawachii grown on barley. The results of DNA microarray and gene ontology analyses showed that the expression of genes involved in the glycerol, trehalose, and pentose phosphate metabolic pathways, which function downstream of glycolysis, was downregulated by shifting the cultivation temperature from 40 to 30°C. In addition, significantly reduced expression of genes related to heat shock responses and increased expression of genes related with amino acid transport were also observed. These results suggest that solid-state cultivation at 40°C is stressful for A. kawachii and that heat adaptation leads to reduced citric acid accumulation through activation of pathways branching from glycolysis. The gene expression profile of A. kawachii elucidated in this study is expected to contribute to the understanding of gene regulation during koji production and optimization of the industrially desirable characteristics of A. kawachii.     

Fermentation of kraft black liquor for the production of citric acid by Candida tropicalis

Summary The production of citric acid by batch fermentation with the yeast strain Candida tropicalis ATCC 20240 was chosen as a potential process for the valorization of kraft black liquor. The effect of nitrogen concentration was studied and direct bioconversion of acetate to citrate was achieved when no nitrogen was supplemented to the medium. The use of kraft black liquor's acetate as a potential substrate for citric acid production was investigated. The acid precipitated liquor was highly inhibitory when its concentration was above 25% of the fermentation broth content. The yields of citric acid at low concentrations of kraft black liquor (5% and 15%) were the same as those recorded in synthetic acetate medium. Other organic acids present in the liquor may affect the yields and rates of citric acid production over acetate. Substrate uptake rates and product formation rates were lower, however, in comparison to synthetic media. The utilization of immobilized biomass improved the process parameters on kraft black liquor and enhanced the fermentation capabilities.     

A comparative evaluation of oxygen mass transfer and broth viscosity using Cephalosporin-C production as a case strategy

The production of Cephalosporin-C (CPC) a secondary metabolite, using a mold Acremonium chrysogenum was studied in a lab scale Internal loop air lift reactor. Cephalosporin-C production process is a highly aerobic fermentation process. Volumetric gas–liquid mass transfer coefficient (k_La) and viscosity (η) were evaluated, during the growth and production phases of the microbial physiology. An attempt has been made to correlate the broth viscosity, η and volumetric oxygen transfer coefficient, k_La during the Cephalosporin-C production in an air lift reactor. The impact of biomass concentration and mycelial morphology on broth viscosity has been also evaluated. The broth exhibits a typical non-Newtonian fermentation broth. Rheology parameters like consistency index and fluidity index are also studied.     

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.     

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.