Biosynthesis of poly (γ-glutamic acid) from l-glutamine,citric acid and ammonium sulfate in Bacillus subtilis IFO3335

Poly(-glutamic acid) (PGA) production in Bacillus subtilis IFO3335 was studied. PGA was only slightly produced from medium (100 ml) containing 2 g citric acid and 0.5 g ammonium sulfate in B. subtilis IFO3335. When 0.01 g/100 ml l-glutamine was added to this medium, a large amount of PGA (0.45 g/100 ml), without any by-products such as polysaccharides, was produced. The changes in cell growth, and PGA, glutamic acid, citric acid and ammonium sulfate concentrations in this medium during cultivation were investigated. It was found that PGA was effectively produced for the short time of 20 h after an induction period and that glutamic acid was scarcely excreted during PGA production. PGA could be effectively produced using this medium containing l-glutamine, citric acid and ammonium sulfate. It is suggested that a small amount of l-glutamine added to the medium activated enzymes in the pathway of PGA synthesis in B. subtilis IFO3335. It can be presumed that the enzyme catalyzing the reaction from 2-oxoglutaric acid to l-glutamic acid was glutamate synthase in this bacterium.     

Biosynthesis of citric acid by Yarrowia lipolytica repeat-batch culture on ethanol

After analysis of batch culture and identification of the ways for prolongation of citric acid active synthesis by yeast, repeat-batch (RB) cultivation was suggested. Yarrowia lipolytica strain RB cultivation was studied and optimal conditions for cultivation selected. It was shown that when applying RB cultivation, better results were obtained than for batch cultivation. The activity of the culture remained stable after cultivation for more than 700 h. Comparative analysis of enzyme activities confirmed the regularity of the effect described, as the activity of practically of all the enzymes participating in ethanol oxidation and citric acid biosynthesis remained stable over time during RB cultivation. Advantages of RB cultivation for the production of citric acid by yeast are discussed. Received: 1 March 1999 / Received revision: 28 June 1999 / Accepted: 5 July 1999     

Zur Kinetik der Nebenproduktbildung bei der Überproduktion von Citronensäure durch Saccharomycopsis lipolytica

After exhaustion of the N-sources the yeast S.l. excretes citric and isocitric acid with high rates without interferring in the postlogarithmic phase the intracellular production of reserve materials like polysaccharides and especially lipids. The synthesis of citric acids and of reserve materials are therefore autonomically proceeding processes inside of the cells With increasing lipid content the ergosterol content increases The utilization of the ergosterol rich yeasts as valuable byproduct of the citric acid production is discussed.     

Biosensor online control of citric acid production from glucose by Yarrowia lipolytica using semicontinuous fermentation

Our study aimed at the development of an effective method for citric acid production from glucose by use of the yeast Yarrowia lipolytica. The new method included an automated bioprocess control using a glucose biosensor. Several fermentation methodologies including batch, fed‐batch, repeated batch and repeated fed‐batch cultivation were tested. The best results were achieved during repeated fed‐batch cultivation: Within 3 days of cycle duration, approximately 100 g/L citric acid were produced. The yields reached values between 0.51 and 0.65 g/g and the selectivity of the bioprocess for citric acid was as high as 94%. Due to the elongation of the production phase of the bioprocess with growth‐decoupled citric acid production, and by operating the fermentation in cycles, an increase in citric acid production of 32% was achieved compared with simple batch fermentation.     

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 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     

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.     

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 Aspergillus niger ATCC 9142 from a treated ethanol fermentation co-product using solid-state fermentation

Aims:  To investigate the ability of the citric acid-producing strain Aspergillus niger ATCC 9142 to utilize the ethanol fermentation co-product corn distillers dried grains with solubles for citric acid production following various treatments.
Methods and Results:  The ability of A. niger ATCC 9142 to produce citric acid and biomass on the grains was examined using an enzyme assay and a gravimetric method, respectively. Fungal citric acid production after 240 h was higher on untreated grains than on autoclaved grains or acid-hydrolysed grains. Fungal biomass production was enhanced after autoclaving and acid-hydrolysis of the grains. Phosphate supplementation to the grains slightly stimulated citric acid production while methanol addition decreased its synthesis. Using the phosphate-supplemented grains, the optimal incubation temperature, initial moisture content of the grains and the length of fermentation time for ATCC 9142 citric acid production were determined to be 25°C, 82% and 240 h, respectively.
Conclusions:  A. niger ATCC 9142 synthesized citric acid on corn distillers dried grains with solubles. The phosphate-treated grains increased citric acid production by the strain.
Significance and Impact of the Study:  The ethanol fermentation co-product corn distillers dried grains with solubles could be useful commercially as a substrate for A. niger citric acid production.     

Morphological regulation of Aspergillus niger to improve citric acid production by chsC gene silencing

Bioprocess and Biosystems Engineering - The mycelial morphology of Aspergillus niger, a major filamentous fungus used for citric acid production, is important for citric acid synthesis during...     

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⁺.     

Induction and Citric Acid Productivity of Fluoroacetate-sensitive Mutant Strains of Candida lipolytica

To establish a novel process for the economical production of citric acid from n-paraffins by yeast, attempts were made to obtain some mutant strains capable of producing citric acid in higher yield without (+)-isocitric acid.

From among the mutant strains derived from Candida lipolytica ATCC 20114, which produced citric acid and (+)-isocitric acid in the ratio of about 60:40 from n-paraffins, a citrate non-utilizing mutant strain, K-20, and a fluoroacetate-sensitive mutant strain , S-22, were selected on the basis of high citric acid and low (+)-isocitric acid productivity.

The mutant strain S-22 showed extremely poor growth in a medium containing sodium citrate as the sole carbon source and extremely high sensitivity to fluoroacetate. The production ratio of citric acid and (+)-isocitric acid by the mutant strain was changed to 97:3, and the yield of the citric acid from n-paraffins, charged to the fermentation medium, reached 145%(w/w).     

Möglichkeiten der Gewinnung von Koppelprodukten der mikrobiellen Eiweißsynthese auf der Basis von Kohlenwasserstoffen

The mass production of SCP on the base of hydrocarbons opens the possibility to get a complex of further substances. It is distinguished between by-products – belonging to essential nonprotein – structures of the growing cell and the so – called coupled products accumulating intra – or extracellularly by means of regulation. An important principle of SCP-coupled synthesis of products is the use and the maintenance of proportionality between the growth rate and cell concentration of the producing cells and the accumulation of products. As examples are discussed: fermentation by yeasts and bacteria on the base of n-alkanes, methane and methanol in connection with products as fatty acids, poly-β-hydroxybutyric acid, citric and gluconic acid.     

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.     

Growth ofMethylomonas methanolica: Factors Influencing growth yield

Summary The activities of ACH¹, NAD-ICDH, NADP-ICDH and CS were determined in cell extracts of high and low citric acid-producing strains of A. niger, cultivated on molasses medium by the surface or submerged method. A high differentiation in the activities of the enzymes studied was found to occur at various accumulation stages of citric acid and during its decomposition by moulds. During intensive citric acid synthesis, the activity of ACH and that of both dehydrogenases decreased significantly (though they did not disappear completely) compared to their initial activities during the growth period of the mycelium. CS activity, however, was maintained at almost the same level over the whole fermentation period or increased slightly, particularly in the case of low citric acid-producing strains. The relationships between the activities of these enzymes were reversed during citric acid decomposition by moulds. Moreover, it was shown that in the period of mycelium growth the maximal activities of the enzymes were much higher in submerged culture than in surface culture.     

Glutamic acid and by-product synthesis by immobilized cells of the bacterium Corynebacterium glutamicum

Summary The time course for the synthesis of glutamic acid and by-products from glucose was investigated using immobilized cell reactor of the bacterium C.glutamicum. Lactic acid, succinic acid, alanine acid and aspartic acid were formed early in the fermentation and during the active growth phase, whereas gluconic acid, -ketoglutaric acid and proline were produced late and during the active phase of glutamic acid synthesis. Oxygen transfer rate in fermentation broth had a pronounced effect on the nature and quantities of fermentation products. In continuous fermentation and at OTR of 102.5 mMO₂/l.h., formation of by-products greatly decreased and up to 58.5 g/l of glutamic acid were produced with a conversion efficiency of 74.6% of the theoretical value and volumetric productivity of 6.2 g/l.h.     

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.     

Improved production of citric acid by Yarrowia lipolytica using oleic acid as the oxygen‐vector and co‐substrate

Yarrowia lipolytica is able to secrete large amounts of citric acid (CA), which is greatly affected by the dissolved oxygen concentration (DOC) in the fermentation medium. In this study, oleic acid was selected as oxygen‐vector to improve DOC during CA fermentation. When 2% (v/v) of oleic acid was added to the culture broth, higher DOC (>42.1%) was determined throughout the CA synthesis phase. The yield of CA reached a maximum of 32.1 g/L (25.4% higher than the control) and the biomass was 8.8 g/L. The substrate uptake rate, products formation rate and key enzyme activities were also determined, and the results indicated that CA synthesis was strengthened with oleic acid addition. Furthermore, it was detected that oleic acid could be assimilated by the cells, which means that oleic acid could be served both as oxygen‐vector and co‐substrate for CA synthesis by Y. lipolytica. In a bioreactor with working volume of 3 L, the highest concentration of CA reached to 36. 4 g/L in the presence of 2% (v/v) oleic acid after 192 h of fermentation. These results confirmed that oleic acid could be applied in the large‐scale production of CA by Y. lipolytica.     

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.