Metabolic precursors enhance the production of poly‐ε‐lysine by Streptomyces noursei NRRL 5126

Poly‐ε‐lysine produced by streptomyces species is a promising biopolymer owing to its antimicrobial activity and safety for humans. A number of nutritional factors influencing poly‐ε‐lysine production by Streptomyces noursei NRRL 5126 were studied. Various metabolic precursors such as amino acids, tricarboxylic acid cycle intermediates and cofactors were investigated for improved production of poly‐ε‐lysine. Results indicated L ‐aspartate (2 mM) and citric acid (5 mM) to substantially increase the poly‐ε‐lysine production from 97.08 to 409.94 mg/L. Addition of citric acid after 24 h and L ‐aspartate after 36 h of fermentation medium further enhanced poly‐ε‐lysine production to 497.67 mg/L after a total fermentation time of 108 h. However, the use of cofactors of enzymes involved in the biosynthesis of poly‐ε‐lysine inhibited its production which is believed to be due to diversion of the flux to other metabolites.     

Control of biogenic H(2)S production with nitrite and molybdate

The effects of the metabolic inhibitors, sodium nitrite and ammonium molybdate, on production of H₂S by a pure culture of the sulfate-reducing bacterium (SRB) Desulfovibrio sp. strain Lac6 and a consortium of SRB, enriched from produced water of a Canadian oil field, were investigated. Addition of 0.1 mM nitrite or 0.024 mM molybdate at the start of growth prevented the production of H₂S by strain Lac6. With exponentially growing cultures, higher levels of inhibitors, 0.25 mM nitrite or 0.095 mM molybdate, were required to suppress the production of H₂S. Simultaneous addition of nitrite and molybdate had a synergistic effect: at time 0, 0.05 mM nitrite and 0.01 mM molybdate, whereas during the exponential phase, 0.1 mM nitrite and 0.047 mM molybdate were sufficient to stop H₂S production. With an exponentially growing consortium of SRB, enriched from produced water of the Coleville oil field, much higher levels of inhibitors, 4 mM nitrite or 0.47 mM molybdate, were needed to stop the production of H₂S. The addition of these inhibitors had no effect on the composition of the microbial community, as shown by reverse sample genome probing. The results indicate that the efficiency of inhibitors in containment of SRB depends on the composition and metabolic state of the microbial community. Journal of Industrial Microbiology & Biotechnology (2001) 26, 350–355. Received 02 August 2000/ Accepted in revised form 17 April 2001     

Use of Energy Crop (Ricinus communis L.) for Phytoextraction of Heavy Metals Assisted with Citric Acid

Ricinus communis L. is a bioenergetic crop with high-biomass production and tolerance to cadmium (Cd) and lead (Pb), thus, the plant is a candidate crop for phytoremediation. Pot experiments were performed to study the effects of citric acid in enhancing phytoextraction of Cd/Pb by Ricinus communis L. Citric acid increased Cd and Pb contents in plant shoots in all treatments by about 78% and 18–45%, respectively, at the dosage of 10 mM kg^?1 soil without affecting aboveground biomass production. Addition of citric acid reduced CEC, weakened soil adsorption of heavy metals and activated Cd and Pb in soil solutions. The acid-exchangeable fraction (BCR-1) of Pb remained lower than 7% and significantly increased with citric acid amendment. Respective increases in soil evaluation index induces by 14% and 19% under the Cd1Pb50 and Cd1Pb250 treatments upon addition of citric acid resulted in soil quality improvement. Ricinus communis L. has great potential in citric acid-assisted phytoextraction for Cd and Pb remediation.     

Effect of nucleosides on interferon production and development of antiviral state induced by poly I.poly C.

Effect of nucleosides both on induction of antiviral state in chick embryo cells (CEC) or rabbit kidney cells (RK13) and on interferon production in RK13 or mouse fibroblast cells (L cells) by polyriboinosinic-polyribocytidylic acid (poly I.poly C) was studied. Addition of inosine or a fifty-fifty mixture of inosine and uridine at a final concentration of 0.1 mM to 10 mM to a growth medium enhanced development of antiviral state in CEC. The nucleoside effect was also observed in RK13 at 0.1 mM but not at a concentration higher than 1 mM. Interferon production in RK13 by superinduction (sequential treatment with metabolic inhibitors after exposure to poly I.poly C) was enhanced 1.5- to 4.0-fold by addition of the nucleoside mixture to the growth medium. When RK13 was pretreated with 10 units per ml of interferon and then superinduced by inhibitors, the enhancing effect of nucleosides on interferon production was not observed. Interferon production in L cells was potentiated a little by addition of 1 mM of the nucleoside mixture to the growth medium. The effect of nucleoside was not observed when the nucleosides were added after exposure to poly I.poly C. The nucleoside effect may be applicable for production of high titered interferon.     

Low- and high-affinity transport systems for citric acid in the yeast Candida utilis.

Citric acid-grown cells of the yeast Candida utilis induced two transport systems for citric acid, presumably a proton symport and a facilitated diffusion system for the charged and the undissociated forms of the acid, respectively. Both systems could be observed simultaneously when the transport was measured at 25 degrees C with labelled citric acid at pH 3.5 with the following kinetic parameters: for the low-affinity system, Vmax, 1.14 nmol of undissociated citric acid s-1 mg (dry weight) of cells-1, and Km, 0.59 mM undissociated acid; for the high-affinity system, Vmax, 0.38 nmol of citrate s-1 mg (dry weight) of cells-1, and Km, 0.056 mM citrate. At high pH values (above 5.0), the low-affinity system was absent or not measurable. The two transport systems exhibited different substrate specificities. Isocitric acid was a competitive inhibitor of citric acid for the high-affinity system, suggesting that these tricarboxylic acids used the same transport system, while aconitic, tricarballylic, trimesic, and hemimellitic acids were not competitive inhibitors. With respect to the low-affinity system, isocitric acid, L-lactic acid, and L-malic acid were competitive inhibitors, suggesting that all of these mono-, di-, and tricarboxylic acids used the same low-affinity transport system. The two transport systems were repressed by glucose, and as a consequence diauxic growth was observed. Both systems were inducible, and not only citric acid but also lactic acid and malic acid may induce those transport systems. The induction of both systems was not dependent on the relative concentration of the anionic form(s) and of undissociated citric acid in the culture medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pretreatment of indian cane molasses for increased production of citric acid

Surface culture citric acid fermentation was carried out by Aspergillus niger T55, a strain isolated from its natural source, using cane molasses, either untreated or treated by various methods. Citric acid biosynthesis is seriously impaired by both organic and inorganic inhibitors. A combined treatment of molasses with tricalcium phosphate, hydrochloric acid, and Sephadex fractionation minimizes the level of inorganic and organic inhibitors in molasses and increases the production of citric acid (65% weight yield based on total reducing sugar). The optimum level of individual metal ions for citric acid production depends on the concentration of other metals in the medium.     

Low- and high-affinity transport systems for citric acid in the yeast Candida utilis.

Citric acid-grown cells of the yeast Candida utilis induced two transport systems for citric acid, presumably a proton symport and a facilitated diffusion system for the charged and the undissociated forms of the acid, respectively. Both systems could be observed simultaneously when the transport was measured at 25 degrees C with labelled citric acid at pH 3.5 with the following kinetic parameters: for the low-affinity system, Vmax, 1.14 nmol of undissociated citric acid s-1 mg (dry weight) of cells-1, and Km, 0.59 mM undissociated acid; for the high-affinity system, Vmax, 0.38 nmol of citrate s-1 mg (dry weight) of cells-1, and Km, 0.056 mM citrate. At high pH values (above 5.0), the low-affinity system was absent or not measurable. The two transport systems exhibited different substrate specificities. Isocitric acid was a competitive inhibitor of citric acid for the high-affinity system, suggesting that these tricarboxylic acids used the same transport system, while aconitic, tricarballylic, trimesic, and hemimellitic acids were not competitive inhibitors. With respect to the low-affinity system, isocitric acid, L-lactic acid, and L-malic acid were competitive inhibitors, suggesting that all of these mono-, di-, and tricarboxylic acids used the same low-affinity transport system. The two transport systems were repressed by glucose, and as a consequence diauxic growth was observed. Both systems were inducible, and not only citric acid but also lactic acid and malic acid may induce those transport systems. The induction of both systems was not dependent on the relative concentration of the anionic form(s) and of undissociated citric acid in the culture medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Strain variability and the effects of organic compounds on the growth of the chemolithotrophic bacterium Thiobacillus ferrooxidans

The effects of naturally-occurring organic compounds on ferrous iron oxidation by the bacterium Thiobacillus ferrooxidans were examined with a view to using these compounds to treat or prevent acid mine/rock drainage. The compounds glucose, cellobiose, galacturonic acid, and citric acid were added to the growth medium of five different strains of the bacterium and growth studies were done to determine whether or not strain differences existed with respect to organic compound sensitivity. The effects of these compounds were compared to the effects of sodium dodecyl sulfate (SDS) an anionic detergent. Each of the compounds tested had an inhibitory effect on the strains of the bacterium and sensitivity to these compounds was strain dependent. All strains appeared to be equally susceptible to SDS. Inhibitory concentrations ranged from 70 mM to >280 mM for glucose, 7.5 mM to 150 mM for cellobiose, 20 mM to 230 mM for galacturonic acid, and 50 mM to 130 mM for citric acid. SDS effectively inhibited iron oxidation for all strains at a concentration of 0.3 mM, the lowest concentration tested. Some naturally-occurring organic compounds, therefore, might be candidates for the growth control of T. ferrooxidans.     

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.     

Effect of sugars, hydrogen ion concentration and ammonium nitrate on the formation of citric acid by Aspergillus niger.

Aspergillus niger Mulder strain when grown on a synthetic medium containing urea as the sole source of nitrogen at pH 5.2, formed a mixture of citric and gluconic acids. On growing the organism at pH 2.0 the gluconic acid content was reduced but citric acid yield remained low. Addition of NH4NO3 to the medium lowered the gluconic acid yields to undetectable levels with a simultaneous increase in the citric acid content. Of the sugars used for the production of citric acid, sucrose in an unautoclaved medium was found to be the best carbon source. Sucrose medium if autoclaved at pH 2.0, or a mixture of glucose and fructose instead of sucrose gave lower yields of citric acid. Under optimum conditions only citric acid was produced and the yield was 66-68 per litre after a growth period of about 10 days.     

Enhanced production of (+)-terrein in fed-batch cultivation of Aspergillus terreus strain PF26 with sodium citrate

(+)-Terrein is a fungal metabolite with multiple biological activities, especially with great value in medicine. However, the mass production of single configuration terrein is still a big challenge. In this study, the effects of acetic acid, sodium acetate, citric acid and sodium citrate on the (+)-terrein production by Aspergillus terreus strain PF26 derived from marine sponge Phakellia fusca were investigated. Sodium citrate was selected for fed-batch cultivation because it showed the best effect on (+)-terrein production among the four regulators tested. As a result, 5.38 g/L (+)-terrein production was achieved by feeding 10 mM sodium citrate on the 3rd day in shake flask, which was 33.8 % higher than the control and represented the highest yield of (+)-terrein. In a 7.5-L stirred bioreactor, 2.58 g/L of (+)-terrein production was achieved by the feeding of 10 mM sodium citrate on the 8th day. The results from this study lay a basis for the high-yield production of (+)-terrein by fermentation.     

Contribution of cyanide-insensitive respiratory pathway, catalyzed by the alternative oxidase, to citric acid production in Aspergillus niger

In Aspergillus niger, a cyanide (CN)- and antimycin A-insensitive and salicylhydroxamic acid (SHAM)-sensitive respiratory pathway exists besides the cytochrome pathway and is catalyzed by the alternative oxidase (AOX). In this study, A. niger WU-2223L, a citric acid-producing strain, was cultivated in a medium containing 120 g/l of glucose, which is the concentration usually needed for citric acid production, and the effects of 2% (v/v) methanol, an inducer of citric acid, 2 microM antimycin A, and 1 mM SHAM on AOX activities and citric acid production were investigated. The AOX activity, measured as duroquinol oxidase, was localized in the purified mitochondria regardless of the presence of any additives. When WU-2223L was cultivated with antimycin A or methanol, both citric acid production and citric acid productivity, shown as the ratio of production per mycelial dry weight, increased with the increase of both the activity of AOX and the rate of CN-insensitive and SHAM-sensitive respiration. On the other hand, when WU-2223L was cultivated with SHAM, an inhibitor of AOX, the CN-insensitive and SHAM-sensitive respiration was not detected and the citric acid production and the productivity drastically decreased, although mycelial growth was not affected. These results clearly indicated that the CN-insensitive and SHAM-sensitive respiration catalyzed by AOX, localized in the mitochondria, contributed to citric acid production by A. niger.     

Ammonium ion & citric acid supplementation in batch cultures ofAspergillus niger B 60

Summary The effect of a single pulse of ammonium sulphate or of citrate upon the progress and final outcome of a batch citric acid fermentation was studied. It was found that the optimum addition time for the supplemental N was in the range of 40 to 75 h. Final citric acid concentration achieved was significantly increased when the concentration of N source added was between 0.25 and 0.5 kg m^–3. The mechanism of the observed stimulation seemed to be an indirect one. Addition of exogenous citric acid to the broth, led to an increase in citrate production by the culture. The optimum time for citric acid addition was around 90 h.Nomenclature Y_p/s Yield of citric acid produced (kg) on sucrose consumed (kg) - P/t Overall citric acid productivity (kg m^–3 h^–1)     

A novel recycling process using the treated citric acid wastewater as ingredients water for citric acid production

In this study, an integrated process coupling citric acid and methane fermentations was proposed to solve severe wastewater pollution problem in cassava-based citric acid production. The accumulation patterns of the potential and major inhibitors in this process, including organic compounds, volatile fatty acids (VFAs), total ions and pigments were investigated. Both simulation and experimental results indicated that these inhibitors could reach their equilibrium levels after 3–7 fermentation runs when reutilizing the treated citric acid wastewater. As a result, the proposed citric acid fermentation process by recycling the wastewater treated in methane fermentation could be stably operated for more than 15 runs, which could save a large amount of fresh water and relieve the severe wastewater pollution in citric acid production potentially.     

Effect of propionic acid on citric acid fermentation in an integrated citric acid–methane fermentation process

In this study, an integrated citric acid-methane fermentation process was established to solve the problem of wastewater treatment in citric acid production. Citric acid wastewater was treated through anaerobic digestion and then the anaerobic digestion effluent (ADE) was further treated and recycled for the next batch citric acid fermentation. This process could eliminate wastewater discharge and reduce water resource consumption. Propionic acid was found in the ADE and its concentration continually increased in recycling. Effect of propionic acid on citric acid fermentation was investigated, and results indicated that influence of propionic acid on citric acid fermentation was contributed to the undissociated form. Citric acid fermentation was inhibited when the concentration of propionic acid was above 2, 4, and 6 mM in initial pH 4.0, 4.5 and, 5.0, respectively. However, low concentration of propionic acid could promote isomaltase activity which converted more isomaltose to available sugar, thereby increasing citric acid production. High concentration of propionic acid could influence the vitality of cell and prolong the lag phase, causing large amount of glucose still remaining in medium at the end of fermentation and decreasing citric acid production.     

The relationship of some metal ions with citric acid production by Aspergillus niger using tamarind seed powder as raw material

Tamarind seed powder (TSP) was used as raw material for microbial production of citric acid. The maximum citric acid production was observed when the TSP-basal medium (TSP with inorganic nitrogen and phosphorus source) was supplemented with Mg²⁺, Fe²⁺, Cu²⁺ and Zn²⁺. The use of methanol and ferrocyanide as a modulator for citric acid production was studied. On methanol supplementation to the medium, ammonium nitrate was found to be better than ammonium carbonate as a source of inorganic nitrogen. Addition of ferrocyanide after sterilization to the medium containing methanol gave the maximum citrate production of 363 μg/ml.     

Osteoclast precursors display dynamic metabolic shifts toward accelerated glucose metabolism at an early stage of RANKL-stimulated osteoclast differentiation.

Mature osteoclasts have an increased citric acid cycle and mitochondrial respiration to generate high ATP production and ultimately lead to bone resorption. However, changes in metabolic pathways during osteoclast differentiation have not been fully illustrated. We report that glycolysis and oxidative phosphorylation characterized by glucose and oxygen consumption as well as lactate production were increased during receptor activator of nuclear factor-kappaB ligand (RANKL)-induced osteoclastogenesis from RAW264.7 and bone marrow-derived macrophage cells. Cell proliferation and differentiation varied according to glucose concentrations (0 to 100 mM). Maximal cell growth occurred at 20 mM glucose concentration and differentiation occurred at 5 mM concentration. Despite the similar growth rates exhibited when cultured cells were exposed to either 5 mM or 40 mM glucose, their differentiation was markedly decreased in high glucose concentrations. This finding suggests the possibility that osteoclastogenesis could be regulated by changes in metabolic substrate concentrations. To further address the effect of metabolic shift on osteoclastogenesis, we exposed cultured cells to pyruvate, which is capable of promoting mitochondrial respiration. Treatment of pyruvate synergistically increased osteoclastogenesis through the activation of RANKL-stimulated signals (ERK and JNK). We also found that osteoclastogenesis was retarded by blocking ATP production with either the inhibitors of mitochondrial complexes, such as rotenone and antimycin A, or the inhibitor of ATP synthase, oligomycin. Taken together, these results indicate that glucose metabolism during osteoclast differentiation is accelerated and that a metabolic shift towards mitochondrial respiration allows high ATP production and induces enhanced osteoclast differentiation.     

Sensory properties of citric acid: psychophysical evidence for sensitization, self-desensitization, cross-desensitization and cross-stimulus-induced recovery following capsaicin

In a first experiment, human subjects used a bipolar scale to rate the irritant sensation elicited by 10 sequentially repeated applications of either 3 ppm capsaicin or 250 mM citric acid on one side of the dorsal surface of the tongue, at 1 min intervals (30 s inter-stimulus interval). Citric acid-evoked irritation significantly increased across trials, consistent with sensitization. With capsaicin there was a large degree of inter- and intra-individual variation in successive ratings with no overall sensitization. Following the sequential stimulation series and a 10 min rest period, self- and cross-desensitization effects were tested in a two-alternative forced choice (2-AFC) paradigm by placing either citric acid or capsaicin on both sides of the tongue and asking subjects to indicate which side of the tongue yielded a stronger irritant sensation. Subjects also gave separate intensity ratings for irritation on each side of the tongue. Capsaicin self-desensitization was confirmed, while cross-desensitization to citric acid was not observed. In addition, citric acid self-desensitization and cross-desensitization to capsaicin were observed. In a second experiment a stronger capsaicin solution (33 ppm) was applied to one side of the tongue using cotton swabs. After the burning sensation elicited by capsaicin had disappeared, citric acid was applied bilaterally and cross-desensitization was observed using the same 2-AFC and rating procedures. This was followed by repeated re-application of citric acid at 1 min intervals to the capsaicin-treated side. The irritant sensation elicited by citric acid increased significantly, indicating a 'cross-stimulus-induced recovery' from capsaicin desensitization. In a final experiment we investigated the effect of the sodium channel blocker amiloride on the perceived irritation elicited by citric acid or capsaicin. Following application of amiloride to one side of the tongue with cotton swabs, either citric acid or capsaicin was applied bilaterally and subjects asked to perform a 2-AFC and intensity ratings. Amiloride significantly, albeit weakly, reduced the irritation elicited by citric acid while it weakly but significantly enhanced capsaicin-evoked irritation. These findings are discussed in terms of involvement of vanilloid and acid-sensitive ion channels in acid-evoked irritation and pain.     

Stimulatory effect of alcohols (methanol and ethanol) on citric acid productivity by a 2-deoxy D-glucose resistant culture of aspergillus niger GCB-47

The present study describes citric acid fermentation by Aspergillus niger GCB-47 in a 15-1 stainless steel stirred fermentor. Among the alcohols tested as stimulating agents, 1.0% (v/v) methanol was found to give maximum amount of anhydrous citric acid (90.02 +/- 2.2 g/l), 24 h after inoculation. This yield of citric acid was 1.96 fold higher than the control. Methanol has a direct effect on mycelial morphology and it promotes pellet formation. It also increases the cell membrane permeability to provoke more citric acid excretion from the mycelial cells. The sugar consumed and % citric acid was 108 +/- 3.8 g/l and 80.39 +/- 4.5%, respectively. The desirable mycelial morphology was in the form of small round pellets having dry cell mass 14.5 +/- 0.8 g/l. Addition of ethanol, however, did not found to enhance citric acid production, significantly. The maximum value of Yp/x (i.e., 5.825 +/- 0.25 g/g) was observed when methanol was used as a stimulating agent. The best results of anhydrous citric acid were observed, 6 days after inoculation when the initial pH of fermentation medium was kept at 6.0.     

Inhibition of fungal NADP+-isocitrate dehydrogenase by citric acid

The variations observed during earlier studies in the activity of NADP+-isocitrate dehydrogenase (EC. 1.1.1.42) in a strain of Aspergillus niger were found to be related to the extent of washing of mycelium. As a result the mycelium washed four times with phosphate buffer (0.05 M, pH 7.5), the enzyme activity present in 4 and 8 days old fungal mycelia increased five- and two-fold, respectively. In vivo studies showed a complete loss of enzyme activity in mycelia resuspended in HCl-KCl buffer (0.02 M, pH 2.2) containing citric acid (13 mM or more). The in vitro studies revealed 50% loss of enzyme activity in presence of 3.6 to 5.2 mM citric acid. However, in case of Aspergillus niger ATCC 1015, which produced less citric acid than the above strain, a much higher citric acid concentration (13 to 26 mM) was required to cause 50% loss of enzyme activity. These findings suggest a correlation between citric acid inhibition of NADP+-isocitrate dehydrogenase and the ability of A. niger to accumulate citric acid in the medium.