Optimization of date syrup for enhancement of the production of citric acid using immobilized cells of Aspergillus niger

Date syrup as an economical source of carbohydrates and immobilized Aspergillus niger J4, which was entrapped in calcium alginate pellets, were employed for enhancing the production of citric acid. Maximum production was achieved by pre-treating date syrup with 1.5% tricalcium phosphate to remove heavy metals. The production of citric acid using a pretreated medium was 38.87% higher than an untreated one that consumed sugar. The appropriate presence of nitrogen, phosphate and magnesium appeared to be important in order for citric acid to accumulate. The production of citric acid and the consumed sugar was higher when using 0.1% ammonium nitrate as the best source of nitrogen. The production of citric acid increased significantly when 0.1 g/l of KH₂PO₄ was added to the medium of date syrup. The addition of magnesium sulfate at the rate of 0.20 g/l had a stimulating effect on the production of citric acid. Maximum production of citric acid was obtained when calcium chloride was absent. One of the most important benefits of immobilized cells is their ability and stability to produce citric acid under a repeated batch culture. Over four repeated batches, the production of citric acid production was maintained for 24 days when each cycle continued for 144 h. The results obtained in the repeated batch cultivation using date syrup confirmed that date syrup could be used as a medium for the industrial production of citric acid.     

Effects of minerals on the production of valine byAerobacter aerogenes

A study has been made on the mineral requirements of a strain ofAerobacter aerogenes for the production of valine. It was observed that K₂HPO₄ and MgSO₄]. 7 H₂O were required at concentrations of 0.1% and 0.05% respectively while the optimum level of each of the trace elements Fe and Mo was 1 μg/ml. NaCl, KCl and trace elements like Mn, Ni, Co, Cu and Zn had an adverse effect on the production of valine. The requirements for metals except Mg for growth of the organism and valine production are different.     

The Effect of CO2-Concentration on the Occurrence of a Number of Acids from the Citric Acid Cycle in Tomato Leaves

Examination of the effect of CO₂-concentration and time of day on the content of malic acid, citric acid, aconitic acid, isocitric acid, succinic acid and fumaric acid in tomato leaves, revealed that the total content of these acids will rise with the CO₂-concentration up to 0.10 vol% CO₂. In the morning up to 0.22 vol% CO₂ was needed for optimal effect. Samples of leaves picked at 1 a.m. showed the lowest content of these acids. At 9 a.m. the content had increased, and at 4 p.m. the increase was considerable. The content of malic and citric acid constituted 36 and 34% of the total acid content. In the afternoon and the night the aconitic acid represented 14% and in the morning 20% of the total acid content. Isocitric acid, fumaric acid and succinic acid occurred only in relatively small concentrations.     

Citric acid production from glucose. I. Growth and excretion kinetics in a stirred fermentor

The growth and citric acid production kinetics of Saccharomycopsis lipolytica on glucose are investigated in an aerated stirred fermentor. Cellular growth first proceeds exponentially until exhaustion of ammonia in the fermentation medium. Cells then continue to grow at a reduced rate with a concomitant decrease in intracellular nitrogen content. Citric and isocitric acid production starts at the end of the growth phase. During about 80 hr excretion proceeds at a constant rate of 0.7 g/liter/hr for citric acid and 0.1 g/liter/hr for isocitric acid. The final citric and isocitric acid concentrations are 95 and 10g/liter, respectively. During acid excretion cellular respiration accounts for 60 and 35% of consumed oxygen and glucose. Both acid and CO₂ production rates follow a Michaelis–Menten-type dependence on oxygen concentration with Michaelis–Menten constants of 0.9 and 0.15 mg/liter for acid and CO₂ productions, respectively.     

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.     

Continuous citric acid fermentation by <Emphasis Type="Italic">Candida oleophila</Emphasis> under nitrogen limitation at constant C/N ratio

Summary The central aspect of this work was to investigate the influence of nitrogen feed rate at constant C/N ratio on continuous citric acid fermentation by Candida oleophila ATCC 20177. Medium ammonia nitrogen and glucose concentrations influenced growth and production. Space-time yield (STY) meaning volumetric productivity, biomass specific productivity (BSP), product concentration, product selectivity and citrate/isocitrate ratio increased with increasing residence time (RT). BSP increased in an exponential mode lowering nitrogen feed rates. Highest BSP for citric acid of 0.13 g/(g h) was achieved at lowest NH₄Cl concentration of 1.5 g/l and highest STY (1.2 g/l h) with 3 g NH₄Cl/l at a RT of 25 h. Citric acid 74.2 g/l were produced at 58 h RT and 6 g NH₄Cl/l. Glucose uptake rate seems to be strictly controlled by growth rate of the yeast cells. Optimum nitrogen concentration and adapted C/N ratio are essential for successful continuous citric acid fermentation. The biomass-specific nitrogen feed rate is the most important factor influencing continuous citric acid production by yeasts. Numerous chemostat experiments showed the feasibility of continuous citrate production by yeasts.     

Cadmium — citric acid — xylem cell wall interactions in tomato plants

Abstract. Mutual interactions between cadmium ions, citric acid and xylem cell walls were examined. Cadmium and citric acid were measured as ¹¹⁵Cd and [1,5-¹⁴C] citric acid, respectively. Xylem cell walls were obtained by bacterial degradation of tomato stem sections (Lycopersicon esculentum Mill, cv. Tiny Tim), and applied as ion-exchange columns. The xylem column material carried 2·4 dm³ H₂O kg^?1 dry weight, and was temporarily capable of buffering perfusates at pH 5·7. Sorbed cadmium and citric acid were determined from H₂O and HCl rinses after perfusion periods. In all experiments, total cadmium and/or citric acid recoveries were better than 98%, indicating both the effectiveness of the rinses applied and the possibility of full regeneration of the xylem column. The results indicate that the presence of 2·45 mol m^?3 citric acid causes an approximately 50% reduction of adsorbed cadmium levels, irrespective of the applied total cadmium concentrations (0·04–0·4 mol m^?3 Cd(NO₃)₂.4H₂O). This reduction is probably related to a corresponding reduction to approximately 2% of the control applied free Cd²⁺ concentration, the latter also independent of the total cadmium concentrations. Furthermore, without inducing positively charged citrate complexes in the applied solution, the presence of cadmium resulted in increased levels of citric acid absorbed in the xylem column. The Donnan Free Space accumulation of citric acid in the presence of Cd(NO₃)₂.4H₂O, observed in the experiments described, could be expressed by its distribution coefficient, as approximately 15 times the control accumulation. These data indicate that the xylem column may operate as a ligand exchanger, suggesting the importance of metal ions for the longitudinal and lateral movement of organic complexing compounds in the xylem.     

Citric Acid Production from Hydrolysate of Pretreated Straw Cellulose by Yarrowia lipolytica SWJ-1b Using Batch and Fed-Batch Cultivation

In this study, crude cellulase produced by Trichoderma reesei Rut-30 was used to hydrolyze pretreated straw. After the compositions of the hydrolysate of pretreated straw were optimized, the study showed that natural components of pretreated straw without addition of any other components such as (NH₄)₂SO₄, KH₂PO₄, or Mg²⁺ were suitable for citric acid production by Yarrowia lipolytica SWJ-1b, and the optimal ventilatory capacity was 10.0 L/min/L medium. Batch and fed-batch production of citric acid from the hydrolysate of pretreated straw by Yarrowia lipolytica SWJ-1b has been investigated. In the batch cultivation, 25.4 g/L and 26.7 g/L citric acid were yields from glucose and hydrolysate of straw cellulose, respectively, while the cultivation time was 120 hr. In the three-cycle fed-batch cultivation, citric acid (CA) production was increased to 42.4 g/L and the cultivation time was extended to 240 hr. However, iso-citric acid (ICA) yield in fed-batch cultivation (4.0 g/L) was similar to that during the batch cultivation (3.9 g/L), and only 1.6 g/L of reducing sugar was left in the medium at the end of fed-batch cultivation, suggesting that most of the added carbon was used in the cultivation.     

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.     

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;     

Histidine decarboxylase production by Lactobacillus hilgardii: Effect of organic acids

Histidine decarboxylase production from Lactobacillus hilgardii 5w, isolated from wine, was inhibited by the presence of l-malic acid in the basal culture medium. The inhibition was related to l-malic acid concentration. The maximal production of the enzyme at 12 h of culture incubated at 30°C was inhibited 71% by 2 g/L l-malic acid and 47% by 0.5 g/L. In these conditions l-malic acid consumption was 16% and 20% respectively. The addition of 300 mg/L citric acid to the basal medium stimulated the enzyme production from 9 to 45 nmoles/min/mg dry weight, and the increase was correlated with citric acid concentration. When different concentrations of l-malic acid were added to the basal medium plus 200 mg/L citric acid, reversion of stimulation was observed, achieving the maximum at a concentration of 2 g/L. In this case, citric acid comsumption was not modified, whereas L-malic acid utilization was higher.     

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     

Influence of pectin and glucose on growth and polygalacturonase production by Aspergillus niger in solid-state cultivation

The solid-state production of endo- and exo-polygalacturonases (PG) by Aspergillus niger was studied in a media containing wheat bran, salts, and different citric pectin and/or glucose concentrations. Kinetic analysis of the process indicated that the formation of PG and the growth of A. niger are associated processes. By increasing citric pectin from 0 to 16% (w/w), the maximum A. niger concentration (X _m) was raised from 94 to 121 mg/g dry medium suggesting that pectin can be used by A. niger as a growth substrate besides its role as an inducer. With 16% (w/w) pectin, 281 U exo-PG/gdm and 152 U endo-PG/gdm were obtained. Otherwise, pectin concentrations from 20 to 30% (w/w) hindered both production and growth. A. niger concentrations of 108–113 mg/gdm were achieved in runs with glucose from 5 to 12% (w/w), whereas at 16 and 20% (w/w) glucose, lower X _m values (ca. 100 mg/gdm) were measured. The addition of glucose to the wheat bran medium, up to 10% (w/w) led to maximum endo-PG titers slightly lower than those found in the absence of glucose. Nevertheless, exo-PG formation in these media was strongly increased and activities over 370 U/gdm were achieved. The results suggest that in experiments with pectin concentrations until 16% (w/w), exo-PG production was repressed by pectin-degradation products although these same substances had favored biomass growth. When glucose concentrations over 10% (w/w) were added to the media, the maximum activities of both enzymes decreased drastically, suggesting that glucose at high concentrations also exerts a repressive effect on PG production.     

Bioconversion of palm oil mill effluent for citric acid production: statistical optimization of fermentation media and time by central composite design

A laboratory-scale study was conducted to evaluate the feasibility of using palm oil mill effluent (POME) as a major substrate and other nutrients for maximum production of citric acid using the potential fungal strain Aspergillus niger (A103). Statistical optimization of medium composition (substrate–POME, co-substrates–wheat flour and glucose, and nitrogen source–ammonium nitrate) and fermentation time was carried out by central composite design (CCD) to develop a polynomial regression model through the effects of linear, quadratic, and interaction of the factors. The statistical analysis of the results showed that, in the range studied, ammonium nitrate had no significant effect whereas substrate, co-substrates and fermentation time had significant effects on citric acid production. The optimized medium containing 2% (w/w) of substrate concentration (POME), 4% (w/w) of wheat flour concentration, 4% (w/w) of glucose concentration, 0% (w/v) of ammonium nitrate and 5 days fermentation time gave the maximum predicted citric acid of 5.37 g/l which was found to be 1.5 g/l in the experimental run. The determination of coefficient (R ²) from the analysis observed was 0.964, indicating a satisfactory adjustment of the model with the response. The analysis showed that the major substrate POME (P < 0.05), glucose (P < 0.01), nutrient (P < 0.05), and fermentation time (P < 0.01) was more significant for citric acid production. The bioconversion of POME for citric acid production using optimal conditions showed the higher removal of chemical oxygen demand (82%) with the production of citric acid (5.2 g/l) on the final day of fermentation process (7 days). The pH and biosolids accumulation were observed during the bioconversion process.     

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.     

柠檬酸和EDTA-Na2对黑麦草吸收Pb及营养元素特性的影响

为探究柠檬酸或EDTA-Na_2对Pb污染下黑麦草(Lolium perenne L.)吸收Pb和营养元素特性的影响,对水培黑麦草进行不同处理,研究黑麦草一些生理生化指标的变化。结果表明,与对照相比,Pb处理降低黑麦草干重,增加质膜透性和根系脱氢酶活性,且在叶和根中积累Pb,而叶和根中6种营养元素含量的变化不尽相同。与Pb处理同时加入低浓度的柠檬酸或EDTA-Na_2对其生长影响较小,且叶片的Pb积累量较低;而同时加入高浓度的柠檬酸或EDTA-Na_2,虽然强化Pb在叶片中的积累,但是加重了生长的抑制作用和营养元素的稳态失衡;1 mmol L~(–1)的柠檬酸强化叶片积累Pb的效应强于同浓度的EDTA-Na_2,而5和10 mmol L~(–1)柠檬酸的强化作用则弱于同浓度的EDTA-Na_2。因此,适当浓度的柠檬酸或EDTA-Na_2在治理Pb污染环境中具有一定作用。     

Effects of citric acid and the siderophore desferrioxamine B (DFO-B) on the mobility of germanium and rare earth elements in soil and uptake in Phalaris arundinacea

Effects of citric acid and desferrioxamine B (DFO-B) on the availability of Ge and selected rare earth elements (REEs) (La, Nd, Gd, Er) to Phalaris arundinacea were investigated. A soil dissolution experiment was conducted to elucidate the effect of citric acid and DFO-B at different concentrations (1 and 10 mmol L^?1 citric acid) on the release of Ge and REEs from soil. In a greenhouse, plants of P. arundinacea were cultivated on soil and on sand cultures to investigate the effects of citric acid and DFO-B on the uptake of Ge and REEs by the plants. Addition of 10 mmol L^?1 citric acid significantly enhanced desorption of Ge and REEs from soil and uptake into soil-grown plants. Applying DFO-B enhanced the dissolution and the uptake of REEs, while no effect on Ge was observed. In sand cultures, the presence of citric acid and DFO-B significantly decreased the uptake of Ge and REEs, indicating a discrimination of the formed complexes during uptake. This study clearly indicates that citric acid and the microbial siderophore DFO-B may enhance phytoextraction of Ge and REEs due to the formation of soluble complexes that increase the migration of elements in the rhizosphere.     

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.     

Citric acid production from sugar cane molasses by 2-deoxyglucose-resistant mutant strain ofAspergillus niger

Citric acid production from sugar cane molasses byAspergillus niger NIAB 280 was studied in a batch cultivation process. A maximum of 90 g/L total sugar was utilized in citric acid production medium. From the parental strainA. niger, mutant strains showing resistance to 2-deoxyglucose in Vogal's medium containing molasses as a carbon source were induced by γ-irradiation. Among the new series of mutant strains, strain RP7 produced 120 g/L while the parental strain produced 80 g/L citric acid (1.5-fold improvement) from 150 g/L of molasses sugars. The period of citric acid production was shortened from 10 d for the wild-type strain to 6–7 d for the mutant strain. The efficiency of substrate uptake rate with respect to total volume substrate consumption rate,Q _s (g per L per h) and specific substrate consumption rate,q _s (g substrate per g cells per h) revealed that the mutant grew faster than its parent. This indicated that the selected mutant is insensitive to catabolite repression by higher concentrations of sugars for citric acid production. With respect to the product yield coefficient (Y _p/x), volume productivity (Q _p) and specific product yields (q _p), the mutant strain is significantly (p≤0.05) improved over the parental strain.     

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.