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Gene transfer between different Trichoderma species and Aspergillus niger through intergeneric protoplast fusion to convert ground rice straw to citric acid and cellulases.

Single-stage direct bioconversion of cellulosic materials to citric acid using intergeneric hybrids obtained from three different Trichoderma species and Aspergillus niger was carried out. The recent results were obtained on the basis of either resistance or sensitivity to one or more of five metal ions, two catabolite repressors, and five antifungal agents, which were used in this study at different concentrations. Sixty-six fusants were isolated after using the three intergeneric protoplast fusion experiments, belonging to two types of intergeneric fusants. Fusants of the first type are heterokaryons (35 fusants). On the other hand, those of the second type are haploids (31 fusants), i.e., they were stable. The present study can be successfully applied in the construction of 14 new genetic fusants, which produced at least 100% more citric acid than the citric acid producer strain A. niger. Out of the fusants, three (1/18, 2/13 and 2/15) showed about a threefold increase of citric acid production in comparison with the parent A. niger strain. Furthermore, studies on DNA content showed that this finding may be submitted on the evidence that citric acid and cellulases production was not correlated with DNA content; however, the productivity depends on specific DNA content.

Antifungal Agents↗

Use of citric acid for heavy metals extraction from contaminated sewage sludge for land application.

Recent studies revealed that organic acids such as citric and oxalic acids seemed to be more promising as chemical extracting agents for removal of heavy metals from contaminated sludge, since they are biodegradable and can attain a higher metal extraction efficiency at mildly acidic pH compared to other extracting agents. Results of a lab-scale study on the efficiency of citric acid in the extraction of chromium (Cr), copper (Cu), lead (Pb), nickel (Ni) and zinc (Zn) from anaerobically digested sludge, revealed that citric acid seemed to be highly effective in extracting Cr (at 100%), Cu (at 88%), Ni (at 98%) and Zn (at 100%) at pH 2.33, mostly at 5 days leaching time except for Cu and Zn, which are at 1 day and 2 h contact times respectively. Lead removal at the same pH was also high at 95% but at a longer leaching time of 11 days. At pH 3, citric acid seemed to be highly effective in extracting Pb (at 100%) at 1 day leaching time, although higher removals were also attained for Ni (70%) and Zn (80%) at only 2 h leaching time. Chemical speciation studies showed that Cr, Cu and Ni in the sludge sample seem to predominate in residual fractions, while Pb and Zn were found mostly bound to organic and inorganic matter forms, hence the potential of the sludge for land application.

Citric Acid↗

[Exudation and accumulation of citric acid in common bean in response to Al toxicity stress].

Significant differences in the exudation and accumulation of citric acid in common bean genotypes were observed in response to Al toxicity stress by hydroponic cultural experiments. Secreted citric acid increased with increasing external concentrations of Al3+ which ranged from 0 to 50 mumol.L-1, while ranged from 50 to 80 mumol.L-1, secreted citric acid decreased with increasing external concentrations of Al3+. Among different genotypic common beans, citric acid secreted in G19842 was the largest, while Al uptake per unit dry weight in G19842 was the least. No difference in the accumulation of citric acid in leaves was found among different genotypic common beans, while the size of the content of citric acid in roots was G19842 > AFR > ZPV > G5273. The amount of citric acid exuded was smaller induced by phosphorus deficiency than that induced by Al toxicity stress. Exposure to 50 mumol.L-1 LaCl3 could not induce the exudation of citric acid, and it implied that the exudation and accumulation of citric acid in common bean was an important physiological response of resistance to Al toxicity stress.

Aluminum↗

Effect of fermentation conditions on the production of citric acid from cheese whey by Aspergillus niger.

The effect of pH value, methanol, and salt concentration on the production of citric acid from cheese whey by two strains of Aspergillus niger, i.e. CAIM 111 and CAIM 167, was investigated. Lactose concentration, utilized lactose, citric acid concentration, conversion coefficient of lactose to citric acid, and mycelial dry weight were measured during the fermentation process. The maximum citric acid concentration (1.06 and 0.82 g/l), and conversion coefficient (5.58 and 7.45%) were obtained at pH 3.5 after 9 days of fermentation for A. niger CAIM 111 and A. niger CAIM 167, respectively. The presence of 4% (v/v) methanol in the fermentation medium increased the amount of citric acid produced by A. niger CAIM 111 and A. niger CAIM 167 by 23% and 18%, respectively. Both strains showed a high ability to utilize lactose for the production of citric acid when grown in the presence of 10% (w/v) salt. The conversion coefficient of lactose to citric acid was 28.24% for A. niger CAIM 111 and 25.60% for A. niger CAIM 167 when the fermentation medium had a 10% (w/v) level of salt. The cumulative effect of fermentation medium pH (3.5), methanol concentration (4%, v/v) and salt concentration (10%, w/v) during the fermentation process of whey did not enhance the production of citric acid by A. niger CAIM 111, while it did increase the production of citric acid by A. niger CAIM 167 by about 4-fold.

Aspergillus niger↗

Root canal irrigation with citric acid solution.

The purpose of this study was to investigate various properties of citric acid and EDTA solution as decalcifying and cleansing agents in root canal irrigation, and antibacterial effects of citric acid and EDTA solution. Powdered dentin-resin mixtures were used for evaluating the decalcifying effect of citric acid and EDTA solution. Twelve bacterial strains isolated from infected root canals, were used to evaluate the antibacterial effects of citric acid and EDTA solution. Powdered dentin-resin mixture was found to be more soluble in a 0.5, 1, and 2 M citric acid solutions than in a 0.5 M EDTA solution. Citric acid solution showed antibacterial effects on all the bacteria used.

Anti-Infective Agents, Local↗

The role of citric acid on the phytoremediation of heavy metal contaminated soil.

Adsorption and hydroponics experiments were conducted to study the role of citric acid on the phytoremediation of heavy metal contaminated soil. The results show that addition of citric acid decreased the adsorption of both lead and cadmium, such an effect was bigger for cadmium than for lead. The decrease in the adsorption of Pb and Cd was mainly due to a decrease of pH in the presence of citric acid. The presence of citric acid could alleviate the toxicity of Pb and Cd to radish, and stimulate their transportation from root to shoot. The studies of heavy metal forms using sequential extraction demonstrated that lead was mainly existed as FHAC (a lower bioavailable form) in the root, while F(HCl) was the dominant form in the leaf. The addition of citric acid to the soil changed the concentration and relative abundance of all the forms. The detoxifying effect of citric acid to Pb in shoots might result from the transformation of higher toxic forms into lower toxic forms. Cadmium was mainly present as F(NaCl), therefore, it had higher toxicity than lead. The addition of citric acid increased the abundance of F(H2O) + F(NaCl), indicating that citric acid treatment could transform cadmium into more transportable forms.

Adsorption↗

[Effect of trace metals on cell morphology, enzyme activation, and production of citric acid in a strain of Aspergillus wentii].

Data concerning the effect of very low concentrations of metals on citric acid production by microorganisms, as well as on the activity of enzymes presumptively involved in the process, are confuse. The bulk of information was obtained mainly studying selected strains of Aspergillus niger. Information concerning other citric acid producer filamentous fungi, such as A. wentii, is scanty. In the present article we report the effect of different cations on the growth pattern of A. wentii P1 as well as on the related citric acid production and the activity of several enzymes. It was found that without any addition to the culture medium the fungus developed a pelleted form of growth, pellets being about 1.5 mm in diameter. The citric acid yield was about 90%. The addition of Cu2+ impaired the sugar uptake, as well as the production of citric acid and biomass. The uptake of sugar increased in the presence of Zn2+, and there was a marked increase of the biomass production, which could account for the low citric acid production. The addition of Fe2+ impaired the citric acid production and, as sulfate, the sugar uptake. The presence of Fe3+ markedly impaired the citric acid production and increased the sugar uptake. There is no agreement about the enzymes involved in the accumulation of citric acid by microorganisms. In spite of this, aconitase (Ac), isocitrate lyase (IL), isocitrate dehydrogenase NAD(+)-dependent (ICDH- NAD+) and isocitrate dehydrogenase NADP(+)-dependent (ICDH-NADP+) are often postulated as key enzymes. In our case, these enzymes were active during the standard fermentation, although with variations, particularly concerning Ac and IL. The behavior of enzymes might be different when tested in vivo or in vitro, mainly from the quantitative point of view. Nevertheless, the activity determined in vitro might give some indication concerning the effect on fermentation of substances present in the medium. It was found that all the enzymes tested increased their activity in the presence of Fe2+ and Fe3+. The addition of Cu2+ improved the activity of Ac, IL and ICDH-NADP+, while that of ICDH-NAD+ was impaired. The presence of Zn2+ decreased the activity of the enzymes excepting that of ICDH-NADP+ which increased. It must be pointed out that according to these results there are differences concerning the behavior of ICDH-NAD+ and ICDH-NADP+ in the presence of Cu2+ or Zn2+.

Aconitate Hydratase↗

Mechanisms of capsaicin- and citric-acid-induced cough reflexes in guinea pigs.

Previously, we developed a novel coughing model that evoked coughs via citric acid microinjection into the larynx of unanesthetized, unrestrained guinea pigs. Here, we compared the effects of capsaicin and citric acid administration into the larynx using this model. Inhalation of capsaicin (30 microM) or citric acid (0.4 M) for 5 min induced cough mimetic responses over a 10-min observation period (mean +/- S.E.M.: 8.85 +/- 1.60 and 10.40 +/- 1.17 coughs, respectively, n = 18). Microinjection of 0.4 M citric acid into the larynx (20 microl in total in 10 aliquots of 2 microl at 30-s intervals) induced 27.29 +/- 2.69 coughs over a 10-min observation period, whereas microinjection of 3 mM capsaicin evoked a maximum of 8.32 +/- 1.82 coughs (n = 18). Desensitization of C-fibers by capsaicin (100 mg/kg, s.c.) abolished the coughs evoked upon inhalation or microinjection of capsaicin and inhalation of citric acid, but had no significant effect on those induced by microinjection of citric acid. These results indicate that there were fewer non-myelinated C-fiber afferents in the larynx area than in other regions (bronchi or alveoli). Consequently, citric acid microinjected into the larynx may induce the cough reflex by stimulating other fibers (for example, Adelta-fibers), rather than C-fibers. In contrast, inhaled citric acid predominantly stimulated C-fibers, and hardly affected Adelta-fibers.

Animals↗

The effects of citric acid on phytate-phosphorus utilization in young chicks and pigs.

Several bioassays were conducted with young chicks and pigs fed phosphorus (P)-deficient corn-soybean meal diets. With diets for chicks containing .62% Ca and .42% P (.10% available P), graded doses of a citric acid + sodium citrate (1:1, wt:wt) mixture (0, 1, 2, 4, or 6% of diet) resulted in linear (P < .01) increases in both weight gain and tibia ash. Relative to chicks fed no citric acid, tibia ash (%) and weight gain (g/d) were increased by 43 and 22%, respectively, in chicks fed 6% citric acid. Additional chick trials showed that 6% citric acid alone or sodium citrate alone was as efficacious as the citric acid + sodium citrate mixture and that 1,450 U/kg of phytase produced a positive response in bone ash and weight gain in chicks fed a diet containing 6% citrate. Varying the Ca:available P ratio with and without citrate supplementation indicated that citric acid primarily affected phytate-P utilization, not Ca, in chicks. Moreover, chicks did not respond to citrate supplementation when fed a P-deficient (.13% available P), phytate-free casein-dextrose diet. Young pigs averaging 10 to 11 kg also were used to evaluate citric acid efficacy in two experiments. A P-deficient corn-soybean meal basal diet was used to construct five treatment diets that contained 1) no additive, 2) 3% citric acid, 3) 6% citric acid, 4) 1,450 U/kg phytase, and 5) 6% citric acid + 1,450 U/kg phytase. Phytase supplementation increased (P < .01) weight gain, gain:feed, and metatarsal ash, whereas citric acid addition increased only gain:feed (P < .05) and metatarsal ash (P < .08). A subsequent 22-d pig experiment was conducted to evaluate the effect of lower levels of citric acid (0, 1, 2, or 3%) or 1,450 U/kg phytase addition to a P-deficient corn-soybean meal diet. Phytase supplementation improved (P < .01) all criteria measured. Weight gain and gain:feed data suggested a response to citric acid addition, but this was not supported by fibula ash results (P > .10). The positive responses to phytase were much greater than those to citric acid in both pig experiments. Thus, dietary citric acid effectively improved phytate P utilization in chicks but had a much smaller effect in pigs.

6-Phytase↗

The effect of citric acid on the calcium and phosphorus requirements of chicks fed corn-soybean meal diets.

Data previously reported from our laboratory indicated that supplementation of a corn-soybean meal diet with citric acid improves P utilization in chicks. The four experiments reported herein were conducted to further evaluate the effects of citric acid on Ca and P utilization for chicks fed a corn-soybean meal diet. Diets in all experiments were fed to chicks from 8 to 21 or 22 d of age. The first experiment evaluated the effect of 6% citric acid on the Ca requirement of chicks. A Ca-deficient basal diet [23% CP, 0.54% Ca, 0.45% available P (AP)] containing 0 to 0.7% supplemental Ca in 0.1% increments was fed with or without 6% citric acid. The results indicated that citric acid did not significantly affect the Ca requirement. A second experiment evaluated different levels of citric acid (0, 2, 4, or 6%) on P utilization, and results indicated that 4 and 6% citric acid produced the largest responses in growth and tibia ash. Experiments 3 and 4 were then conducted to determine whether 4 or 6% citric acid would reduce the level of supplemental P required. Dietary treatments were a P-deficient basal diet (23% CP, 1.0 or 1.3% Ca, 0.20% AP) supplemented with 0 to 0.25% inorganic P with or without 4 or 6% citric acid. When diets contained citric acid, weight gain and tibia ash were maximized at lower AP levels than when diets contained no citric acid. The results of this study indicate that citric acid increases P utilization in corn-soybean meal diets and reduces the AP requirement by approximately 0.10% of the diet.

Animal Feed↗

Connective tissue attachment to periodontally diseased roots after citric acid demineralization.

PREVIOUS INVESTIGATIONS have demonstrated that application of citric acid to a root surface results in exposure of dentin and/or cementum matrix collagen fibrils. Several studies have suggested a rapid and consistent connective tissue reattachment to citric acid treated roots. This study was initiated to determine if such an attachment was obtainable on human periodontally diseased teeth, in vivo, and could be confirmed through observations using the scanning electron and light microscopes. Full thickness flaps were raised on 18 single rooted teeth with moderate to advanced periodontitis. Citric acid (pH = 1.0) was applied to nine teeth with contralateral teeth serving as controls. Six to fifteen weeks later, the teeth with attached periodontal tissue were removed. Sagittal sections were obtained, with one-half of the root being processed for light microscopy and the remaining half studied by scanning electron microscopy. Six of nine citric acid treated roots provided evidence of fibrous attachment. Connective tissue was apposed directly to old or newly formed cementum, but never directly to dentin. Fibrous attachment was usually functionally oriented, i.e., perpendicular to the root surface. No evidence of fibrous attachment was found among the control specimens.

Citrates↗

Thermal characterization of citric acid solid dispersions with benzoic acid and phenobarbital.

The glass transition temperatures of citric temperatures of citric acid glass were determined by differential scanning calorimetry to be 10.2 and 13.5 degrees for in situ and bulk-prepared samples, respectively. Mechanical stress on citric acid glass induced foci for crystallization. Benzoic acid addition to citric acid glass decreased its glass transition temperature while phenobarbital addition increased its glass transition temperature, the latter forming a glass solution.

Benzoates↗

Phase behavior of binary and ternary amorphous mixtures containing indomethacin, citric acid, and PVP.

PURPOSE: To better understand the nature of drug-excipient interactions we have studied the phase behavior of amorphous binary and ternary mixtures of citric acid, indomethacin and PVP, as model systems. METHODS: We have prepared amorphous mixtures by co-melting or coprecipitation from solvents, and have measured glass transition temperatures with differential scanning calorimetry. RESULTS: Citric acid and indomethacin in the amorphous state are miscible up to 0.25 weight fraction of citric acid, equivalent to about 2 moles of citric acid and 3 moles of indomethacin. Phase separation, as reflected by two Tg values, occurs without crystallization leading to a saturated citric acid-indomethacin amorphous phase and one essentially containing only citric acid. PVP-citric acid and PVP-indomethacin form non-ideal miscible systems at all compositions. A ternary system containing 0.3 weight fraction of PVP produces a completely miscible system at all citric acid-indomethacin compositions. The use of 0.2 weight fraction of PVP, however, only produces miscibility up to a weight fraction of 0.4 citric acid relative to indomethacin. The two phases above this point appear to contain citric acid in PVP and citric acid in indomethacin, respectively. CONCLUSIONS: Two components of an amorphous solid mixture containing citric acid and indomethacin with limited solid state miscibility can be solubilized as an amorphous solid phase by the addition of moderate levels of PVP.

Citric Acid↗

Hydroxyapatite formation on cellulose cloth induced by citric acid.

Hydroxyapatite (HAp) formation on cellulose cloth with the aid of citric acid was investigated. The cellulose cloths were soaked in simulated body fluid (SBF) solutions (1.5 SBF) with ion concentrations 1.5 times that of SBF (1.0 SBF) with and without citric acid and carbonate containing HAp crystals were found to form only in the 1.5 SBF solution that contained citric acid. The results were explained in terms of hydrogen bonding of citric acid to the cellulose cloth and its chelating ability of calcium ions. Practical application may involve the inclusion of citric acid in the 1.5 SBF solution to promote formation of HAp on previously bioinert cellulose cloth.

Journal Article↗

Properties of citric acid at the glass transition.

To better understand the properties of citric acid when used in solid dosage forms as an acid-base buffer, we initiated a study of its properties in the amorphous state. Such a state often arises during processes such as lyophilization and wet granulation. In view of inconsistencies in the literature concerning the glass transition temperature, Tg, of citric acid in the dry and hydrated states, we measured the Tg of samples formed by a melt-quench cool sequence in a DSC. We also used DSC to measure Tg', the glass transition temperature of the maximally freeze-concentrated solution. It was shown that dry citric acid has a Tg of 11 degrees C, while that containing 8.6% water (equimolar) has a value of -25 degrees C. The Tg' of a frozen solution of citric acid is -53 degrees C. Measuring Tg for the dry and hydrated samples at various scanning rates allowed measurement of the activation energy for enthalpy relaxation at Tg and enabled estimation of the degree of fragility (or the strength parameter) for both samples. It was shown that citric acid is a fairly fragile liquid expected to exhibit non-Arrhenius dynamic behavior and that the presence of residual water at a level of 8.6% causes a decrease in fragility.

Calorimetry, Differential Scanning↗