Organic acids production byStreptomyces spp. isolated from soil,rhizosphere and mycorrhizosphere of pine (Pinus sylvestris L.)

Summary The streptomycetes studied released into the medium the following organic acids: pyruvic, α-ketoglutaric, lactic, malic (or citric), succinic and oxalic. Soil streptomycetes produced more α-ketoglutaric-, lactic- and succinic acid than the root zone microorganisms. Mean indices of the total production of organic acids were in the following order: soil>rhizosphere>mycorrhizosphere. Amounts of pyruvic acid excreted by the soil streptomycetes were inversely proportional to their biomass, whereas those of α-ketoglutaric acid were directly proportional to dry weight. Small repeatability of the results of α-keto acids determinations in these organisms was stated.     

Synthesis of organic acids by immobilized propionic bacteria in the flow system and stabilization of the process

The capacity of immobilized cells of propionic bacteria to synthesize organic acids was examined. Propionibacterium shermanii cells incorporated into polyacrylamide gel were capable to synthesize propionic, acetic and pyruvic acids in the flow system. As a carbon source glucose, lactate-Na or whey lactose was used. The greatest amount of the acids was synthesized with the use of lactate-Na. The life-time of the biocatalyst (immobilized cells) can be increased by its reactivation with a nutrient medium required for optimal cell proliferation.     

Root exudates of wetland plants influenced by nutrient status and types of plant cultivation

The present study investigated the amounts of root exudates and composition of organic acids released from two wetland plants (Typha latifolia and Vetiver zizanioides) under two nutrient treatments: low level (0.786 mM N and 0.032 mM P) and high level (7.86 mM N and 0.32 mM P) and two types of plant cultivation: monoculture and co-culture of the two plants. Low nutrient treatment significantly (p < 0.05) increased the root exudates of T. latifolia during the initial growth period (1-21 d) and those of V. zizanioides and the co-culture during the whole growth period. The concentrations of dissolved organic carbon in the root exudates of the co-culture in the low nutrient treatment were 3.23-7.91 times of those in the high nutrient treatment during the medium growth period (7-28 d). The compositions of organic acids varied between the two plant species and between the two nutrient treatments. The pattern of organic acids was also different between the co-culture and the monoculture. Oxalic acid was by far the major organic acid exuded from the two wetland plants. The present study on root exudates suggests that co-culture of wetland plant species would be more useful in the reclamation of waste water than a monoculture system.     

Organic Acids of Ditylenchus triformis and Turbatrix aceti

Pyruvic acid, lactic acid and several tricarboxylic acid cycle acids were extracted from Ditylenchus triformis and Turbatrix aceti and identified. Fumaric acid was predominant in both nematodes. Small amounts o f malic and α-ketoglutaric acids and intermediate quantities o f lactic, citric, succinic, and pyruvic acids occurred in D. triformis. In T. aceti citric, lactic, and α-ketoglutaric acids were less abundant than succinic, malic and pyruvic acids. Only traces of aconitic and oxalacetic acids occurred in both nematodes. All the organic acids detected accounted for only about one per cent of the dry weight of nematodes o f the two species.     

Physicochemical properties of the microbial exopolysaccharide ethapolan synthesized on a mixture of growth substrates

Some physicochemical properties of the microbial exopolysaccharide (EPS) ethapolan synthesized by Acinetobacter sp. 12S depended on whether the producer was grown on a mixture of ethanol and glucose or on single substrates. Irrespective of the carbon source in the nutrient medium, the contents of carbohydrates, pyruvic acid, uronic acids, and mineral components in the EPS remained unchanged. The EPS were also identical in their monosaccharide composition: the molar ratio of glucose, mannose, galactose, and rhamnose was 3:2:1:1. EPS with a higher proportion of fatty acids was synthesized during growth on the mixture of ethanol and glucose. Average molecular weight and the proportion of high-molecular (over two million) fractions were greater in ethapolan produced on the substrate mixture. In the presence of 0.1 M KCl, after transformation into the H+ form, and in the Cu(2+)-glycine system, solutions of these EPS showed higher viscosity than solutions of EPS synthesized on single substrates. The reasons for the improved rheological properties of the EPS produced on the substrate mixture are discussed.     

End products of glucose and glutamine metabolism by L929 cells

Products of glucose and glutamine metabolism by L929 cells were detected and quantitated by gas chromatography and mass spectrometry of the oxime-trimethylsilyl derivatives. This method allowed detection and identification of all major carboxylic and amino acids produced in the system. Although lactic acid was expected to be the major product, alanine, citric, glutamic, aspartic, and pyruvic acids were also released into the culture medium at significant rates. Incorporation of labeled carbon from D-[U-13C]glucose showed that the alanine, lactic, and pyruvic acids were derived from glucose as was one-third of the citric acid carbon. The rate of glucose utilization for production of these end products was 29-fold greater than the rate of glucose oxidation to CO2, and calculated ATP production from alanine and pyruvate synthesis exceeded that from lactate synthesis by nearly 2-fold. Utilization of glutamine for synthesis of aspartic, glutamic, and citric acids also exceeded the rate of glutamine oxidation, thereby making end-product synthesis from glucose and glutamine the dominant cellular metabolic activity. In the absence of glucose, synthesis and intracellular levels of aspartic and glutamic acids increased, whereas synthesis and cell content of the other acids decreased markedly. This response is consistent with the metabolic pattern proposed by Moreadith and Lehninger (Moreadith, R.W., and Lehninger, A.L. (1984) J. Biol. Chem. 259, 6215-6221) in which much of the glutamine used by these cells is converted to aspartate in the absence of a pyruvate source and to aspartate or citrate in the presence of pyruvate.     

Physicochemical Properties of the Microbial Exopolysaccharide Ethapolan Synthesized on a Mixture of Growth Substrates

Some physicochemical properties of the microbial exopolysaccharide (EPS) ethapolan synthesized by Acinetobacter sp. 12S depended on whether the producer was grown on a mixture of ethanol and glucose or on a single substrate. Irrespective of the carbon source in the nutrient medium, the contents of carbohydrates, pyruvic acid, uronic acids, and mineral components in the EPS remained unchanged. The EPS were also identical in their monosaccharide composition: the molar ratio of glucose, mannose, galactose, and rhamnose was 3 : 2 : 1 : 1. EPS with a higher content of fatty acids was synthesized during growth on the mixture of ethanol and glucose. The average molecular mass and the content of high-molecular (M > 2 MDa) fractions were greater in ethapolan produced on the substrate mixture. In the presence of 0.1 M KCl, after transformation into the H⁺ form, and in the Cu²⁺–glycine system, solutions of these EPS showed higher viscosity than solutions of EPS synthesized on single substrates. The reasons for the improved rheological properties of the EPS produced on the substrate mixture are discussed.     

Quantitative evaluation of the role of organic acid exudation in the mobilization of rock phosphate by rape

Phosphorus-deficient rape plants appear to acidify part of their rhizosphere by exuding malic and citric acid. A simulation model was used to evaluate the effect of measured exudation rates on phosphate uptake from Mali rock phosphate. The model used was one on nutrient uptake, extended to include both the effect of ion uptake and exudation on rhizosphere pH and the effect of rhizosphere pH on the solubilization of rock phosphate. Only the youngest zones of the root system were assumed to exude organic acids. The transport of protons released by organic acids was described by mass flow and diffusion. An experimentally determined relation was used describing pH and phosphate concentration in the soil solution as a function of total soil acid concentration. Model parameters were determined in experiments on organic acid exudation and on the uptake of phosphate by rape from a mixture of quartz sand and rock phosphate. Results based on simulation calculations indicated that the exudation rates measured in rape plants deficient in phosphorus can provide the roots with more phosphate than is actually taken up. Presence of root hairs enhanced the effect of organic acid exudation on calculated phosphate uptake. However, increase of root hair length without exudation as an alternative strategy to increase phosphate uptake from rock phosphate appeared to be less effective than exudation of organic acids. It was concluded that organic acid exudation is a highly effective strategy to increase phosphate uptake from rock phosphate, and that it unlikely that other rhizosphere processes play an important role in rock phosphate mobilization by rape.     

Estimations of Uronic Acids as Quantitative Measures of Extracellular and Cell Wall Polysaccharide Polymers from Environmental Samples

The extracellular polysaccharide polymers can bind microbes to surfaces and can cause physical modification of the microenvironment. Since uronic acids appear to be the components of these extracellular films that are most concentrated in a location outside the cell membrane, a quantitative assay for uronic acids was developed. Polymers containing uronic acids are resistant to quantitative hydrolysis, and the uronic acids, once released, form lactones irreproducibly and are difficult to separate from the neutral sugars. These problems were obviated by the methylation of the uronic acids and their subsequent reduction with sodium borodeuteride to the corresponding alcohol while they were in the polymer and could not form lactones. This caused the polymers to lose the ability to adhere to their substrates, so they could be quantitatively recovered. The hydrolysis of the dideuterated sugars was reproducible and could be performed under conditions that were mild enough that other cellular and extracellular polymers were not affected. The resulting neutral sugars were readily derivatized and then were separated and assayed by glass capillary gas-liquid chromatography. The dideuterated portion of each pentose, hexose, or heptose, identified by combined capillary gas-liquid chromatography and mass spectrometry, accurately provided the proportion of each uronic acid in each carbohydrate of the polymer. Examples of the applications of this methodology include the composition of extracellular polymers in marine bacteria, invertebrate feeding tubes and fecal structures, and the microfouling films formed on titanium and aluminum surfaces exposed to seawater.     

Differences in cluster-root formation and carboxylate exudation in Lupinus albusL. under different nutrient deficiencies

In contrast with the well document role of proteoid root formation and carboxylate exudation in acclimation to P deficiency in white lupin (Lupinus albus L.), their role under other nutrient deficiencies and their ecological significance are still poorly understood. In the present work, differences in proteoid root formation, exudation of carboxylates by root clusters, non-proteoid and proteoid root tips by using a non-destructive method, and concentrations of organic acids in the tissues of plants grown in the absence of P, Fe or K were studied. Proton release from roots increased soon after withdrawing Fe from the medium; within three days the solution pH decreased from 6 to about 4, and this increased release in protons continued until the end of the experiment. Acidification appeared much later, on the 10th day and the 14th day after withdrawal of P and K, respectively; the extent of the acidification was also weaker than under –Fe (5.2 for –P and 5.7 for control on the 10th day; 6.0 for –K and 6.1 for control on the 14th day). Root clusters formed when plants were grown under –P and –Fe, but not under –K conditions. The root clusters developed sooner under –Fe conditions, but the number of clusters was far less than under –P. Under P deficiency, root clusters released mainly citrate, but also some malate; while the major organic acid released by root tips of both non-proteoid and proteoid roots was malate. However, under Fe deficiency, the majority of the organic acids exuded both by the root clusters and root tips was malate, whereas only a small amount of citrate was detected. The release rate of citrate by – P root clusters was greater than that by – Fe root clusters. Moreover, the release rate of malate was greater in –Fe root clusters than in –P root clusters, but the opposite was found in proteoid root tips, i.e. faster in –P than in –Fe proteoid root tips. The significances of proteoid root formation and release of organic acids in acclimation to different nutrient deficiencies for white lupin plants are discussed.     

Genotypic variability in phosphorus solubilizing activity of root exudates by pigeonpea grown in low-nutrient environments

A pot experiment confirmed that pigeonpea could efficiently utilize various sources of phosphorus (P) (aluminium phosphate, iron phosphate and apatite), irrespective of genotype. A qualitative assay method for iron (Fe)-P solubilizing activity showed that root exudates collected from P-deficient pigeonpea contained Fe-P solubilizing substances and that they were released mainly from root tips. Citric, malic, malonic, succinic and piscidic acids were identified in root exudates. Citric and piscidic acids release from roots was increased by low-P treatment in all the genotypes tested. The release rates of citric and piscidic acids were affected by the P concentration of shoots rather than that of roots. The pigeonpea roots released approximately 5–100 times more piscidic acid than citric acid depending on P stress status, plant age and genotype. When organic acids were added to Alfisols, citric acid was most capable of mobilizing P from the soil, followed by piscidic acid and malic acid. No correlation was found between genotypic variability in the release rates of citric and piscidic acids from the roots under low-P treatment at hydroponic culture and in the growth and P uptake of plants on Alfisols. Although citric and piscidic acids released from pigeonpea roots may play a partial role in solubilizing unavailable insoluble P in soils, the releases were thought to be an unsatisfactory strategy for explaining genotypic variation in low P availability of pigeonpea.     

Gas chromatographic assay of iduronic and glucuronic acids as aldonic acid butaneboronates

A gas chromatographic procedure has been developed for the analysis of uronic acids as aldonic acid butaneboronates. With these derivatives, aldoses frequently accompanying uronic acids in polysaccharide hydrolyzates are readily separated and measured. The method has been applied to the assay of iduronic and glucuronic acid released by enzymes associated with various mucopolysaccharidoses.     

The Amino Acid Content in Gamma-irradiated Rice†

High phosphate accumulating bacteria were isolated by autoradiography. One isoate, Arthrobacter globiformis PAB-6 accumulated phosphate intracellularly at 20% of dry cell mass in a simple synthetic medium. This amount was 3~7 times higher than type cultures examined. Almost no phosphate was released into the medium after cessation of growth. Fifty percent of total intracellular phosphate was fractionated as nucleic acids, while 20% each was recovered from cold PCA soluble fractions and polyphosphate fractions. The large content of nucleic acids in this bacterium appeared due to increased RNA content, specifically 4 S RNA fraction.     

Exopolysaccharide: a novel important factor in the microbial dissolution of tricalcium phosphate

Four strains (Enterobacter sp. EnHy-401, Arthrobacter sp.ArHy-505, Azotobacter sp.AzHy-510 and Enterobacter sp.EnHy-402) which have the ability to solubilize tricalcium phosphate (TCP) were used to study the mechanism of P-solubilization. It was found that three phosphate solubilizing bacteria (EnHy-401, ArHy-505 and AzHy-510) producing exopolysaccharide (EPS) have a stronger ability for P-solubilization than isolate EnHy-402 without EPS production, of those, the strain EnHy-401 with the highest EPS production and efficient organic acids on P-solubilization had a stronger capacity for P-solubilization than the others. Further studies demonstrated that addition of EPS into medium could increase the amount of phosphorus solubilized by organic acid, but failed to release phosphorus from TCP alone. The synergistic effects of EPS and organic acid on TCP solubilization varied with the origin and the concentration of EPS in medium. EPS produced by EnHy-401 was most effective in promoting phosphorus release at an optimal concentration in medium. The increase of P-solubilization brought by EPS attributed to the participation of EPS led to the change in homeostasis of P-solubilization, pushing it towards P dissolved by holding free phosphorus in the medium, consequently resulting in greater phosphorus released from insoluble phosphate. We therefore suggest that EPS with ability of phosphorus-holding may be a novel important factor in the microbial dissolution of TCP except for organic acid.     

Excretion of alpha-keto acids by strains of Streptomyces venezuelae

Cultures of Streptomyces venezuelae released acidic metabolites during nitrogen-limited growth on glucose. The main products were pyruvic acid and alpha-ketoglutaric acid. Variation in the extent of acid production was observed; spores of the parental strain 13s gave approximately 10% of low-producing colonies when plated on acid-base indicator medium. Examination of one low producer, strain PC 51-5, showed that differences in acid production became apparent only in low-glucose media containing manganese. In both strains PC 51-5 and 13s, uptake of alpha-keto-[5-14C]glutaric acid occurred by diffusion and no marked differences in permeability to alpha-ketoglutarate were detected. However, differences were observed in the activity of alpha-ketoglutarate dehydrogenase. In cultures of strain PC 51-5, the specific activity of the enzyme increased throughout growth, whereas in the parental strain activity decreased and could not be detected in older mycelium. Loss of enzyme activity was accompanied by excretion of alpha-ketoglutaric acid and failure to assimilate the product after glucose exhaustion. The results suggest that accumulation of pyruvic and alpha-ketoglutaric acids in S. venezuelae cultures grown in glucose-containing media may be due to regulatory suppression of the dehydrogenases by this carbon source.     

Iron-regulated excretion of alpha-keto acids by Salmonella typhimurium.

Excretion of alpha-keto acids by clinical isolates and laboratory strains of Salmonella typhimurium was determined by high-performance liquid chromatography analysis of culture supernatants. The levels of excretion increased markedly with increasing iron stress imposed by the presence of alpha,alpha'-dipyridyl or conalbumin in the medium. The major product was pyruvic acid, but significant concentrations of alpha-ketoglutaric acid, alpha-ketoisovaleric acid, and alpha-ketoisocaproic acid were also observed. Maximal excretion occurred at iron stress levels that initially inhibited bacterial growth; the concentration of alpha,alpha'-dipyridyl at which this was observed differed between strains depending on their ability to secrete and utilize siderophores, suggesting that the intracellular iron status was important in determining alpha-keto acid excretion. However, prolonged incubation of the siderophore-deficient S. typhimurium strain enb-7 under conditions of high iron stress resulted in significant delayed bacterial growth, promoted by tonB-dependent uptake of iron complexed with the high accumulated levels of pyruvic acid and other alpha-keto acids. Strain RB181, a fur derivative of enb-7, excreted massive amounts of alpha-keto acids into the culture medium even in the absence of any iron chelators (the concentration of pyruvic acid, for example, was >25 mM). Moreover, RB181 was able to grow and excrete alpha-keto acids in the presence of alpha,alpha'-dipyridyl at concentrations threefold greater than that which inhibited the growth of enb-7.     

Modified alkaloid pattern in developing tobacco callus

Developing Nicotiana tabacum L. cv. Wisconsin-38 callus grown on modified Murashige-Skoog (MS) medium with Kao organic acids (pyruvic, citric, malic and fumaric acids) contains abnormally high levels of nornicotine and total alkaloids when compared with the leaves of the donor plant. Nornicotine/nicotine ratios observed during callus development suggest that nicotine is converted into nornicotine in the callus, with subsequent movement of alkaloids into roots formed on the callus and into the agar medium. Addition of Kao organic acids to the medium increases alkaloid levels, but cannot account for the abnormal increase in nicotine demethylation. This study thus reports two new findings: (a) that the total alkaloid content of tobacco callus can be greatly enhanced to 3.75% on a dry weight basis by exogenous organic acids, and (b) that endogenous nornicotine can accumulate in tobacco tissue cultures.     

The use of 13C-n.m.r. spectroscopy to monitor alginate biosynthesis in mucoid Pseudomonas aeruginosa.

The biosynthesis of alginate by a mucoid strain of Pseudomonas aeruginosa, isolated from a cystic-fibrosis patient, was monitored by using 13C-n.m.r. spectroscopy of bacterial cultures incubated with 1-13C- or 2-13C-enriched fructose. When 1-13C- or 2-13C-enriched fructose was used as the precursor of alginate, enrichment with 13C in the constituent uronic acid monomers of the polysaccharide could only be detected in C-1 or C-2 respectively, indicating that alginate is synthesized in Ps. aeruginosa directly from fructose, with the hexose molecule being retained intact; this rules out the involvement of C3 intermediates, which occurs when glucose is the alginate precursor. The absence of detectable poly-L-gluluronate block sequences from the alginate of Ps. aeruginosa was confirmed, and it was shown that there is no modification of the arrangement of the constituent uronic acids between polymerization to form alginate and the appearance of the mature alginate in the extracellular medium. The 13C-n.m.r. data also provided independent evidence for acetylation on D-mannuronate residues and for the ratio of D-mannuronate to L-guluronate residues in newly synthesized alginate, which had previously been determined only for material secreted from bacteria into the extracellular medium.     

Root-induced nitrogen mineralisation: A nitrogen balance model

The possibility is examined that carbon (C) released into the soil from a root could enhance the availability of nitrogen (N) to plants by stimulating microbial activity. Two models are described, both of which assume that C released from roots is used by bacteria to mineralise and immobilise soil organic N and that immobilised N released when bacteria are grazed by bacterial-feeding nematodes or protozoa is taken up by the plant. The first model simulates the individual transformations of C and N and indicates that root-induced N mineralisation could supply only up to 10% of the plant's requirement, even if unrealistically ideal conditions are assumed. The other model is based on evidence that about 40% of immobilised N is subsequently taken up by the plant. A small net gain of N by the plant is shown (i.e. the plant takes up more N than it loses through exudation), although with exudate of up to C:N 33:1 less than 6% of the plant's requirement is supplied by root-induced N mineralisation. It is argued, however, that rhizosphere bacteria do not use plant-derived C to mineralise soil organic N to any great extent and that in reality root-induced N mineralisation is even less important than these models indicate.     

The determination of iduronic acid and glucuronic acid in heparins by a new technique: Re-evaluation of the variable hexuronic acid composition of mammalian heparins

A new technique for the quantitative determination of the uronic acid components of heparin is described. Heparin was deamination with organic nitrite and methanolyzed. The monomeric derivatives of uronic acids were quantitated by gas chromotography. Depolymerization to monomeric units appeared essentially complete as judged from the yield of uronic acid derivatives. By applying the method the relative contents of iduronic acid and glucuronic acid in a number of heparin samples were estimated. In all samples examined the content of iduronic acid was larger than that of glucuronic acid. A species specific difference in the iduronic acid to glucuronic acid ration of heparin was noted. Noticeable difference of this ratio was observed also between different organs of a species of animal and among heparin fractions obtained from an organ.