Isolation and characterization of nickel-resistant microflora from serpentine soils of Andaman

Serpentine soils of Andaman Islands, India characteristically contain high levels of nickel, cobalt and chromium and are colonized by indigenous nickel-hyperaccumulating plants. Attempts have been made to isolate and characterize nickel-resistant microorganisms from these hitherto unexplored naturally nickel-percolated soils. The majority of the nickel-resistant organisms showed a minimum inhibitory concentration (MIC) of Ni²⁺ ranging from 300 to 400 mg/l and about 3.4% of the total 89 isolates representing bacterial strains were able to grow at 400 mg/l Ni²⁺. The potent Ni²⁺-resistant strains AND305 and AND603 were tentatively identified as Pseudomonas spp. and strain AND408 as Bacillus sp. following detailed analysis of morphological and physio-biochemical characteristics. Growth kinetics of these Ni²⁺-resistant bacteria showed a prolonged lag phase in Ni²⁺-containing media, which extended with increasing nickel concentration. In addition to Ni²⁺, these isolates were also resistant to Co²⁺, Cd²⁺, Cr⁶⁺, Fe³⁺, Cu²⁺, Mg²⁺, Mn²⁺(50–200 mg/l) and Hg²⁺ (0.5–2.0 mg/l) and the multiple metal-resistance of the isolates were also associated with the resistance to antibiotics ampicillin, cycloserine and penicillin G.     

Studies on citric acid production with immobilized Yarrowia lipolytica in repeated batch and continuous air-lift bioreactors

Citric acid was produced from glucose in repeated-batch shake-flask and continuous air-lift cultivations by calcium-alginate-immobilized Yarrowia lipolytica A-101 yeast. The medium composition was systematically studied in a batch system by using experimental design and empiric modelling. The highest citric acid product concentration of 39 g/l was reached with a medium containing 150 g/l of glucose, 0.105 g/l of potassium dihydrogen phosphate, 0.84 g/l of magnesium sulphate and 21 mg/l of copper sulphate (5.2 mg/l of copper). The results were further improved by hardening the alginate carrier beads with glutaraldehyde, and by activation of the immobilized biocatalyst in a nutrient solution. In continuous air-lift bioreactors with varying height-to-diameter ratio the highest productivity of 350 mg/l per hour with a dilution rate of 0.023 l/h and a citric acid product concentration of 12 g/l was reached with a ratio of 3. Correspondence to: H. Kautola     

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.     

Continuous <Emphasis Type="SmallCaps">d</Emphasis>-lactic acid production by a novelthermotolerant <Emphasis Type="Italic">Lactobacillus delbrueckii</Emphasis> subsp. <Emphasis Type="Italic">lactis</Emphasis> QU 41

We isolated and characterized a d-lactic acid-producing lactic acid bacterium (d-LAB), identified as Lactobacillus delbrueckii subsp. lactis QU 41. When compared to Lactobacillus coryniformis subsp. torquens JCM 1166 ^T and L. delbrueckii subsp. lactis JCM 1248 ^T, which are also known as d-LAB, the QU 41 strain exhibited a high thermotolerance and produced d-lactic acid at temperatures of 50 °C and higher. In order to optimize the culture conditions of the QU 41 strain, we examined the effects of pH control, temperature, neutralizing reagent, and initial glucose concentration on d-lactic acid production in batch cultures. It was found that the optimal production of 20.1 g/l d-lactic acid was acquired with high optical purity (>99.9% of d-lactic acid) in a pH 6.0-controlled batch culture, by adding ammonium hydroxide as a neutralizing reagent, at 43 °C in MRS medium containing 20 g/l glucose. As a result of product inhibition and low cell density, continuous cultures were investigated using a microfiltration membrane module to recycle flow-through cells in order to improve d-lactic acid productivity. At a dilution rate of 0.87 h⁻¹, the high cell density continuous culture exhibited the highest d-lactic acid productivity of 18.0 g/l/h with a high yield (ca. 1.0 g/g consumed glucose) and a low residual glucose (<0.1 g/l) in comparison with systems published to date.     

Influence of Glucose and Saturated Free-Fatty Acid Mixtures on Citric Acid and Lipid Production by Yarrowia lipolytica

In the present report, the effect of glucose and stearin (substrate composed by saturated free-fatty acids) on the production of biomass, reserve lipid, and citric acid by Yarrowia lipolytica ACA-DC 50109 was investigated in nitrogen-limited cultures. Numerical models that were used in order to quantify the kinetic behavior of the above Yarrowia lipolytica strain showed successful simulation, while the optimized parameter values were similar to those experimentally measured and the predictive ability of the models was satisfactory. In nitrogen-limited cultures in which glucose was used as the sole substrate, satisfactory growth and no glucose inhibition occurred, although in some cases the initial concentration of glucose was significantly high (150 g/l). Citric acid production was observed in all trials, which was in some cases notable (final concentration 42.9 g/l, yield 0.56 g per g of sugar consumed). The concentration of unsaturated cellular fatty acids was slightly lower when the quantity of sugar in the medium was elevated. In the cases in which stearin and glucose were used as co-substrates, in spite of the fact that the quantity of cellular lipid inside the yeast cells varied remarkably (from 0.3 to 2.0 g/l – 4 to 20% wt/wt), de novo fatty acid biosynthesis was observed. This activity increased when the yeast cells assimilated higher sugar quantities. The citric acid produced was mainly derived from the catabolism of sugar. Nevertheless, citric acid yield on sugar consumed and citrate specific production rate, as evaluated by the numerical model, presented substantially higher values in the fermentation in which no fat was used as glucose co-substrate compared with the cultures with stearin used as co-substrate.     

Diversity of the Carbohydrate Composition of the Antigenic Polysaccharides of Proteobacteria of the Genera Pseudoalteromonasand Marinomonas

The sugar analysis of the glycans of the type strains of marine proteobacteria of the genera Pseudoalteromonasand Marinomonas—Pseudoalteromonas atlanticaIAM12927^T, P. aurantiaNCIMB 2033^T, P. citreaATCC 29719^T, P. elyakoviiKMM 162^T, P. espejianaATCC 29659^T, P. piscicidaNCIMB 645^T, P. tetraodonisIAM 14160^T, Marinomonas communisATCC 27118^T, and M. vagaATCC 27119^T—showed that they contain glucose, galactose, galactosamine, glucosamine, fucose, rhamnose, mannose, heptose, 2-keto-3-deoxyoctonate (KDO), uronic acids, colitose (3,6-dideoxy-L-xylo-hexose), and 6-deoxy-L-talose. The carbohydrate composition of the antigenic polysaccharides (PSs) of P. elyakoviiKMM 162^Tand P. espejianaATCC 29659^Tdepended on the type and the concentration of carbohydrate substrates in the nutrient media. The molar proportion between rhamnose, glucose, and galactose (ca. 1 : 0.3 : 2) in the PS of P. elyakoviiKMM 162^Twas almost the same in the media lacking carbohydrates or containing glucose or galactose at a concentration of 1 g/l. At the same time, the molar proportion between fucose, glucose, galactose, galactosamine, and glucosamine (ca. 1 : 1 : 1 : 2 : 0.5) in the PS of P. espejianaATCC 29659^Tdepended on the presence and the concentration of carbohydrate substrates in the medium. A high concentration of glucose in the medium (30 g/l) brought about a rise in the content of glucose in PSs (9-fold for the PS of P. elyakoviiKMM 162^Tand 4.6-fold for the PS of P. espejianaATCC 29659^T) and led to a decrease in the content of other carbohydrates. The cultivation of these two strains at a lactose concentration of 30 g/l resulted in their PSs containing glucose and galactose in about equal proportions (ca. 1 : 1 in the case of P. espejianaATCC 29659^Tand ca. 2.1 : 1.7 in the case of P. elyakoviiKMM 162^T).     

Continuous ethanol production at high glucose concentrations by a passively immobilized Zymomonas mobilis system

Summary The effect of high glucose concentrations on continuous ethanol production by passively immobilized Zymomonas mobilis cells has been studied. High effluent ethanol concentrations always led to low productivities. The maximum ethanol concentration attained was 92.8 g/l (98% glucose conversion) at a dilution rate of 0.14 h^-1 with 200 g/l glucose medium. The observed enhancement of cell immobilization in the fibrous support at high glucose concentrations in the feed input seems to be related to the formation of bacterial filaments.Preliminary results from this work were previously presented at the Second Spanish Conference on Biotechnology (Barcelona, 1988)     

Influence of some metal ions on the lipid content and arachidonic acid production byMortierella sp.

The influence of Ca²⁺, Mg²⁺, Mn²⁺, and Fe²⁺ ions on lipid accumulation, fatty acid composition and arachidonic acid (ARA) production byMortierella sp. S-17 was investigated. A beneficial effect of Mn²⁺ in the concentration range of 2–500 mg/L on lipogenesis was observed. The other elements at about 1 g/L repressed lipid accumulation and ARA yield. The highest yield of ARA (723 mg per liter or 148 mg per gram of dry mycelium) after incubation of the fungus in a glucose medium in the presence of 2 mg Mn²⁺ per liter was obtained. A strong inhibitory effect of Fe²⁺ (above 40 mg/L) on ARA formation was observed.     

Citric acid production by Yarrowia lipolytica: Effect of nitrogen and biomass concentration on yield and productivity

Summary Citric acid yields of Yarrowia lipolytica (NRRL Y-1095) grown on glucose ranged from 0.38–0.77 g/g and were dependent on both biomass and nitrogen concentration (as NH₄Cl and yeast extract). Increasing the biomass concentration by 3% (w/v) increased fermentor productivities from 0.6 to 1.22 g citric acid/L h.     

Action of Cu2+ on Bacillus thuringiensis Growth Investigated by Microcalorimetry

By using an LKB-2277 Bioactivity Monitor, ampoule mode, the heat output of Bacillus thuringiensis growth metabolism is determined at 28°C and the effect of Cu²⁺ on B. thuringiensis growth is studied. Copper is regarded as an essential trace element for life. Its deficiency may be the cause of diseases. Cu²⁺ at different concentrations has different effects on B. thuringiensis growth metabolism: a low concentration (0–30 g/ml) of Cu²⁺ can promote the growth of B. thuringiensis, a high concentration (40–120 g/ml) can inhibit growth of the bacteria, and a concentration of Cu²⁺ of up to 130 g/ml completely inhibits B. thuringiensis growth.     

Production and partial purification of naringinase by Penicillium decumbens PTCC 5248

Penicillium decumbens PTCC 5248 produced naringinase when grown in a medium contained naringin as a source of carbon. Rhamnose also induced production of naringinase. Prunin disappeared as the time of enzymatic reaction increased. On fractionation with isopropanol 24-fold purification was achieved. Optimum pH and temperature for naringinase activity were determined to be 4.5 and 55 °C respectively. The K_m value of the enzyme with respect to naringin was found to be 1.7 mM. Citric acid, glucose, Ca²⁺, Mg²⁺, Zn²⁺ all inhibited naringinase activity.     

Overproduction of poly(β-malic acid) (PMA) from glucose by a novel Aureobasidium sp. P6 strain isolated from mangrove system

After over 100 strains of Aureobasidium spp isolated from mangrove system were screened for their ability to produce poly(β-malic acid) (PMA), it was found that Aureobasidium sp. P6 strain among them could produce high level of Ca²⁺-PMA. Fourteen percent glucose and 6.5 % CaCO₃ in the medium were the most suitable for Ca²⁺-PMA production. Then, 100.7 g/l of Ca²⁺-PMA was produced using Aureobasidium sp. P6 strain within 168 h at flask level. During 10-l batch fermentation, when the medium contained 12.0 % glucose, 98.7 g/l of Ca²⁺-PMA in the culture and 14.7 g/l of cell dry weight were obtained within 156 h, leaving 0.34 % reducing sugar in the fermented medium. When glucose concentration in the fermentation medium was 14.0 %, 118.3 g/l of Ca²⁺-PMA in the culture and 16.4 g/l of cell dry weight were obtained within 168 h, leaving 0.4 % reducing sugar in the fermented medium. After purification of Ca²⁺-PMA from the culture and acid hydrolysis of the pure Ca²⁺-PMA, analysis of HPLC showed that Aureobasidium sp. P6 strain only produced two main components of Ca²⁺-PMA and minor amount of calcium malate and that the hydrolysate of PMA was mainly composed of calcium malate. This is the first time to report that the novel yeast strain Aureobasidium sp. P6 strain isolated from the mangrove systems can produce such high amount of Ca²⁺-PMA.     

Effect of calcium on synthesis of dipicolinic acid inPenicillium citreoviride and its feedback resistant mutant

Dipicolinic acid synthesis inPenicillium citreoviride strain 3114 was inhibited by Ca²⁺ ions, but not by Ba²⁺, Cu²⁺or Fe²⁺. Among the metals tested, only Zn²⁺ inhibited the synthesis of dipicolinic acid and promoted sporulation. None of these metals reversed the inhibition by Ca²⁺ or Zn²⁺. A mutant 27133-dpa-ca selected for resistance to feedback inhibition by dipicolinic acid: Ca²⁺ complex showed cross-resistance to inhibition by dipicolinic acid: Zn²⁺. Both 3114 and271 33-dpa-ca excreted a number of aliphatic and amino acids during secondary metabolism of dipicolinic acid. In the presence of 1000 ppm of Ca²⁺, accumulation of citric acid and α-aminoadipic acid was completely inhibited under conditions of inhibition of dipicolinic acid in parent strain 3114 but not in the mutant. Citric acid with or without Ca²⁺ did not inhibit thede novo synthesis of dipicolinic acid in the strain 3114. In fact, citric acid in the presence of Ca²⁺ improved significantly rate of dipicolinic acid synthesis. Apart from resistance to feed back inhibition by dipicolinic acid: Ca²⁺ complex, mutant differed from the parent in three other aspectsviz. (i) dipicolinic acid synthesis was not subject to catabolite repression by glucose, (ii) sporulation as well as dipicolinic acid synthesis was dependent on the presence of Ca²⁺ ions in the medium and (iii) Mg²⁺ requirement for the mutant increased three fold. Higher requirement of the Mg²⁺ could be partially relieved by Ca²⁺ during secondary metabolism. The results support the inference thatde novo synthesis of dipicolinic acid is regulated through feedback inhibition by dipicolinic acid: Ca²⁺complex.     

Non-competitive product inhibition in lactic acid fermentation from glucose

Summary A kinetic study regarding product inhibition in lactic acid fermentation by Streptococcus faecalis, which produces l-lactic acid, was performed in a chemostat at various feed concentrations of glucose (10, 20, and 30 g/l) at pH 7.0. Steady-state kinetic constants for the specific consumption rate of glucose and the specific production rate of lactic acid were determined at a residual glucose concentration below 2 g/l, which was accomplished in a chemostat. All the parameters, the specific growth rate, the specific consumption rate of glucose, and the specific production rate of lactic acid, were definitely related to non-competitive inhibition with regard to the concentration of the product, lactic acid.Offprint requests to: K. Hiyama     

Growth and phycocyanin formation of Spirulina platensis in photoheterotrophic culture

Summary Glucose and acetate enhanced cell growth and phycocyanin production of S. platensis. The highest specific growth rate, cell concentration and phycocyanin production were respectively 0.62 d^-1, 2.66 g/l and 322 mg/l on glucose and 0.52 d^-1, 1.81 g/l and 246 mg/l on acetate. The specific growth rate of the alga on 2.5 g glucose/l was markedly increased with increasing light intensity up to 2 klux. Further increasing light intensity to 4 klux only resulted in a very slight increase in specific growth rate. At a light intensity above 4 klux, photoinhibition occurred. Light favoured phycocyanin formation. The highest phycocyanin content was obtained at a light intensity of 4 klux. When the light intensity decreased to 2 klux or less, the optimal glucose concentration for biomass production shifted from 2.5 g/l to 5.0 g/l.     

Growth and butyric acid production by Clostridium populeti

The growth of Clostridium populeti in 2% (w/v) glucose medium containing 0.2% (w/v) yeast extract was optimal with 10 mM NH₄Cl as the nitrogen source. Although the maximum specific growth rate (=0.32 h^-1) with 5 mM NH₄Cl was similar, the biomass yield was about 30% lower than that at the optimum. Either sodium sulphide or cysteine-HCl at an optimum concentration of 0.33 mM and 5.0 mM respectively, could serve as the sole sulphur source for growth. The growth rate was unaffected by initial glucose concentrations of up to 10% (w/v), but in the presence of 15% glucose it declined by about 35%. The molar yield of butyric acid (mol/mol glucose) declined from 0.70 in 1% (w/v) initial glucose medium to 0.39 in 10% glucose medium. In 5.7% initial glucose medium, butyric acid levels of 6.3 g/l were obtained (0.56 mol butyrate/mol glucose) after 72 h of incubation in 2.5 l batch cultures. A decrease of about 50% in the maximum specific growth rate of C. populeti was observed in the presence of an initial concentration of either 1.2 g/l of butyric acid or 18.9 g/l of acetic acid.This paper is issued as NRCC No. 29032     

Sorption of Copper and Zinc to the Plasma Membrane of Wheat Root

Sorption of Cu²⁺ and Zn²⁺ to the plasma membrane (PM) of wheat root (Triticum aestivum Lcv. Scout 66) vesicles was measured at different pH values and in the presence of organic acids and other metals. The results were analyzed using a Gouy-Chapman-Stem model for competitive sorption (binding and electrostatic attraction) to a negative binding site. The binding constants for the two investigated cations as evaluated from the sorption experiments were 5 M^–1 for Zn²⁺ and 400 M^–1 for Cu²⁺. Thus, the sorption affinity of Cu²⁺ to the PM is considerably larger than that of Ca²⁺, Mg²⁺ or Zn²⁺. The greater binding affinity of Cu²⁺ was confirmed by experiments in which competition with La³⁺ for sorption sites was followed. The amount of sorbed Cu²⁺ decreased with increasing K⁺, Ca²⁺, or La³⁺ concentrations, suggesting that all these cations competed with Cu²⁺ for sorption at the PM binding sites, albeit with considerable differences among these cations in effectiveness as competitors with Cu²⁺. The sorption of Cu²⁺ and Zn²⁺ to the PM decreased in the presence of citric acid or malic acid. Citric acid (as well as pH) affected the sorption of Cu²⁺ or Zn²⁺ to PM more strongly then did malic acid.     

Mass production of poly-β-hydroxybutyric acid by fully automatic fed-batch culture of methylotroph

Summary Fifty-one methylotrophs were checked with respect to their ability of poly--hydroxybutyric acid (PHB) production from methanol. One of them, Pseudomonas sp. K, was chosen from its good growth on a minimum synthetic medium. Optimal temperature and pH for its growth were 30° C and 7.0, respectively. Concentrations of PO ₄ ^3- and NH ₄ ⁺ in the medium should be kept at low levels. PHB formation was stimulated by deficiency of nutrient such as NH ₄ ⁺ , SO ₄ ^2- , Mg²⁺, Fe²⁺ or Mn²⁺. Among them, nitrogen deficiency was chosen from its effectiveness and easiness for PHB accumulation.The microorganism was cultivated to produce a large amount of poly--hydroxybutyric acid (PHB) from methanol by means of microcomputer-aided fully automatic fed-batch culture technique. During the cultivation, temperature, dissolved oxygen concentration (DO), and methanol concentration in the culture broth were maintained at 30° C 2.5±0.5 ppm and 0.5±0.2 g/l, respectively. Other nutrients, nitrogen source and mineral ions, were also controlled to maintain their initial concentrations in the medium during cell growth phase. When the high cell concentration was achieved (160 g/l), feedings of ammonia and minerals were stopped and only methanol was supplied successively to accumulate PHB. At 175 h, high concentration of PHB (136 g/l) was obtained and total cell concentration became 206 g/l. DO must be maintained above the critical level during the PHB formation phase, too. PHB yield from methanol (g PHB/g methanol) was 0.18 and the maximum PHB content reached 66% of dry weight. Solid PHB produced by the strain had the melting point of 176° C and the average molecular weight of 3.0x10⁵.     

Continuous production of itaconic acid by Aspergillus terreus immobilized in a porous disk bioreactor

Summary Aspergillus terreus NRRL 1960 was grown on porous disks rotating intermittently in and out of the liquid phase. This immobilized fungal cell bioreactor was used to produce itaconic acid from glucose in a continuous operation. The effect of temperature, pH, disk rotation speed, and feed rate on the itaconic acid concentration and volumetric productivity were studied. The highest itaconic acid concentration and volumetric productivity obtained were 18.2 g/l and 0.73 g/l·h, respectively, under the following conditions: temperature at 36°C, pH 3.0, disk rotation speed at 8 rpm, and feed rate at 60 ml/h. These results are better than those by conventional fermentation or by other immobilized method.Nomenclature F feed rate (l/h) - K _1s saturation constant for immobilized cells (g/l) - K _2s saturation constant for suspended cells (g/l) - M ₁ increased mass of immobilized cells (g) - M ₂ total mass of immobilized cells (g) - P concentration of itaconic acid (g/l) - S substrate concentration in and out of the reactor (g/l) - S ₀ substrate concentration in the feed (g/l) - V liquid volume of the reactor (1) - X concentration of the suspended cells (g/l) - Y ₁ apparent yield of the immobilized cells (g cells/g substrate) - Y ₂ apparent yield of the suspended cells (g cell/g substrate) - Y ₃ apparent yield of itaconic acid (g itaconic acid/g substrate) - m ₁ maintenance and by-products coefficient of the immobilized cells (g substrate/g cell·h) - m ₂ maintenance and by-products coefficient of the suspended cells (g substrate/g cell·h) - µ_1max maximum specific growth rate of the immobilized cells (h^-1) - µ_2max maximum specific growth rate of the suspended cells (h^-1)     

Channelling of glucose by methanol for citric acid production from Aspergillus niger

Citric acid produced by Aspergillus niger was increased from 4.6g l^-1 to 7.8gl^-1 by supplementing basal medium with methanol (30mll^-1). While stimulating citric acid production, methanol did not improve membrane permeability of the fungus for citric acid. Methanol inhibited the germination of Aspergillus spores. An increase in glucose concentration from 50gl^-1 to 100gl^-1 in the presence of methanol (30mll^-1) improved citric acid production (1.6-fold) while at higher levels of glucose concentration methanol had no effect on citic acid production.