Influence of a supplementary carbon source on biodegradation of pyridine by freely suspended and immobilizedPimelobacter sp.

The effect of the presence of supplementary glucose or acetate on the growth and pyridine-degrading activity of freely suspended and calcium-alginate-immobilizedPimelobacter sp. was investigated. Although the supplementary carbon sources could be degraded simultaneously with pyridine,Pimelobacter sp. exhibited a preference for pyridine over supplementary carbon sources. Thus, the pyridine-degrading activity of the freely suspended cells was not decreased significantly by the addition of either glucose (1.5–6 mM) or acetate (6–24 mM) to the pyridine (6–24 mM). In the semi-continuous immobilized cell culture, immobilized cells also exhibited a preference for pyridine over supplementary carbon sources and did not switch their substrate preference throughout the culture. Owing to a high cell concentration, the volumetric pyridine degradation rate at 24 mM pyridine in the immobilized cell culture was approximately six times higher than that in the freely suspended cell culture. Furthermore, the immobilized cells could be reused 16 times without losing their pyridine-degrading activity during the culture period tested. Taken together, the use of immobilizedPimelobacter sp. for the degradation of pyridine is quite feasible because of the preference for pyridine over supplementary carbon sources, the high volumetric pyridine degradation rate, and the reusability of immobilized cells.     

Biodegradation of pyridine by freely suspended and immobilized Pimelobacter sp.

Freely suspended and Ca-alginate-immobilized cells of Pimelobacter sp. were used for degradation of pyridine. When the pyridine concentration was up to 2 g l^–1, freely suspended cells completely degraded pyridine regardless of the initial cell concentrations used. However, when the pyridine concentration increased to 4 g l^–1, the initial cell concentration in freely suspended cell culture should be higher than 1.5 g dry cell weight l^–1 for complete degradation of pyridine. In addition, a freely suspended cell culture with a high initial cell concentration resulted in a high volumetric pyridine-degradation rate, suggesting the potential use of immobilized cells for pyridine-degradation. When the immobilized cells were used for pyridine-degradation, neither specific pyridine-degradation rate nor tolerance against pyridine was improved. However, a high volumetric pyridine-degradation rate in the range 0.082–0.129 g l^–1 hr^–1 could be achieved by the immobilized cells because of the high cell concentration. Furthermore, when the immobilized cells were reused in degrading pyridine at a concentration of 2–4 g l^–1 they did not lose their pyridine-degrading activity for 2 weeks. Taken together, the data obtained here showed the feasibility of using immobilized cells for pyridine-degradation.     

Production of mannitol by a novel strain of Candida magnoliae

A novel microorganism was isolated which is able to produce mannitol when grown in the presence of fructose and glucose as carbon sources. In flask culture in a medium containing 150 g fructose l^–1, it yielded 67 g mannitol l^–1 after 168 h. In fed-batch culture with 3–12% (w/v) fructose, production reached a maximum of 209 g mannitol l^–1 after 200 h, corresponding to an 83% yield and a 1.03 g l^–1 h^–1 productivity. The isolated strain was identified as Candida magnoliae based on identical sequences in the D1/D2 domain of its 26S rDNA and a similar carbon source utilization pattern with C. magnoliae reference strains.     

The growth of Geotrichum candidum on whiskey distillery spent wash

Summary A strain of the imperfect fungus Geotrichum candidum was selected for its ability to utilize the low-pH, high biochemical oxygen demand (BOD) waste generated by an Irish malt whiskey distillery. Growth of the organism on this complex medium, in both batch and continuous culture, showed a sequential assimilation of the major components with the most complete carbon assimilation (63.7%) being achieved in batch culture. Optimum temperature for the continuous culture of the organism was found to be 22°C; at this temperature and at a dilution rate of 0.125 h^–1 a productivity of 2.24 g l^–1 h^–1 was obtained. Variations in organism morphology, produced by varying growth rate and nutritional or environmental factors, as well as the potential of the process for the production of single cell protein from carbohydrate-rich waste are discussed.     

Induction of trehalase activity on a nitrogen-free medium: a sporulation-specific event in the fission yeast, Schizosaccharomyces pombe

Summary Kinetic experiments with synchronously sporulating cultures of a homothallic h⁹⁰ strain of Schizosaccharomyces pombe showed that trehalase activity abruptly increased in the late sporulation process, coinciding with the appearance of visible spores. Trehalase activity was absent in vegetative cells. A set of strains different in genetic constitution at the mating type loci was tested for induction of trehalase on nitrogen-free sporulation medium. The appearance of trehalase activity on the sporulation medium was observed only in sporulating cultures; cultures of homothallic strains (h⁹⁰) and diploid strains heterozygous for mating type (h⁺/h^–), and mixed cultures of heterothallic h⁺ and h^– strains. Trehalase activity was not induced in nonsporogenic strains: heterothallic haploid strains (h⁺ and h^–), diploid strains homozygous for mating type (h⁺/h⁺ and h^–/h^–) and the homothallic strain harboring the mutation in the mat2 gene, which was unable to undergo the first meiotic division. Trehalose accumulation on the sporulation medium was observed solely in the sporulating cultures. These results led us to conclude that the induction of trehalase activity as well as the accumulation of trehalose in the medium lacking nitrogen sources was a sporulation-specific event under the control of the mating type genes.     

Kinetic analysis of ethanol production by an acetate-resistant strain of recombinant Zymomonas mobilis

Zymomonas mobilis ZM4/Ac^R (pZB5), a mutant recombinant strain with increased acetate resistance, has been isolated following electroporation of Z. mobilis ZM4/Ac^R. This mutant strain showed enhanced kinetic characteristics in the presence of 12 g sodium acetate l^–1 at pH 5 in batch culture on 40 g glucose, 40 g xylose l^–1 medium when compared to ZM4 (pZB5). In continuous culture, there was evidence of increased maintenance energy requirements/uncoupling of metabolism for ZM4/Ac^R (pZB5) in the presence of sodium acetate; a result confirmed by analysis of the effect of acetate on other strains of Z. mobilis. Nomenclature m Cell maintenance energy coefficient (g g^–1 h^–1)Maximum overall specific growth rate (1 h^–1)Maximum specific ethanol production rate (g g^–1 h^–1)Maximum specific total sugar utilization rate (g g^–1 h^–1)Biomass yield per mole of ATP (g mole^–1 Ethanol yield on total sugars (g g^–1)Biomass yield on total sugars (g g^–1)True biomass yield on total sugars (g g^–1)     

Morphology,physiology and infectivity of twoFrankia isolates An 1 and An 2 from root nodules ofAlnus nitida

Summary Two different strains, An 1 and An 2, were obtained from root nodules ofAlnus nitida Endl., collected from one locality in the area of its natural habitat near Bahrin, District Swat, Pakistan. The light and electron microscopy of the isolates revealed the occurrence of septate and branched hyphae bearing sporangia and vesicles. The strains differed in their growth requirements, nitrogen-fixing ability and production of extracellular pigments, thus indicating the existence of more than oneFrankia strain in the same locality. In the absence of combined nitrogen in the medium strain An 1 formed vesicles and fixed N₂ (up to 200 nmol C₂H₄. mg protein^–1.h^–1), while strain An 2 under the experimental conditions formed only few vesicles and fixed N₂ at a very low rate (ca 10 nmol C₂H₄. mg protein^–1 .h^–1). The nitrogenase activity of strain An 1 was strongly affected by the O₂ concentration.Frankia An 1 and An 2 were infective and effective onA. nitida andA. glutinosa but not onDatisca cannabina andElaeagnus umbellata. Both An 1 and An 2 strains were more infective and effective onA. glutinosa thanFrankia strains AvcIl and CpI1.     

Purification and properties of p-hydroxybenzoate hydroxylases from Rhodococcus strains

Gram-positive bacteria of the genus Rhodococcus catabolize p-hydroxybenzoate (PHB) through the initial formation of 3,4-dihydroxybenzoate. High levels of p-hydroxybenzoate hydroxylase (PHBH) activity are induced in six different Rhodococcus species when these strains are grown on PHB as sole carbon source. The PHBH enzymes were purified to apparent homogeneity and appeared to be homodimers of about 95 kD with each subunit containing a relatively weakly bound FAD. In contrast to their counterparts from gram-negative microorganisms, the Rhodococcus PHBH enzymes prefer NADH to NADPH as external electron donor. All purified enzymes were inhibited by Cl^– and for five of six enzymes more pronounced substrate inhibition was observed in the presence of chloride ions.     

Morphological,Physiological, and Molecular Characterization of a Newly Isolated Steroid-Degrading Actinomycete,Identified as <Emphasis Type="Italic">Rhodococcus ruber</Emphasis> Strain Chol-4

The aerobic degradation of cholesterol, testosterone, androsterone, progesterone, and further steroid compounds as sole carbon source has been observed in the newly isolated bacterial Gram-positive strain Chol-4. The 16S rRNA gene sequence shares the greatest similarity with members of the genus Rhodococcus, with the closest shared nucleotide identities of 98–99% with Rhodococcus ruber (DSM 43338^T) and Rhodococcus aetherivorans (DSM 44752^T). Phylogenetic analysis of Rhodococcus 16S rRNA gene sequences consistently places strain Chol-4 in a clade shared with those both type strains within the Rhodococcus rhodochrous subclade. The results of DNA–DNA hybridization against its two phylogenetically closest neighbors as well as the results of morphological, physiological, and biochemical tests allowed genotypic and phenotypic differentiation of strain Chol-4 from Rhodococcus ruber (DSM 43338^T) on the species level and from the other validly described Rhodococcus species on the genus level. Strain Chol-4 therefore merits recognition as a novel strain of the species Rhodococcus ruber and demonstrates for the first time the capability of this species to utilize a great variety of steroid compounds as growth substrates never shown for other species of this genus so far. The genome of strain Chol-4 harbors at least one gene cluster that may be responsible for the degradation of steroid compounds. This gene cluster was identified in a cloned 5458 bp BamHI–EcoRV DNA fragment and compared to similar genes from other Gram-positive and Gram-negative bacteria described so far.     

Biofiltration of waste gases with the fungi Exophiala oligosperma and Paecilomyces variotii

Two biofilters fed toluene-polluted air were inoculated with new fungal isolates of either Exophiala oligosperma or Paecilomyces variotii, while a third bioreactor was inoculated with a defined consortium composed of both fungi and a co-culture of a Pseudomonas strain and a Bacillus strain. Elimination capacities of 77 g m^–3 h^–1 and 55 g m^–3 h^–1 were reached in the fungal biofilters (with removal efficiencies exceeding 99%) in the case of, respectively, E. oligosperma and Paecilomyces variotii when feeding air with a relative humidity (RH) of 85%. The inoculated fungal strains remained the single dominant populations throughout the experiment. Conversely, in the biofilter inoculated with the bacterial–fungal consortium, the bacteria were gradually overgrown by the fungi, reaching a maximum elimination capacity around 77 g m^–3 h^–1. Determination of carbon dioxide concentrations both in batch assays and in biofiltration studies suggested the near complete mineralization of toluene. The non-linear toluene removal along the height of the biofilters resulted in local elimination capacities of up to 170 g m^–3 h^–1 and 94 g m^–3 h^–1 in the reactors inoculated, respectively, with E. oligosperma and P. variotii. Further studies with the most efficient strain, E. oligosperma, showed that the performance was highly dependent on the RH of the air and the pH of the nutrient solution. At a constant 85% RH, the maximum elimination capacity either dropped to 48.7 g m^–3 h^–1 or increased to 95.6 g m^–3 h^–1, respectively, when modifying the pH of the nutrient solution from 5.9 to either 4.5 or 7.5. The optimal conditions were 100% RH and pH 7.5, which allowed a maximum elimination capacity of 164.4 g m^–3 h^–1 under steady-state conditions, with near-complete toluene degradation.     

Degradation of sodium anthraquinone sulphonate by free and immobilized bacterial cultures

Aerobic biodegradation of a xenobiotic recalcitrant compound sodium anthraquinone-2-sulphonate (SAS), was investigated using as an inoculum a mixed microbial culture, which was activated sludge from industrial and domestic waste-water treatment plants. The difference in SAS degradation was examined using two main systems: (1) suspended cells and (2) immobilized cells, both in batch and in continuous culture. In the suspended cell system, under continuous culture conditions using SAS as a unique source of carbon and energy, it was possible to degrade about 95% of this substrate after 6 days. Maximal SAS removal rates in the suspended-cell system were 593 mg SAS l^–1 h^–1 and 88.7 mg SAS l^–1 h^–1 for dilution rates (D) of 0.05 h^–1 and 0.075 h^–1, respectively. In the immobilized-cell system, almost all SAS was degraded in 6 days and the maximal removal rate reached 88.7 mg SAS l^–1 h^–1 at D=0.05 h^–1. Application of a continuous-flow enrichment procedure resulted in selection of several kinds of micro-organisms and led to a progressive elimination of some species of Aeromonas. A stable microbial community of 11 strains has been established and characterized at D=0.075 h^–1. Most of them were Gram-negative and belonged to the genus Pseudomonas.     

The isolation and characterisation of a salicylate-hydroxylase-producing strain of Pseudomonas putida

Summary A salicylate-hydroxylase-producing strain of Pseudomonas putida with an unusual capability to grow at toxic levels of salicylate up to 10 g l^–1 has been isolated. It grew well under continuous culture conditions, with optimum growth at pH 6.5 and a temperature of 25° C. The use of an ammonium salt as a nitrogen source, instead of nitrate, resulted in a 30–40% increase in its biomass yield coefficient. Optimum growth under continuous culture conditions was achieved using 4 g l^–1 salicylate at 25° C, pH 6.5 and 0.2 h^–1 dilution rate. High salicylate hydroxylase enzyme activity [236 units (U) l^–1] and productivity (424.8 U h^–1) were obtained at a dilution rate of 0.45 h^–1 using a mineral medium containing 4 g l^–1 of salicylate. Operating under continuous culture conditions with oxygen limitation and a slight accumulation of residual salicylate (0.2 g l^–1) resulted in a decrease in culture performance and enzyme productivity. Correspondence to: R. Marchant     

High efficiency styrene biodegradation in a biphasic organic/water continuous reactor

Styrene was degraded as sole source of carbon and energy by a selected bacterial community in a two-phase aqueous-organic medium (80%:20%, vol/vol). Silicone oil was used to solubilize styrene, which is sparingly soluble in water and to prevent its toxicity toward microorganisms. Preliminary studies with the mixed population in batch cultures indicate that the specific activity and the maximum growth rate at optimal 3H 6.0 were 46 mg·g^–1·h^–1 and 0.15 h^–1, respectively. In pH-regulated chemostat cultures, styrene was degraded at dilution rates ranging from 0.05 to 0.20 h^–1. Kinetic parameters and the proportion of each strain in the mixed culture were followed. At 0.20 h^–1, only one strain as compared to four initially present, remained in the medium. This strain Pseudomonas aeruginosa, degrades styrene with a specific activity of 293 mg·g^–1·h^–1. Such results could lead to industrial treatment of waste gas or water polluted with styrene. Correspondence to: J,-M. Lebeault     

Production of cholesterol oxidase by a newly isolated Rhodococcus sp.

Fifteen strains of microorganisms with ability to degrade cholesterol were isolated. Among them a Gram-positive, non-motile, non-sporing bacterium with meso-DAP in the cell wall and with a rod-coccus cycle showed the highest ability for cholesterol degradation. It was identified as Rhodococcus sp. strain 2C and was deposited by code 1633 in Persian type culture collection (PTCC). This strain was able to produce high levels of both extracellular and cell-bound cholesterol oxidases in media containing cholesterol as a sole carbon source. The effects of medium composition and physical parameters on cholesterol oxidase production were studied. The optimized medium was found to contain cholesterol 0.15% (w/v), yeast extract 0.3% (w/v), diammonium hydrogen phosphate 0.1% (w/v), Tween 80 (0.05%). The optimum pH and temperature for cholesterol oxidase production in optimized medium were found to be 8–30 °C respectively. Triton X-100 showed the greatest effect in releasing the cell-bound enzyme. The first and most probably the main metabolite of cholesterol degradation was purified and identified as 4-cholestene-3-one.     

Production of poly-γ-glutamic acid by fed-batch culture of Bacillus licheniformis

Fed-batch cultures of Bacillus licheniformis produced poly--glutamic acid (PGA), a water-soluble biodegradable polymer. PGA reached 35 g l^–1 with a productivity of 1 g l^–1 h^–1 by pulsed-feeding of citric acid (1.44 g h^–1) and l-glutamic acid (2.4 g h^–1) when citric acid was depleted from the culture medium.     

Anaerobic,alkaliphilic, saccharolytic bacterium <Emphasis Type="Italic">Alkalibacter saccharofermentans</Emphasis> gen. nov., sp. nov. from a soda lake in the Transbaikal region of Russia

Three strains of new obligately anaerobic alkaliphilic bacteria have been isolated as a saccharolytic component from the cellulolytic community of alkaline Lake Nizhnee Beloe (Transbaikal region, Russia), a lake with low salt concentration. DNA analysis of these strains showed an interspecies level of DNA similarity of 96–100%. Strain Z-79820 was selected for further investigations. Cells were Gram-positive, asporogenous, nonmotile short rods with pointed ends. The strain was a true alkaliphile: growth occurred from pH 7.2 to 10.2 with the optimum at pH 9.0. Strain Z-79820 was halotolerant and could grow in medium with up to 10% (w/v) NaCl, with the optimum between 0 and 4% NaCl. The new isolate obligately depended on Na⁺ ions in the form of carbonates or chlorides. Total Na⁺ content needed for optimal growth was 0.46 M Na⁺, with a wide range from 0.023–0.9 M Na⁺ at which growth also occurred. The isolate was a mesophile and grew at temperatures from 6 to 50°C (slow growth at 6 and 15°C) with an optimum at 35°C. The organotrophic organism fermented ribose, xylose, glucose, mannose, fructose, sucrose, mannitol, and peptone. The products of glucose fermentation were acetate, ethanol, formate, H₂, and CO₂. Yeast extract was required for some anabolic needs. The DNA G+C content of the type strain Z-79820 was 42.1 mol%. The new bacterium fell into the 16S rRNA gene cluster XV of the Gram-positive bacteria with low G+C content, where it formed an individual branch. Based on its growth characteristics and genotype traits, we propose the new genus and species named Alkalibacter saccharofermentans with the type strain Z-79820 (=DSM14828), Uniqem-218 (Institute Microbiology, RAS; ).     

Mechanisms for tolerance to diatomaceous earth between strains of Tribolium castaneum

Fourteen strains of Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) had mortalities ranging from 5 to 100% when exposed to diatomaceous earth at 600 ppm for seven days. The most tolerant strain had a lethal dose for 50% of the population (LD₅₀) of 413 ppm and the most susceptible strain had a LD₅₀ of 238 ppm. Adults of the tolerant strain were lighter (2.0 mg) than the susceptible strain (2.6 mg). Tolerant adults lost water at lower rate (6 g h^–1 than susceptible adults (12 g h^–1), when held in wheat treated with 600 ppm diatomaceous earth for 24 h, than held at 5% r.h. with no food. Tolerant adults that were not exposed to diatomaceous earth lost water at a lower rate (3 g h^–1) than susceptible adults (5 g h^–1). Both strains, exposed and not exposed to diatomaceous earth died when their water content was between 33 and 37% of their total weight. Insects taken directly from the cultures had 52% (tolerant) and 53% (susceptible) of their total weight as water. Tolerant adults moved slower through grain and across filter paper than susceptible adults. Tolerant adults avoided wheat treated with diatomaceous earth at concentrations as low as 75 ppm, whereas the adults from the susceptible strain did not avoid diatomaceous earth, even at 600 ppm. The consequences of a strain tolerant to diatomaceous earth is discussed with respect to the use of diatomaceous earth to control stored-product insect infestations.     

Reduction of chromate by microorganisms isolated from metal contaminated sites of Karachi, Pakistan

Three bacterial strains, two identified as Pseudomonas stutzeri and one as a strain of cucurbit yellow vine disease bacterium, isolated from a foundry soil and a tannery, respectively, in Pakistan, were resistant to up to 1 mM chromate and anaerobically reduced Cr(VI) up to 100 M. The highest removal was by P. stutzeri CMG463: 88 mol l^–1 (88% of that supplied; specific rate was 3.0 nmol mg^–1 protein h^–1), while 58 and 76 mol l^–1 (58% and 76%) were removed by P. stutzeri CMG462 and cucurbit yellow vine disease bacterium CMG480, respectively. These isolates were compared to strains isolated from an uncontaminated coastal site in the UK and designated as K2 (Pseudomonas synxantha) K3 (Bacillus sp.), and J3 (unidentified Gram-positive strain). Strain K3 was Cr-sensitive, partially lysed by Cr(VI), but had the highest removal of chromate anaerobically: 92 mol l^–1 (92% of that supplied) at a specific rate of 71 nmol mg^–1 protein h^–1. Analysis of cell sections using transmission electron microscopy with energy dispersive X-ray analysis showed intracellular chromium in P. stutzeri but the cucurbit yellow vine disease bacterium and the Bacillus sp. precipitated chromium extracellularly. The isolates from the Cr-contaminated sites did not remove more Cr(VI), overall, than Cr-unstressed bacteria, but their tolerance to Cr(VI) is potentially useful for bioremediation, particularly since other studies have shown that the two P. stutzeri strains can bioaccumulate Cu²⁺.     

Poly-3-hydroxybutyrate (P3HB) production by bacteria from xylose,glucose and sugarcane bagasse hydrolysate

Fifty-five bacterial strains isolated from soil were screened for efficient poly-3-hydroxybutyrate (P3HB) biosynthesis from xylose. Three strains were also evaluated for the utilization of bagasse hydrolysate after different detoxification steps. The results showed that activated charcoal treatment is pivotal to the production of a hydrolysate easy to assimilate. Burkholderia cepacia IPT 048 and B. sacchari IPT 101 were selected for bioreactor studies, in which higher polymer contents and yields from the carbon source were observed with bagasse hydrolysate, compared with the use of analytical grade carbon sources. Polymer contents and yields, respectively, reached 62% and 0.39 g g^–1 with strain IPT 101 and 53% and 0.29 g g^–1 with strain IPT 048. A higher polymer content and yield from the carbon source was observed under P limitation, compared with N limitation, for strain IPT 101. IPT 048 showed similar performances in the presence of either growth-limiting nutrient. In high-cell-density cultures using xylose plus glucose under P limitation, both strains reached about 60 g l^–1 dry biomass, containing 60% P3HB. Polymer productivity and yield from this carbon source reached 0.47 g l^–1 h^–1 and 0.22 g g^–1, respectively.     

Accumulation of astaxanthin and lutein in<Emphasis Type="Italic"> Chlorella zofingiensis</Emphasis> (Chlorophyta)

When grown photoautotrophically, Chlorella zofingiensis strain CCAP 211/14 accumulates a significant amount of valuable carotenoids, namely astaxanthin and lutein, of increasing demand for use as feed additives in fish and poultry farming, as colorants in food, and in health care products. Under standard batch-culture conditions, this microalgal strain exhibits high values of both growth rate (about 0.04 h^–1) and standing cell population (over 10¹¹ cells l^–1, or 7 g dry weight l^–1). Lutein, in a free (unesterified) form, was the prevalent carotenoid during early stages of cultivation (over 0.3 pg cell^–1, equal to 4 mg g^–1 dry weight, or 20 mg l^–1 culture), whereas esterified astaxanthin accumulated progressively, to reach a maximum (over 0.1 pg cell^–1, equal to 1.5 mg g^–1 dry weight, or 15 mg l^–1 culture) in the late stationary phase. A differential response of lutein and astaxanthin accumulation was also recorded with regard to the action of some environmental and nutritional factors. C. zofingiensis CCAP 211/14 represents a unique model system for analyzing the differential regulation of the levels of primary (lutein) and secondary (astaxanthin) carotenoids. Relevant also from the biotechnological viewpoint, this photosynthetic organism, with outstanding attributes for fast photosynthetic growth and carotenoid accumulation, might prove most valuable for its application to the mass production of either or both lutein and astaxanthin.