Long-term survival of the plant-growth-promoting bacteria Azospirillum brasilense and Pseudomonas fluorescens in dry alginate inoculant

Two plant-growth-promoting bacteria, Azospirillum brasilense Cd and Pseudomonas fluorescens 313, immobilized in 1983 in two types of alginate-bead inoculant (with and without skim-milk supplement) and later dried and stored at ambient temperature for 14 years, were recovered in 1996. The population in each type of bead had decreased, yet significant numbers survived (10⁵–10⁶ cfu/g beads). Population numbers depended on the bead type and the three independent bacterial counting methods: the conventional plate-count method, indirect enzyme-linked immunosorbent assay and the limited-enrichment technique. Both bacterial species retained several of their original physiological features. When inoculated onto wheat plants, both species colonized and produced plant-growth effects equal to those of the contemporary strain from a culture collection or to their own 1983 records. This study showed that bacteria can survive in alginate inoculant over long periods. Received: 1 May 1998 / Received revision: 24 August 1998 / Accepted: 3 September 1998     

Bioconversion of limonene to α-terpineol by immobilized Penicillium digitatum

Bioconversion of (4R)-(+)-limonene to (4R)-(+)-α-terpineol by immobilized fungal mycelia of Penicillium digitatum was investigated in batch, repeated-batch and continuously fed systems. The fungi were immobilized in calcium alginate beads. These beads remained active for at least 14 days when they were stored at 4 °C. Three different aeration rates were tested. The highest yield was obtained at a dissolved oxygen level of 50.0 μmol/l. α-Terpineol production by this fungus was 12.83 mg (g beads)⁻¹ day⁻¹, producing a 45.81% bioconversion of substrate. Repeated-batch bioconversion showed yield decreases in the second and the third cycles. Regeneration with nutrient media after the third cycle improved the bioconversion yields. With continuous bioconversion, the half-life was dependent on the aeration. The optimum conditions with a continuous reactor were at an aeration rate of 0.3 standard l/min and a dilution rate of 0.0144 h⁻¹. Received: 10 June 1997 / Received revision: 18 August 1997 / Accepted: 11 September 1997     

Reactions of pentachlorophenol with laccase from Coriolus versicolor

Laccase, purified from Coriolus versicolor, removed pentachlorophenol (PCP) from solution at pH 5, depending on initial PCP concentration and amount of laccase. With 100 units of laccase, 100% of 25 μg ml⁻¹ PCP and 60% of 200 μg ml⁻¹ PCP were removed respectively over 72 h. No free chloride was released in the reaction. In reaction with 100 μg PCP, products were primarily polymers (about 80,000 MW) with only 2–3 pg of o- and p-chloranils formed. Polymers were stable to acid hydrolysis and no release of PCP, or other low-molecular-weight products, was detected over several weeks. Laccase has a potential use in the biotreatment of aqueous effluents containing PCP, with polymerised products being removed from solution due to their high molecular weight. Received: 7 June 1999 / Received revision: 18 August 1999 / Accepted: 2 September 1999     

Effect of phosphate and oxygen concentrations on alginate production and stoichiometry of metabolism of Azotobacter vinelandii under microaerobic conditions

Alginate production by Azotobacter vinelandii was studied in batch and continuous cultures under microaerobic conditions. In batch culture at a pO₂ of 2–3% (air saturation) alginate production was enhanced by decreasing the PO³⁻ ₄ level in the medium. Alginate yield from biomass (Y _P/X) reached the highest value of 0.66 g/g at the lowest phosphate level (100 mg/l), compared to 0.40 g/g and 0.25 g/g at higher phosphate levels (200 mg/l and 400 mg/l, respectively). In contrast, biomass formation behaved differently and the growth yield (Y _X/S) decreased with decreasing PO₄ ³⁻ concentrations. Moreover, the respiratory quotient (RQ) of the culture was dependent on the initial phosphate concentration, especially in the phosphate-limited phase of growth. As the initial phosphate level decreased from 400 mg/l to 100 mg/l, the average RQ value of the culture declined from 1.46 to 0.89. The low RQ value is very close to the theoretical optimum RQ, calculated to be 0.8 on the basis of the stoichiometry of the metabolic pathways for alginate formation from sucrose. This optimum RQ was also confirmed in continuous culture at different dilution rates. Independent of the dilution rate, a pO₂ value of 2–5% (air saturation) was found to be optimal for alginate production, the corresponding RQ values being 0.80–0.84. In addition, the molecular mass and composition of alginate were also found to be affected by both phosphate and oxygen concentrations. In conclusion, the RQ appears to be a useful parameter for optimum control of alginate production with this microorganism. Received: 31 March 1999 / Received revision: 2 July 1999 / Accepted: 5 July 1999     

Glucoamylase production in batch, chemostat and fed-batch cultivations by an industrial strain of Aspergillus niger

The Aspergillus niger strain BO-1 was grown in batch, continuous (chemostat) and fed-batch cultivations in order to study the production of the extracellular enzyme glucoamylase under different growth conditions. In the pH range 2.5–6.0, the specific glucoamylase productivity and the specific growth rate of the fungus were independent of pH when grown in batch cultivations. The specific glucoamylase producivity increased linearly with the specific growth rate in the range 0–0.1 h⁻¹ and was constant in the range 0.1–0.2 h⁻¹. Maltose and maltodextrin were non-inducing carbon sources compared to glucose, and the maximum specific growth rate was 0.19 ± 0.02 h⁻¹ irrespective of whether glucose or maltose was the carbon source. In fed-batch cultivations, glucoamylase titres of up to 6.5 g l⁻¹ were obtained even though the strain contained only one copy of the glaA gene. Received: 5 May 1999 / Received revision: 7 September 1999 / Accepted: 17 September 1999     

A neutral carrier-based liquid membrane microelectrode for divalent putrescine cations

A new ion-selective liquid membrane microelectrode, based on the neutral carrier 1,1′-bis(2,3-naphtho-18-crown-6), is described that shows the dependence of EMF on the activity of divalent putrescine cations a _Put, with the linear slope s _Put = 26 ± 3 mV/decade (mean ± SD, N = 18), in the range 10⁻⁴–10⁻¹ M at 25 ± 1 °C. Values of potentiometric putrescine cation selectivity coefficients of logK ^Pot _{Put j} (mean ± SD, N) are obtained by the separate solution method for the ions K⁺ (1.0 ± 0.4, 10), Na⁺ (−1.2 ± 0.4, 8), Ca²⁺ (−2.3 ± 0.5, 10) and Mg²⁺ (−2.5 ± 0.5, 7). The microelectrode can be applied for the direct analysis of the activities of free divalent putrescine cations in the range 5 × 10⁻⁴ to 10⁻¹ M in an extracellular ionic environment. Established analytical methods, e.g. high performance liquid chromatography, determine the total concentration of the derivatives of free and bound putrescine. Received: 20 December 1998 / Revised version: 7 May 1999 / Accepted: 27 May 1999     

Degradation of the metal-cyano complex tetracyanonickelate (II) by Fusarium oxysporum N-10

A fungus with the ability to utilize a metal-cyano compound, tetracyanonickelate (II) {K₂[Ni (CN)₄]; TCN}, as its sole source of nitrogen was isolated from soil and identified as Fusarium oxysporum N-10. Both intact mycelia and cell-free extract of the strain catalyzed hydrolysis of TCN to formate and ammonia and produced formamide as an intermediate, thereby indicating that a hydratase and an amidase sequentially participated in the degradation of TCN. The enzyme catalyzing the hydration of TCN was purified approximately ten-fold from the cell-free extract of strain N-10 with a yield of 29%. The molecular mass of the active enzyme was estimated to be 160 kDa. The enzyme appears to exist as a homotetramer, each subunit having a molecular mass of 40 kDa. The enzyme also catalyzed the hydration of KCN, with a cyanide-hydrating activity 2 × 10⁴ times greater than for TCN. The kinetic parameters for TCN and KCN indicated that hydratase isolated from F. oxysporum was a cyanide hydratase able to utilize a broad range of cyano compounds and nitriles as substrates. Received: 9 August 1999 / Received revision: 13 September 1999 / Accepted: 24 September 1999     

High-frequency plant regeneration via somatic embryogenesis and organogenesis and in vitro flowering of regenerated plantlets in Panax ginseng

 The morphogenesis ability of light yellowish globular callus derived from cotyledons of mature zygotic embryos of Panax ginseng was investigated. The optimal media for somatic embryogenesis and shoot organogenesis were MS medium containing 0.5 mg l^–1 2,4-dichlorophenoxyacetic acid, 0.1 mg l^–1 6-benzyladenine (BA), and 500 mg l^–1 lactoalbumin hydrolysate, and SH medium supplemented with 0.5 mg l^–1 α-naphthaleneacetic acid, 0.1 mg l^–1 BA, and 500 mg l^–1casein hydrolysate. The influences of glucose, mannose, fructose, and sorbose in the media on somatic embryogenesis and shoot organogenesis were revealed as differences in the numbers of somatic embryos and adventitious shoots per gram of morphogenic callus. The best regeneration of somatic embryos was obtained on medium containing glucose, with a mean of 8.7 somatic embryos per gram of callus. The best regeneration of shoots was observed on medium containing fructose, with an average of 12.2 adventitious shoots per gram of callus. Of the somatic embryos 95% were converted into regenerated plantlets, and 100% of adventitious shoots rooted to form regenerated plantlets. Regenerated plants were successfully established in soil. Flowering was observed in 5.7% of the regenerated plants derived from shoot organogenesis and in 1.4% of the regenerated plants derived from somatic embryogenesis. Received: 1 December 1998 / Revision received: 13 September 1999 / Accepted: 20 September 1999     

Biochemical basis for carbon monoxide tolerance and butanol production by Butyribacterium methylotrophicum

The biochemical mechanisms for growth tolerance to a 100% CO headspace in cultures, and butanol plus ethanol production from CO by Butyribacterium methylotrophicum were assessed in the wild-type and CO-adapted strains. The CO-adapted strain grew on glucose or CO under a 100% CO headspace, whereas, the growth of the wild-type strain was severely inhibited by 100% CO. The CO-adapted strain, unlike the wild-type, also produced butyrate, from either pyruvate or CO. The CO-adapted strain was a metabolic mutant having higher levels of ferredoxin–NAD oxidoreductase activity, which was not inhibited by NADH. Consequently, only the CO-adapted strain can grow on CO because CO oxidation generates reduced ferredoxin which, via the mutated ferredoxin–NAD reductase activity, forms reduced NADH required for catabolism. When the CO-adapted strain was grown at pH 6.0 it produced butanol (0.33 g/l) and ethanol (0.5 g/l) from CO and the cells contained the following NAD-linked enzyme activities (μmol min⁻¹ mg protein⁻¹): butyraldehyde dehydrogenase (227), butanol dehydrogenase (686), acetaldehyde dehydrogenase (82) and ethanol dehydrogenase (129). Received: 15 September 1998 / Received revision: 12 February 1999 / Accepted: 19 February 1999     

Microbial production, purification, and characterization of (S)-specific N-acetyl-2-amino-1-phenyl-4-pentene amidohydrolase from Rhodococcus globerulus K1/1

Rhodococcus globerulus K1/1 was found to express an inducible (S)-specific N-acetyl-2-amino-1-phenyl-4-pentene amidohydrolase. Optimal bacterial growth and amidohydrolase expression were both observed at about pH 6.5. Purification of the enzyme to a single band in a Coomassie blue-stained SDS-PAGE gel was achieved by nucleic acid and ammonium sulfate precipitation of Rhodococcus globerulus K1/1 crude extract and column chromatography on TSK Butyl-650(S) Fractogel and Superose 12HR. The amidohydrolase was purified to a homogeneity leading to a tenfold increase of the specific activity with a recovery rate of 65%. At pH 7.0 and 23 °C the enzyme showed no loss of activity after 30 days incubation. The amidohydrolase was stable up to 55 °C. The enzyme was inhibited strongly only by 10 mM Zn²⁺ among the tested metal cations and was inhibited 100% by 0.01 mM phenylmethanesulfonyl fluoride. The molecular weight of the native enzyme was estimated to be 92 kDa by gel filtration and 55 kDa by SDS-PAGE, suggesting a homodimeric structure. Received: 8 February 1999 / Received revision: 3 May 1999 / Accepted: 7 May 1999     

High alcohol production by repeated batch fermentation using an immobilized osmotolerant Saccharomyces cerevisiae

A repeated batch fermentation system was used to produce ethanol using an osmotolerant Saccharomyces cerevisiae (VS₃) immobilized in calcium alginate beads. For comparison free cells were also used to produce ethanol by repeated batch fermentation. Fermentation was carried for six cycles with 125, 250 or 500 beads using 150, 200 or 250 g glucose L⁻¹ at 30°C. The maximum amount of ethanol produced by immobilized VS₃ using 150 g L⁻¹ glucose was only 44 g L⁻¹ after 48 h, while the amount of ethanol produced by free cells in the first cycle was 72 g L⁻¹. However in subsequent fed batch cultures more ethanol was produced by immobilized cells compared to free cells. The amount of ethanol produced by free cells decreased from 72 g L⁻¹ to 25 g L⁻¹ after the fourth cycle, while that of immobilized cells increased from 44 to 72 g L⁻¹. The maximum amount of ethanol produced by immobilized VS₃ cells using 150, 200 and 250 g glucose L⁻¹ was 72.5, 93 and 87 g ethanol L⁻¹ at 30°C. Journal of Industrial Microbiology & Biotechnology (2000) 24, 222–226. Received 16 September 1999/ Accepted in revised form 22 December 1999     

Solid matrix characterization of immobilized Pseudomonas putida MTCC 1194 used for phenol degradation

Characterization studies of calcium alginate beads with encapsulated Pseudomonas putida MTCC 1194, used for the biodegradation of phenol, were carried out to investigate the reactivity, reusability and structural strength of the solid matrix. Various techniques were employed to improve the structural stability of the immobilized solid necessary for its use in commercial reactors like packed bed flow reactor, fluidized bed and CSTR systems. Experiments were performed to establish the optimum conditions for durability, strength and steady biochemical reactivity. During a batch run of 40 h a gradual decline in the rate of phenol degradation was observed with the immobilized system. The calcium alginate beads with high structural strength yielded decreased activity. Treatment with a hardening agent like glutaraldehyde for different concentrations and treatment times led to variations in structural stability, reusability and the extent of phenol degradation. Scanning electron microscope studies of the immobilized solid indicated the internal distribution pattern of the cells encapsulated in a calcium alginate bead. Received: 13 November 1998 / Received revision: 27 January 1999 / Accepted: 31 January 1999     

Production of transgenic gentian plants by particle bombardment of suspension-culture cells

 Cell suspension cultures were established from leaf explants of gentian (Gentiana triflora×G. scabra) for the generation of transgenic plants by particle bombardment. The parameters for the bombardment of suspension culture cells with a particle gun were examined by monitoring the transient expression of a gene for β-glucuronidase driven by the cauliflower mosaic virus (CaMV) 35S promoter. We found that prior culture of suspension culture cells for 5 days on solid medium was optimum for successful particle bombardment. Putative transformed calli were obtained from bombarded cells after a two-step selection procedure. Cells were cultured first with 30 mg l^–1 hygromycin in liquid MS medium that contained 10 mg l^–1 N-phenyl-N′-1,2,3-thiadiazol-5-yl urea, 1 mg/l 1-naphthaleneacetic acid and 30 g l^–1 sucrose and then on solid medium prepared from the same liquid medium plus 2 g l^–1 gellan gum. After 12 weeks of selection on solid medium that contained 30 mg l^–1 hygromycin, two transgenic gentian plants were regenerated from each selected callus. Analysis by the polymerase chain reaction and Southern blotting revealed the stable integration of transferred DNA. Received: 3 June 1999 / Revision received: 21 September 1999 / Accepted: 20 September 1999     

Polymerization of guaiacol by lignin-degrading manganese peroxidase from Bjerkandera adusta in aqueous organic solvents

 Lignin-degrading manganese (II) peroxidase (MnP) purified from the culture of a wood-rotting basidiomycete, Bjerkandera adusta, was used in the polymerization of guaiacol. MnP was found to catalyze polymerization of guaiacol in 50% aqueous acetone, dimethyl formamide, methanol, ethanol, dioxane, acetonitrile, ethylene glycol and methylcellosolve. Maximum yield of polyguaiacol was achieved in 50% aqueous acetone. The weight average molecular weight (M _w) of the polymer was estimated to be 30 300 by gel permeation chromatography. However, matrix-assisted laser desorption ionization time of flight mass spectroscopy (MALDI-TOF-MS) analysis gave a more reliable M _w of 1690. IR, ¹³C-NMR, MALDI-TOF-MS and pyrolysis GC-MS analyses showed the presence of C–C and C–O linkages and quinone structure in polyguaiacol. It was also indicated that polyguaiacol has a methoxy-phenyl group as the terminal moiety. This suggests that polyguaiacol is a branched polymer in which guaiacol units are cross-linked at the phenolic group. Thermal gravimetric and differential scanning calorimetric analyses were also carried out. MnP also catalyzed the polymerization of o-cresol, 2,6-dimethoxyphenol and other phenolic compounds and aromatic amines. M _w of these polymers ranged from around 1000 to 1500. Received: 2 August 1999 / Received revision: 10 December 1999 / Accepted: 4 January 2000     

Keratinase of Doratomyces microsporus

 The fungus Doratomyces microsporus produced an extracellular keratinase during submerged aerobic cultivation in a medium containing a protein inducer for enzyme synthesis. The keratinase was purified to homogeneity using hydrophobic interaction chromatography followed by gel chromatography. The molecular weight was estimated to be 33 kDa (from SDS-PAGE analysis) or 30 kDa (by gel chromatography), suggesting a monomeric structure. The isoelectric point of the enzyme was determined to be around 9. The optimal pH and temperature for keratinolytic activity were pH 8–9 and 50 °C, respectively. The serine protease inhibitor PMSF totally inhibited the keratinase. The enzyme was not glycosylated. It was capable of hydrolysing different keratinous materials as well as some non-keratinous proteins. Hydrolysis of some synthetic substrates, specific for known proteinases, suggested that the keratinase of D. microsporus is close to proteinase K. Received: 9 July 1999 / Received revision: 13 September 1999 / Accepted: 17 September 1999     

Removal of organic pollutants and of nitrate from wastewater from the dairy industry by denitrification

The aim of this work was to remove nitrate-N and organic pollutants from wastewater of the dairy industry by denitrification. An artificially prepared wastewater, containing 250 mg/l nitrate-N and 1.5 g/l whey powder, was completely denitrified with removal of 90%–93% of the chemical oxygen demand (COD) of the whey powder by suspended or immobilized mixed cultures and by a suspended or immobilized pure culture that was isolated from the mixed culture inoculum. For the above COD/nitrate-N ratio of 6:1, the results indicated that the organic compounds of the wastewater served as electron donors for complete denitrification and that there was no need to add an external carbon source. In batch denitrification assays the suspended or immobilized mixed cultures proved to be more active and reacted faster than the isolated pure cultures. In continuous denitrification processes with immobilized pure or mixed cultures, the alginate beads, used for immobilization, were not stable for more than 12 days of incubation. The mixed free cultures removed the nitrate-N and COD continuously with no change of their activity for at least 15 days at an optimum hydraulic retention time of 0.27 days with a loading rate of 900 mg nitrate-N l⁻¹ day⁻¹. Received: 13 October 1997 /  Received revision: 16 December 1997 / Accepted: 19 December 1997     

Utilization of sorbed compounds by microorganisms specifically isolated for that purpose

A bacterium obtained by enrichment on nonsorbed phenanthrene was unable to degrade phenanthrene sorbed to polyacrylic beads and had little activity on phenanthrene sorbed to lake-bottom sediment. A bacterium obtained by enrichment on phenanthrene sorbed to polyacrylic beads readily mineralized the compound sorbed to the beads or the sediment. Degradation by the second bacterium of phenanthrene sorbed to beads 38–63 μm or 63–150 μm in diameter was more rapid than the rate of desorption of the hydrocarbon in the absence of the bacterium. Little degradation of sorbed, nonleachable phenanthrene in soil was effected by another isolate obtained by enrichment with the nonsorbed hydrocarbon, but a mixed culture and the bacterium obtained by enrichment on the sorbed compound extensively degraded phenanthrene. Because microorganisms specifically obtained for their capacity to degrade sorbed phenanthrene are more active than species not specialized for use of the bound compound, we suggest that microorganisms enriched on nonsorbed compounds may not be appropriate for evaluation of biodegradation and bioremediation of sorbed compounds. Received: 3 June 1997 / Received revision: 2 September 1997 / Accepted: 15 September 1997     

The kinetics of calcium and magnesium entry into mycorrhizal spruce roots

 The entry of calcium and magnesium from external sources into mycorrhizal roots of 3-year-old Norway spruce trees (Piceaabies [L.] Karst.) was monitored. Roots of intact plants were exposed for various periods of time, ranging from 2 min to 48 h, to nutrient solutions which contained the stable-isotope tracers ²⁵Mg and ⁴⁴Ca. After labelling, samples of roots were excised from the plants, shock-frozen, cryosubstituted and embedded. The resulting isotope composition in this material was analysed by a laser-microprobe-mass-analyser (LAMMA) at relevant positions within cross-sections of the roots. For both elements, we determined (i) the fractions of the isotopes originating from the plant prior to labelling, and (ii) the fraction of isotopes originating from the corresponding tracer that penetrated into the root. Both divalent cations rapidly penetrated across the cortical apoplast and reached the endodermis. After 2 min of exposure to the labelling solution, an initial transient gradient of the tracers could be observed within the root cortex. Subsequently, calcium as well as magnesium equilibrated between the apoplast of the entire cortex and the external tracer with a half-time, t_1/2, of about 3 min. In contrast, the kinetics of radial movement into the vascular stele showed a delay with a t_1/2 of 100–120 min. We take this as strong evidence that there exists a free apoplastic path for divalent cations in the cortex and that the endodermis is a major barrier to the further passage of Mg and Ca into the xylem. While ²⁵Mg in the labelling solution exchanged rapidly with Mg in the cortical apoplast, the exchange across the plasma membrane with Mg present in the protoplasm of the same cortical cells was almost 2 orders of magnitude slower. The kinetics of Ca and Mg entry at +6 °C were similar to those obtained at a root temperature of +22 °C. Received: 23 December 1998 / Accepted: 17 September 1999     

Improvement of mechanical and biological properties of freeze-dried denitrifying alginate beads by using starch as a filler and carbon source

Freeze-dried, alginate-based beads, used for the immobilization of a denitrifying bacterium (Pseudomonas sp.), were filled with different concentrations (10%, 20%, 30% and 40%, w/w) of granular starch. The beads were incubated under denitrifying conditions in laboratory-scale, flow-through columns and monitored for changes in their physical and denitrifying properties. Freeze-dried beads containing high concentrations of starch were found to have better mechanical and denitrifying properties than beads containing low concentrations of this filler. Nitrate removal by the beads was found to be correlated with their starch content. Nitrite accumulation, as a result of incomplete denitrification, increased with the decrease in starch content of the beads. Nitrite in the outlet of the columns was measured in all types of beads during the initial phase of incubation but was undetectable, with the exception of beads with the lowest starch content, at later stages of incubation. Received: 9 November 1998 / Received revision: 3 February 1999 / Accepted: 5 February 1999     

Characterization of an extracellular poly(3-hydroxy-5-phenylvalerate) depolymerase from Xanthomonas sp. JS02

 A bacterium, JS02, capable of degrading an aromatic medium-chain-length polyhydroxyalkanoate (PHA_MCL), poly(3-hydroxy-5-phenylvalerate) (PHPV), was isolated from wastewater-treatment sludge (Ju et al. 1998), and was identified as a Xanthomonas species. An extracellular PHPV depolymerase was purified from the concentrated culture broth of Xanthomonas sp. JS02 by using a chromatography series on Sephadex G-75, QAE-Sephadex A-50 and hydroxyapatite. The molecular mass of the purified enzyme was estimated to be 41.7 kDa. The purified enzyme could hydrolyse PHPV and p-nitrophenyl (PNP)-esters of fatty acids, but did not hydrolyse short-chain-length PHAs, though the culture supernatant could hydrolyse them. The optimum pH range was 8.0–9.0 and the optimum temperature was 60 °C for PNP-octanoate hydrolysis. The K _m values for PNP-hexanoate and PNP-octanoate were 10.9 and 0.88 μM, respectively. Received: 3 June 1999 / Received revision: 24 August 1999 / Accepted: 24 September 1999