Evaluation of solid substrates for enzyme production by Coriolus versicolor, for use in bioremediation of chlorophenols in aqueous effluents

In the development of a system for the removal of chlorophenols from aqueous effluents, a range of solid substrates for the growth of Coriolus versicolor were investigated. Substrates included wood chips, cereal grain, wheat husk and wheat bran. Suitability for transformation of chlorophenols depended on laccase production by the fungus. The greatest amount of laccase (<25 Units g⁻¹ substrate) was produced on wheat husk and wheat bran over 30 days colonisation. Aqueous extracts of laccase from wheat husk and wheat bran cultures removed 100% of 2,4-dichlorophenol (50 ppm) from solution within 5 h and 75–80% of pentachlorophenol (50 ppm) within 24 h. Wheat bran was formulated into pellets with biscuit flour to provide a compact substrate for fungal immobilisation. Addition of 8–12% yeast extract to the pellets increased laccase production five-fold. Colonised pellets were added to chlorophenol solutions in 200–4000-ml bioreactors, resulting in >90% removal of chlorophenols within 100 min. Received: 10 April 2000 / Received revision: 4 July 2000 / Accepted: 10 July 2000     

Production and characterization of ferulic acid esterase activity in crude extracts by Streptomyces avermitilis CECT 3339

Streptomyces avermitilis CECT 3339 produces extracellular ferulic acid esterase (FAE) activity during growth on a range of lignocellulose substrates. Maximal levels of FAE activity were detected in culture filtrates from S. avermitilis CECT 3339 grown in media containing wheat bran and yeast extract as carbon and nitrogen sources respectively. Biochemical characterization of this enzyme activity revealed that it was 100-fold higher when wheat bran was pretreated with Celluclast (a mix of hydrolytic enzymes). FAE was found to be end-product-inhibited. Characterization of the properties of the enzyme showed that FAE exhibited an activity optimum pH at 6 with pH stability between pH 6 and 8. The optimum temperature was 50 °C while the temperature stability was between 30 °C and 40 °C, with rapid inactivation at 60 °C and above. The characteristics and stability of FAE from S. avermitilis CECT 3339 suggest a potential role for this enzyme in combination with endoxylanases for the upgrading of plant-residue silage and for biopulping. Received: 17 November 1997 / Received revision: 13 March 1998 / Accepted: 13 April 1998     

Purification and properties of a novel raw starch degrading cyclomaltodextrin glucanotransferase from Bacillus firmus

A novel raw starch degrading cyclomaltodextrin glucanotransferase (CGTase; E.C. 2.4.1.19), produced by Bacillus firmus, was purified to homogeneity by ultrafiltration, affinity and gel filtration chromatography. The molecular weight of the pure protein was estimated to be 78 000 and 82 000 Da, by SDS-PAGE and gel filtration, respectively. The pure enzyme had a pH optimum in the range 5.5–8.5. It was stable over the pH range 7–11 at 10 °C, and at pH 7.0 at 60 °C. The optimum temperature for enzyme activity was 65 °C. In the absence of substrate, the enzyme rapidly lost its activity above 30 °C. K _m and k _cat for the pure enzyme were 1.21 mg/ml and 145.17 μM/mg per minute respectively, with soluble starch as the substrate. For cyclodextrin production, tapioca starch was the best substrate used when gelatinized, while wheat starch was the best substrate used when raw. This CGTase could degrade raw wheat starch very efficiently; up to 50% conversion to cyclodextrins was obtained from 150 g/l starch without using any additives. The enzyme produced α-, β- and γ-cyclodextrins in the ratio of 0.2:9.2:0.6 and 0.2:8.6:1.2 from gelatinized tapioca starch and raw wheat starch with 150 g/l concentration respectively, after 18 h incubation. Received: 25 September 1998 / Received revision: 15 December 1998 / Accepted: 21 December 1998     

Production of high activity thermostable phytase from thermotolerant Aspergillus niger in solid state fermentation

The thermotolerant fungus, Aspergillus niger NCIM 563, was used for production of extracellular phytase on agricultural residues: wheat bran, mustard cake, cowpea meal, groundnut cake, coconut cake, cotton cake and black bean flour in solid state fermentation (SSF). Maximum enzyme activity (108 U g⁻¹ dry mouldy bran, DMB) was obtained with cowpea meal. During the fermentation phytic acid was hydrolysed completely with a corresponding increase in biomass and phytase activity within 7 days. Phosphate in the form of KH₂PO₄ (10 mg per 100 g of agriculture residue) increased phytase activity. Among various surfactants added to SSF, Trition X-100 (0.5%) exhibited a 30% increase in phytase activity. The optimum pH and temperature of the crude enzyme were 5.0 and 50°C respectively. Phytase activity (86%) was retained in buffer of pH 3.5 for 24 h. The enzyme retained 75% of its activity on incubation at 55°C for 1 h. In the presence of 1 mM K⁺ and Zn²⁺, 95% and 55% of the activity were retained. Scanning electron microscopy showed a high density growth of fungal mycelia on wheat bran particles during SSF. Journal of Industrial Microbiology & Biotechnology (2000) 24, 237–243. Received 07 June 1999/ Accepted in revised form 18 December 1999     

Improvement of α-l-arabinofuranosidase production by Talaromyces thermophilus and agro-industrial residues saccharification

This study is an application of an experimental design methodology for the optimization of the culture conditions of α-l-arabinofuranosidase production by Talaromyces thermophilus. Wheat bran and yeast extract were first selected as the best carbon and nitrogen sources, respectively, for enzyme production. A Plackett–Burman design was then used to evaluate the effects of eight variables. Statistical analyses showed that while pH had a negative effect on α-l-arabinofuranosidase production, wheat bran and MgSO₄ had a significantly positive effect. The values of the latter three parameters were further optimised using a central composite design and a response surface methodology. The experimental results were fitted to a second-order polynomial model that yielded a determination coefficient of R ² = 0.91. The statistical output showed that the linear and quadric terms of the three variables had significant effects. Using optimal conditions, the experimental value of α-l-arabinofuranosidase activity produced was very close to the model-predicted value. The optimal temperature and pH of enzyme activity were 55 °C and 7.0, respectively. This enzyme was very stable over a considerable pH range from 4 to 9. The crude enzyme of T. thermophilus rich in α-l-arabinofuranosidase was also used for saccharification of lignocellulosic materials and arabinose production.     

Amino acid production from rice straw and wheat bran hydrolysates by recombinant pentose-utilizing <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Corynebacterium glutamicum wild type lacks the ability to utilize the pentose fractions of lignocellulosic hydrolysates, but it is known that recombinants expressing the araBAD operon and/or the xylA gene from Escherichia coli are able to grow with the pentoses xylose and arabinose as sole carbon sources. Recombinant pentose-utilizing strains derived from C. glutamicum wild type or from the l-lysine-producing C. glutamicum strain DM1729 utilized arabinose and/or xylose when these were added as pure chemicals to glucose-based minimal medium or when they were present in acid hydrolysates of rice straw or wheat bran. The recombinants grew to higher biomass concentrations and produced more l-glutamate and l-lysine, respectively, than the empty vector control strains, which utilized the glucose fraction. Typically, arabinose and xylose were co-utilized by the recombinant strains along with glucose either when acid rice straw and wheat bran hydrolysates were used or when blends of pure arabinose, xylose, and glucose were used. With acid hydrolysates growth, amino acid production and sugar consumption were delayed and slower as compared to media with blends of pure arabinose, xylose, and glucose. The ethambutol-triggered production of up to 93 ± 4 mM l-glutamate by the wild type-derived pentose-utilizing recombinant and the production of up to 42 ± 2 mM l-lysine by the recombinant pentose-utilizing lysine producer on media containing acid rice straw or wheat bran hydrolysate as carbon and energy source revealed that acid hydrolysates of agricultural waste materials may provide an alternative feedstock for large-scale amino acid production.     

Antimould activity of sourdough lactic acid bacteria: identification of a mixture of organic acids produced by Lactobacillus sanfrancisco CB1

Sourdough lactic acid bacteria, cultivated in wheat flour hydrolysate, produced antimould compounds. The antimould activity varied greatly among the strains and was mainly detected within obligately heterofermentative Lactobacillus spp. Among these, Lb. sanfrancisco CB1 had the largest spectrum. It inhibited moulds related to bread spoilage such as Fusarium, Penicillium, Aspergillus and Monilia. A mixture of acetic, caproic, formic, propionic, butyric and n-valeric acids, acting in a synergistic way, was responsible for the antimould activity. Caproic acid played a key role in inhibiting mould growth. Received: 20 January 1998 / Received revision: 17 April 1998 / Accepted: 27 April 1998     

Comparison of separate hydrolysis and fermentation and simultaneous saccharification and fermentation processes for ethanol production from wheat straw by recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> strain FBR5

Ethanol production by recombinant Escherichia coli strain FBR5 from dilute acid pretreated wheat straw (WS) by separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) was studied. The yield of total sugars from dilute acid (0.5% H₂SO₄) pretreated (160 °C, 10 min) and enzymatically saccharified (pH 5.0, 45 °C, 72 h) WS (86 g/l) was 50.0 ± 1.4 g/l. The hydrolyzate contained 1,184 ± 19 mg furfural and 161 ± 1 mg hydroxymethyl furfural per liter. The recombinant E. coli FBR5 could not grow at all at pH controlled at 4.5 to 6.5 in the non-abated wheat straw hydrolyzate (WSH) at 35 °C. However, it produced 21.9 ± 0.3 g ethanol from non-abated WSH (total sugars, 44.1 ± 0.4 g/l) in 90 h including the lag time of 24 h at controlled pH 7.0 and 35 °C. The bioabatement of WS was performed by growing Coniochaeta ligniaria NRRL 30616 in the liquid portion of the pretreated WS aerobically at pH 6.5 and 30 °C for 15 h. The bacterium produced 21.6 ± 0.5 g ethanol per liter in 40 h from the bioabated enzymatically saccharified WSH (total sugars, 44.1 ± 0.4 g) at pH 6.0. It produced 24.9 ± 0.3 g ethanol in 96 h and 26.7 ± 0.0 g ethanol in 72 h per liter from bioabated WSH by batch SSF and fed-batch SSF, respectively. SSF offered a distinct advantage over SHF with respect to reducing total time required to produce ethanol from the bioabated WS. Also, fed-batch SSF performed better than the batch SSF with respect to shortening the time requirement and increase in ethanol yield.     

The formation of 1-hydroxymethylnaphthalene and 6-hydroxymethylquinoline by both oxidative and reductive routes in Cunninghamella elegans

Extraction of medium after incubation of the fungus, Cunninghamella elegans, with 0.03% (w/v) 1-methylnaphthalene produced mainly 1-hydroxymethylnaphthalene together with some 1-naphthoic acid and hydroxynaphthoic acid. Higher concentrations of substrate were inhibitory to biotransformation. Similar incubations with 1-naphtoic acid as substrate resulted in reduction of the carboxyl group to give 1-hydroxymethylnaphthalene. When 6-methylquinoline was used, the main product was 6-hydroxymethylquinoline but also some quinoline-6-carboxylic acid and some 6-methylquinoline-N-oxide were identified. In a 2-l fermenter 2.5 g substrate was transformed in 324 h. The 6-hydroxymethylquinoline was also produced by reduction of quinoline-6-carboxylic acid by the organism. Received: 9 March 1998 / Received revision: 15 June 1998 / Accepted: 19 June 1998     

Solid-state fermentation for xylanase production by Thermoascus aurantiacus using response surface methodology

We investigated xylanase production by Thermoascus aurantiacus using semisolid fermentation. Multivariant statistical approaches were employed to evaluate the effects of several variables (initial moisture in the medium, cultivation time, inoculum level, and bagasse mass) on xylanase production. The initial moisture content and bagasse mass were the most important factors affecting xylanase activity. The xylanase activity produced by the fungus under the optimized conditions (81% moisture content and 17 g bagasse) was found to be 2700 U per gram of initial dry matter, whereas its value predicted by a polynomial model was 2400 U per gram of initial dry matter. Received: 4 December 1998 / Received revision: 15 March 1999 / Accepted: 16 May 1999     

The colonization of a simulator of the human intestinal microbial ecosystem by a probiotic strain fed on a fermented oat bran product: effects on the gastrointestinal microbiota

The effects of Lactobacillus-GG-fermented oat bran product on the microbiota and its metabolic activity in the human gut were investigated, using a simulator of the human intestinal microbial ecosystem (SHIME), by analysing the bacterial population, short-chain fatty acids and gas production. In addition, the effects of fermented oat bran supernatant and supernatant samples from reactors 4, 5 and 6 (large intestine) on the growth of Escherichia coli IHE 13047, Enterococcus faecalis VTT E-93203, Lactobacillus rhamnosus VTT E-94522 (Lactobacillus GG) and Lactococcus lactis subsp. lactis VTT E-90414 were monitored to ascertain possible stimulatory/inhibitory effects by an in vitro turbidometric method. Our experiments showed that Lactobacillus GG colonized the SHIME reactor and this colonization could be maintained for several weeks without extra supplementation. Oat bran feeding also favoured the growth of bifidobacteria and caused an increase in the production of acetic, propionic and butyric acid as well as CH₄ and CO₂. However, the effects of oat bran, either on bacterial populations or on their metabolic activity, were not directly dose-dependent. In turbidometric measurements, the supernatant of fermented oat bran exerted an inhibitory effect of Lactobacillus GG, but stimulated the growth of enterococci. Received: 19 January 1998 / Received revision: 6 April 1998 / Accepted: 13 April 1998     

Optimization of <Emphasis Type="Italic">Verticillium lecanii</Emphasis> spore production in solid-state fermentation on sugarcane bagasse

Verticillium lecanii is an entomopathogen with high potential in biological control of pests. We developed a solid-state fermentation with sugarcane bagasse as carrier absorbing liquid medium to propagate V. lecanii spores. Using statistical experimental design, we optimized the medium composition for spore production. We first used one-factor-at-a-time design to identify corn flour and yeast extract as the best carbon and nitrogen sources for the spore production of V. lecanii. Then, we used two-level fractional factorial design to confirm corn flour, yeast extract, and KH₂PO₄ as important factors significantly affecting V. lecanii spore production. Finally, we optimized these selected variables using a central composite design and response surface method. The optimal medium composition was (grams per liter): corn flour 35.79, yeast 8.69, KH₂PO₄ 1.63, K₂HPO₄ 0.325, and MgSO₄ 0.325. Under optimal conditions, spore production reached 1.1 × 10¹⁰ spores/g dried carrier, much higher than that on wheat bran (1.7 × 10⁹ spores/g initial dry matter).     

L-NAME administration prevents the inhibition of nucleotide hydrolysis by rat blood serum subjected to hyperargininemia

Summary. The main objective of the present study was to evaluate the in vivo and in vitro effect of Arg on serum nucleotide hydrolysis. The action of N^ω-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase, on the effects produced by Arg was also examined. Sixty-day-old rats were treated with a single or a triple (with an interval of 1 h between each injection) intraperitoneal injection of saline (group I), Arg (0.8 g/kg) (group II), L-NAME (2.0 mg/kg or 20 mg/kg) (group III) or Arg (0.8 g/kg) plus L-NAME (2.0 mg/kg or 20 mg/kg) (group IV) and were killed 1 h later. The present results show that a triple Arg administration decreased ATP, ADP and AMP hydrolysis. Simultaneous injection of L-NAME (20 mg/kg) prevented such effects. Arg in vitro did not alter nucleotide hydrolysis. It is suggested that in vivo Arg administration reduces nucleotide hydrolysis in rat serum, probably through nitric oxide or/and peroxynitrite formation. Both are first authors.     

Genes controlling hydroxylations of phytosiderophores are located on different chromosomes in barley (Hordeum vulgare L.)

Phytosiderophores, mugineic acids, have been demonstrated to be involved in Fe acquisition in gramineous plants. In this study, chromosomal arm locations of genes encoding for biosynthesis of various phytosiderophores were identified in a cultivar of barley (Hordeum vulgare L. cv. Betzes). Using wheat (Triticum aestivum L. cv. Chinese Spring)-barley (cv. Betzes) ditelosomic addition lines for 4HS and 4HL, a gene for hydroxylation of 2′-deoxymugineic acid to mugineic acid was localized to the long arm of barley chromosome 4H. To locate the gene for hydroxylation of mugineic acid to 3-epihydroxymugineic acid, hybrids between the 4H addition line and other wheat-barley addition lines were studied. Only a hybrid between 4H and 7H addition lines produced 3-epihydroxymugineic acid. The gene was further localized to the long arm of chromosome 7H by feeding mugineic acid to ditelosomic addition lines for 7HS and 7HL. A new phytosiderophore was discovered in both 7H and 7HL addition lines, which was identified to be 3-epihydroxy-2′-deoxymugineic acid by detailed nuclear magnetic resonance studies. These results revealed that in barley there are two pathways from 2′-deoxymugineic acid to 3-epihydroxymugineic acid: 2′-deoxymugineic acid → mugineic acid → 3-epihydroxymugineic acid and 2′-deoxymugineic acid → 3-epihydroxy-2′-deoxymugineic acid → 3-epihydroxymugineic acid. Barley genes encoding for the hydroxylations of phytosiderophores are located in different chromosomes and each gene hydroxylates different C-positions: the long arm of chromosome 4H carries the gene for hydroxylating the C-2′ position and the long arm of chromosome 7H carries the gene for hydroxylating the C-3 position of the azetidine ring. Received: 10 August 1998 / Accepted: 30 September 1998     

The composition of the cutin of the caryopses and leaves ofTriticum aestivum L.

The outer layers (bran) of white wheat (Triticum aestivum L. cv. Jubilar) caryopses contain several layers of lipophilic materials. It was the objective of the present work to establish the nature, composition and amounts of the lipid polymers of wheat bran and to compare it with leaf cutin. Prior to analysis, the bran was isolated and divided into two fractions: (i) the inner bran containing the remnants of the nucellus, the seed coat and the inner layers of the pericarp, and (ii) the outer bran consisting of the peripheral layers of the pericarp. Following depolymerization, a total number of 14 long-chain monobasic, dibasic, ω-hydroxymonobasic, α-hydroxymonobasic, dihydroxymonobasic, trihydroxymonobasic and epoxyhydroxymonobasic alkanoic acids have been identified as constituents of bran lipid polymeres. The most abundant single constituent was 9,10-epoxy-18-hydroxyoctadecanoic acid. The qualitative and quantitative compositions of depolymerisates from the inner and outer bran fractions were similar except for the absence of 9,10,18-trihydroxyoctadecanoic acid and of long-chain (C₂₂−C₂₆ ω-hydroxyalkanoic acids in the outer bran. The composition of bran depolymerisates closely resembled the constitution of the BF₃/CH₃OH susceptible fraction of wheat leaf cutin. Only less than 2% of the total amount of monomers released from inner bran were indicative for the presence of suberin. The total cutin content of wheat bran amounted to 4.2 g per kg of dry caryopses. Most of it (96.6%) was contributed by the cuticles of the seed coat and the nucellus while the cuticle of the pericarp made up only 3.4%.     

Enhanced transformation of malachite green by laccase of <Emphasis Type="Italic">Ganoderma lucidum</Emphasis> in the presence of natural phenolic compounds

In this study, we investigated the efficacy of phenolic extract of wheat bran and lignin-related phenolic compounds as natural redox mediators on laccase-mediated transformation of malachite green (MG) using purified laccase from the white-rot fungus Ganoderma lucidum. G. lucidum laccase was able to decolorize 40.7% MG dye (at 25 mg l⁻¹) after 24 h of incubation. Whereas, the addition of phenolic extract of wheat bran enhanced the decolorization significantly (p < 0.001) by two- to threefold than that of purified laccase alone. Among various natural phenolic compounds, acetovanillone, p-coumaric acid, ferulic acid, syringaldehyde, and vanillin were the most efficient mediators, as effective as the synthetic mediator 1-hydroxybenzotriazole. Characterization of MG transformation products by HPLC, UV–Vis, and liquid chromatography-mass spectrometry-electrospray ionization analysis revealed that N-demethylation was the key mechanism of decolorization of MG by laccase. Growth inhibition test based on mycelial growth inhibition of white rot fungus Phanerochaete chrysosporium revealed that treatment with laccase plus natural mediators effectively reduced the growth inhibitory levels of MG than that of untreated one. Among all the tested compounds, syringaldehyde showed the highest enhanced decolorization, as a consequence reduced growth inhibition was observed in syringaldehyde-treated samples. The results of the present study revealed that the natural phenolic compounds could alternatively be used as potential redox mediators for effective laccase-mediated decolorization of MG.     

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     

Production of 2,3-butanediol by newly isolated Enterobacter cloacae

Enterobacter cloacae NRRL B-23289 was isolated from local decaying wood/corn soil samples while screening for microorganisms for conversion of l-arabinose to fuel ethanol. The major product of fermentation by the bacterium was meso-2,3-butanediol (2,3-BD). In a typical fermentation, a BD yield of 0.4 g/g arabinose was obtained with a corresponding productivity of 0.63 g/l per hour at an initial arabinose concentration of 50 g/l. The effects of initial arabinose concentration, temperature, pH, agitation, various monosaccharides, and multiple sugar mixtures on 2,3-BD production were investigated. BD productivity, yield, and byproduct formation were influenced significantly within these parameters. The bacterium utilized sugars from acid plus enzyme saccharified corn fiber and produced BD (0.35 g/g available sugars). It also produced BD from dilute acid pretreated corn fiber by simultaneous saccharification and fermentation (0.34 g/g theoretical sugars). Received: 17 December 1998 / Revision received: 9 March 1999 / Accepted: 20 March 1999     

Agrobacterium-mediated genetic transformation of a phalaenopsis orchid

 Genetically transformed plants of a phalaenopsis orchid [Doritaenopsis Coral Fantasy×Phalaenopsis (Baby Hat×Ann Jessica)] were regenerated after cocultivation of cell clumps with Agrobacterium tumefaciens strains LBA4404 (pTOK233) and EHA101 (pIG121Hm) that harbored genes for β-glucuronidase (GUS) and hygromycin resistance. The efficiency of transformation was markedly increased by 10 h cocultivation of cell clumps with A. tumefaciens that had been induced with 200 μm acetosyringone, and by inclusion of 500 μm acetosyringone in the cocultivation medium. Hygromycin-resistant cell clusters (0.5–3 mm in diameter) were selected from the infected cell clumps after 4–6 weeks of culture on agar (8 g/l)-solidified new Dogashima medium (NDM) containing 20 g/l sucrose, 0.1 mg/l naphthaleneacetic acid, 1.0 mg/l benzyladenine (BA), 50 mg/l hygromycin and 300 mg/l cefotaxime. The cell clusters proliferated 4 weeks after transfer onto the same medium. To induce callus greening, the carbon source was changed from sucrose to maltose. The green calli obtained produced protocorm-like bodies (PLBs) after 4 weeks of culture on phytohormone-free NDM medium. Regeneration of transgenic plantlets was enhanced by incubating PLBs on NDM medium supplemented with 0.1 mg/l abscisic acid, followed by partial desiccation for 10–30 min. Successful transformation was confirmed by histochemical GUS assay, PCR analysis and Southern hybridization of transformants. With this transformation system, more than 100 hygromycin-resistant phalaenopsis plantlets were produced about 7 months following infection of the cell aggregates. Received: 10 November 1998 / Revision received: 4 June 1999 / Accepted: 22 June 1999     

Rapid atrazine mineralisation in soil slurry and moist soil by inoculation of an atrazine-degrading Pseudomonas sp. strain

The evaluation of pesticide-mineralising microorganisms to clean-up contaminated soils was studied with the widely applied and easily detectable compound atrazine, which is rapidly mineralised by several microorganisms including the Pseudomonas sp. strain Yaya 6. The rate of atrazine removal was proportional to the water content of the soil and the amount of bacteria added to the soil. In soil slurry, 6 mg atrazine kg soil⁻¹ was eliminated within 1 day after application of 0.3 g dry weight inoculant biomass kg soil⁻¹ and within 5 days when 0.003 g kg soil⁻¹ was used. In partially saturated soil (60% of the maximal water-holding capacity) 15 mg atrazine kg soil⁻¹ was eliminated within 2 days by 1 g biomass kg soil⁻¹ and within 25 days when 0.01 g biomass kg soil⁻¹ was used. In unsaturated soil, about 60% [U-ring-¹⁴C]atrazine was converted to ¹⁴CO₂ within 14 days. Atrazine was very efficiently removed by the inoculant biomass, not only in soil that was freshly contaminated but also in soil aged with atrazine for up to 260 days. The bacteria exposed to atrazine in unsaturated sterile soil were still active after a starvation period of 240 days: 15 mg newly added atrazine kg soil⁻¹ was eliminated within 5 days. Received: 31 October 1997 / Received revision: 16 January 1998 / Accepted: 18 January 1998