Efficient mediator-independent hydrogenation of carbon-carbon double bonds,using the obligate anaerobe,Acetobacterium woodii,with fructose as an electron donor

Fructose and H₂ were compared as electron donors for hydrogenation of carbon-carbon double bonds using Acetobacterium woodii. Caffeate was used as a model substrate. An electron donor was required and both fructose and H₂ were suitable. With fructose as the donor, the K _s for caffeate was 0.5 mM and the V _max was 678 mmol kg_dry weight ⁻¹ h⁻¹.␣Fructose oxidation was coupled very efficiently to caffeate reduction by an alteration in the fructose fermentation so that acetate was no longer produced. Received: 24 June 1996 / Accepted: 1 July 1996     

Distribution and diving of harbour seals (Phoca vitulina) in Svalbard

The distribution, movements and diving of high-arctic harbour seals (Phoca vitulina) were studied in Svalbard, Norway, from 1992 to 1995. A total of 14 seals were equipped with satellite transmitters at Prins Karls Forland (ca. 78°30′N 12°E). These gave data on position, but ten also gave information on dive depths (N ∼ 160,000) and dive durations (N ∼ 162,000). Dive-depth frequencies show that ∼50% of the diving is shallower than 40 m, and that 95% of the diving is shallower than 250 m. Based on dive-duration frequencies, ∼50% of the dives lasted 2–4 min, 90% of the dives lasted less than 7 min, and 97% were shorter than 10 min. All but three seals stayed in the tagging area. Accepted: 6 October 2000     

Isolation and characterization of highly (R)-specific N-acetyl-1-phenylethylamine amidohydrolase, a new enzyme from Arthrobacter aurescens AcR5b

A new amidohydrolase deacetylating several N-acetyl-1-phenylethylamine derivatives (R)-specifically was found in Arthrobacter aurescens AcR5b. The strain was isolated from a wet haystack by enrichment culture with (R)-N-acetyl-1-phenylethylamine as the sole carbon source. (R) and (S )-N-acetyl-1-phenylethylamine do not serve as inducers for acylase formation. By improving the growth conditions the enzyme production was increased 47-fold. The amidohydrolase was purified to homogeneity leading to a 5.2-fold increase of the specific activity with a recovery of 67%. A molecular mass of 220 kDa was estimated by gel filtration. Sodium dodecyl sulfate/polyacrylamide gel electrophorosis shows two subunits with molecular masses of 16 kDa and 89 kDa. The optimum pH and temperature were pH 8 and 50 °C, respectively. The enzyme was stable in the range of pH 7–9 and at temperatures up to 30 °C. The enzyme activity was inhibited by Cu²⁺, Co²⁺, Ni²⁺, and Zn²⁺, and this inhibition was reversed by EDTA.M Received: 20 September 1996 / Received version: 23 December 1996 / Accepted: 30 December 1996     

Cloning and expression of the Bacillus sphaericus 2362 mosquitocidal genes in a non-toxic unicellular cyanobacterium, Synechococcus PCC6301

Genes encoding the mosquitocidal binary toxin of Bacillus sphaericus 2362 were introduced into Synechococcus PCC6301, a cyanobacterium that can tolerate a number of potential variations in the mosquito breeding environment, and can serve as a food source for mosquito larvae. The toxin genes, preceded by a Synechococcus rbcL promoter, were located on a mobilizable Escherichia coli Synechococcus shuttle vector, which was introduced into Synechococcus PCC6301 at frequencies of 10⁻⁵–10⁻⁷ exconjugants/recipient, depending on the selective conditions used. Recombinant Synechococcus exhibited significant toxicity against 2-day-old and 6-day-old Culex quinquefasciatus larvae, the concentration required to kill 50 % of larvae (LC₅₀) being 2.1 × 10⁵ and 1.3 × 10⁵ cells/ml respectively. Mosquitocidal activity decreased tenfold after 20 generations of non-selective growth. Received: 23 July 1996 / Received revision: 11 November 1996 / Accepted: 15 November 1996     

Isolation and characterization of a thermophilic bacillus strain,that degrades phenol and cresols as sole carbon source at 70 °C

A phenol-degrading thermophilic bacterium, designated Bacillus sp. A2, was isolated from a water and mud sample from a hot spring in Iceland. The aerobic isolate grew optimally on phenol at 65 °C. At 70 °C, 85% of the optimal growth rate was still observed. No growth was observed at 40 °C and 75 °C. Bacillus sp. A2 is a gram-positive spore-forming rod. According to 16S rDNA analysis Bacillus sp. A2 is closely related to Bacillus stearothermophilus, Bacillus kaustophilus and Bacillus thermoleovorans. Bacillus sp. A2 degraded phenol completely in concentrations up to 5 mM. In addition, all three isomers of cresol were utilized as sole carbon and energy sources. The degradation of phenols proceeds via the meta-cleavage pathway and the enzymes involved in its degradation are constitutively expressed. Received: 13 May 1996 / Received revision: 29 July 1996 / Accepted: 12 August 1996     

Intertidal zone of Svalbard 3. Littoral of a subarctic, oceanic island: Bjornoya

Twenty-two stations in the intertidal and shallow sublittoral of Bjornoya (74 °N, 19 °E) were studied in August 1994 revealing a large and diverse standing crop of macro-algae (16 species) and littoral macrofauna (at least 17 species). In most places the biomass of littoral macroorganisms exceeded 100 g ww/m². In the shallow sublittoral, between 2 and 20 m, 45 animal taxa and 23 algae species were collected. Littoral coarse sand meiofauna was dominated by Turbellaria, while, on algae, Halacaridae and Harpacticoida predominated. Meiofauna densities ranged from 0 to 169 ind./10 cm² and biomass from 0 to 0.4 g dw/m². The abundance of littoral species and their zoogeographic origin resemble that of Spitsbergen more than that of the northern Scandinavian coast, although both are of equal distance from Bjornoya. The first record of the boreal bivalve Mytilus edulis from the island is presented. Another striking feature was the presence of the arctic amphipod Gammarus setosus and the absence of its boreal sibling species G.oceanicus. Received: 19 March 1996 / Accepted: 1 December 1996     

Degradation of styrene by white-rot fungi

Degradation of styrene in the gaseous phase was investigated for white-rot fungi Pleurotus ostreatus (two strains), Trametes versicolor, Bjerkandera adusta and Phanerochaete chrysosporium. Fungi were grown in liquid culture and the gas/mycelium contact surface was enhanced with the help of perlite. The influence of various inducers on styrene degradation was studied. The best inducers for styrene degradation were lignosulphonate for P. ostreatus and T. versicolor and wood meal for B. adusta and P. chrysosoporium. Under these conditions all fungi were able to degrade styrene almost completely in 48 h at a concentration of 44 μmol/250 ml total culture volume; one strain of P. ostreatus was able to remove 88 μmol styrene under these conditions. Three transformation products of [¹⁴C]styrene in cultures of P. ostreatus were identified: phenyl-1,2-ethanediol, 2-phenylethanol and benzoic acid; 4% of the styrene was metabolised to CO₂ in 24 h and no other volatile products were found. Received: 16 July 1996 / Received revision: 23 September 1996 / Accepted: 29 September 1996     

Production, purification, and characterization of a highly enantioselective (S )-N-acetyl-1-phenylethylamine amidohydrolase from Rhodococcus equi Ac6

Rhodococcus equi Ac6 was found to express an inducible (S )-specific N-acetyl-1-phenylethylamine amidohydrolase. Optimal bacterial growth and amidohydrolase expression were both observed around pH 6.5. Purification of the enzyme to a single band in a Coomassie-blue-stained sodium dodecyl sulfate/polyacrylamide gel (SDS-PAGE) was achieved by ammonium sulfate precipitation of R. equi Ac6 crude extract and column chromatographies on Fractogel TSK Butyl-650(S) and Superose 12HR. At pH 7.0 and 30 °C the amidohydrolase had a half-life of around 350 days; at 44 °C it was only 10 min. Except for Ni²⁺ and, to some extent, Zn²⁺ and Co²⁺, the enzyme was neither strongly influenced by metal cations nor by chelating agents, but was inhibited by 95% at 0.1 mM phenylmethylsulfonyl fluoride. The molecular mass of the native enzyme was estimated to be 94 kDa by gel filtration and 50 kDa by SDS-PAGE, suggesting a dimeric structure. Specificity experiments revealed a spectrum of related N-acetylated compounds being hydrolyzed with variable enantiomeric selectivities. Received: 20 September 1996 / Received revision: 23 December 1996 / Accepted: 30 December 1996     

Application of the Plackett-Burman experimental design to evaluate nutritional requirements for the production of Colletotrichum coccodes spores

Colletotrichum coccodes is being examined as a biological weed control agent for velvetleaf (Abutilon theophrasti). A modified Richard's solution containing V-8 juice has been used to produce C. coccodes spores for growth-chamber and field experiments. Although C. coccodes sporulates well in this medium, V-8 is not available as a bulk commodity and is too expensive for commercial production. Eight substrates were evaluated as replacements for V-8 juice in modified Richard's solution. Soy protein and casamino acids were equal to V-8 juice for sporulation of C. coccodes. The Plackett-Burman experimental design was used to test the relative importance of various components of a complex medium based on soy protein on mycelium biomass production and sporulation of C. coccodes. A new medium composed of sucrose (20 g/l), soy protein (5 g/l), KNO₃ (5 g/l), KH₂PO₄ (5 g/l), MgSO₄ (2 g/l), CaCl₂ (0.5 g/l), and CuSO₄ (0.05 g/l) was selected as the base medium for further study in the development of a low-cost and effective medium for C. coccodes spore production. Received: 29 July 1996 / Received revision: 21 October 1996 / Accepted: 10 November 1996     

Isochorismate hydroxymutase from a cell-suspension culture of Galium mollugo L.

Isochorismate hydroxymutase (i.e. isochorismate synthase, EC 5.4.99.6) was purified from an anthraquinone-producing cell-suspension culture of Galium mollugo L. Although attempts to stabilize the labile enzyme met with little success, a substantial increase in enzyme activity was observed in the presence of glycine betaine (500 mM). Column chromatography on solid supports other than diethylaminoethyl (DEAE)-Sephacel, Phenylsepharose Cl-4B or Cibacron Blue 3G-A did not give active enzyme preparations. In spite of these drawbacks the enzyme was purified 573-fold. Enzyme activity depended strictly on the presence of Mg²⁺. Kinetic data for chorismate in the forward reaction (K _m = 807 μM, V _max = 6.2 pkat · mg⁻¹) and for isochorismate in the reverse reaction (K _m = 675 μM, V _max = 5.9 pkat · mg⁻¹) were determined. Received: 18 November 1996 / Accepted: 28 December 1996     

Enhanced mineralization of pentachlorophenol by κ-carrageenan-encapsulated Pseudomonas sp. UG30

A pentachlorophenol(PCP)-degrading Pseudomonas sp. strain UG30 was encapsulated in κ-carrageenan for use in PCP degradation. Free and encapsulated cells were compared for their ability to dechlorinate and mineralize 100–800 μg/ml sodium pentachlorophenate in broth. Dechlorination was measured with a chloride ion electrode, and mineralization was measured by ¹⁴CO₂ evolution from radiolabelled [U-¹⁴C]PCP. Free and encapsulated Pseudomonas sp. UG30 cells mineralized up to 200 μg/ml and 600 μg/ml PCP, respectively, after 21 days. Encapsulation of UG30 cells provided a protective effect, allowing dechlorination and mineralization of high levels of PCP to occur. Received: 3 May 1996 / Received revision: 4 September 1996 / Accepted: 13 September 1996     

Extracellular pH affects regulation of the pcbAB gene in Penicillium chrysogenum

The effect of extracellular pH and dissolved oxygen on regulation of the pcbAB gene in P.␣chrysogenum was examined, using Northern analysis and a reporter gene fusion. It was found that ambient pH markedly affected levels of pcbAB mRNA whereas maintenance of dissolved oxygen concentration above 10 % had no detectable effect. The presence of a DNA-binding protein, which binds upstream of the pcbAB translational start codon, was also related to ambient pH. In all fermentations, pcbAB mRNA was most abundant at around the late exponential/early stationary phase of a culture. Received: 10 May 1996 / Received revision: 14 October 1996 / Accepted: 25 October 1996     

Localization and characterization of inclusion bodies in recombinant Escherichia coli cells overproducing penicillin G acylase

Various concentrations of isopropyl β-d-thiogalactopyranoside (IPTG) were used to induce production of the enzyme penicillin G acylase by recom binant Escherichia coli harboring plasmid pQEA11. The plasmid pQEA11 carries a wild-type pga gene, which is under the control of the tac promoter and lacI ^q. At low IPTG concentrations (0.025 – 0.1 mM), enzyme activity increased with increasing IPTG concentrations. At higher IPTG concentrations (0.2 and 0.5 mM), enzyme activity declined progressively. Examination of induced recombinant E. coli cells by transmission electron microscopy showed the presence of only periplasmic inclusion bodies at low IPTG concentrations (up to 0.1 mM) and both periplasmic and cytoplasmic inclusion bodies at high IPTG concentrations (0.2 mM and 0.5 mM). Results from sodium dodecyl sulfate/polyacrylamide gel electrophoresis and immunoblots of whole-cell proteins, membrane proteins and inclusion body proteins in these cells indicated that cytoplasmic inclusion bodies constituted an accumulation of preproenzyme (i.e., precursor polypeptide containing a signal peptide) and that periplasmic inclusion bodies constituted an accumulation of proenzyme (i.e., precursor polypeptide lacking a signal peptide). Received: 27 March 1996 / Received revision: 2 July 1996 / Accepted: 10 November 1996     

Molecular mass of poly[(R )-3-hydroxybutyric acid] produced in a recombinant Escherichia coli

 Poly[(R)-3-hydroxybutyric acid] (PHB) was produced at 37 °C by a recombinant Escherichia coli harboring the Alcaligenes eutrophus biosynthesis phbCAB genes in Luria-Bertani media containing glucose at 10–30 g/l at different pH values and the time-dependent changes in the molecular mass of PHB were studied. PHB polymers accumulated within cells while glucose was present in the medium. The number-average molecular mass of PHB decreased with time during the course of PHB accumulation, and the values for PHB were markedly dependent on the cultivation conditions of the E. coli, ranging from 0.5 MDa to 20 MDa. Under specific conditions (pH 6.0), E. coli produced PHB with an extremely high molecular mass (20 MDa). It has been suggested that a chain-transfer agent is generated in E. coli cells during the accumulation of PHB. Received: 18 July 1996 / Received revision: 4 November 1996 / Accepted: 4 November 1996     

Plasmids for efficient single-copy gene cloning into gdh2 and trpC of Bacillus megaterium DSM319 and QM B1551

We constructed integrative plasmids to place xylA-lacZ indicator gene fusions into two different loci of the Bacillus megaterium chromosome, gdh2 and trpC, in lac mutants of strains DSM 319 and QM B1551, which differ markedly. Single-crossover integration was achieved in all cases while double crossovers occurred only in gdh2 of DSM 319 and QM B1551 and in trpC of QM B1551. Neither of the loci affected regulation of the xylA-lacZ fusions. These results confirm the suitability of the two gene loci for single-copy cloning. Received: 28 October 1996 / Received revision: 29 December 1996 / Accepted: 4 January 1997     

Multi-substrate growth kinetics of Pseudomonas putida for phenol removal

The biodegradation of phenol by a pure culture of Pseudomonas putida was investigated in a continuously fed stirred-tank reactor, under aerobic conditions. The dilution rate was varied between 0.0174 h⁻¹ and 0.278 h⁻¹, covering a wide range of dissolved oxygen and the inhibition region of phenol. Through non-linear analysis of the data, a dual-substrate growth kinetics, Haldane kinetics for phenol and Monod kinetics for oxygen, was derived with high correlation coefficients. Respective biokinetic parameters were evaluated as μ_m = 0.569 h⁻¹, K _p = 18.539 mg/l, K _i = 99.374 mg/l, K _o = 0.048 mg/l, Y _x/p = 0.521 g microorganism/g phenol and Y _x/o = 0.338 g microorganism/g oxygen, being in good agreement with other studies in the literature. Maintenance factors for both phenol and oxygen were calculated for the first time for P. putida while the saturation coefficient for oxygen, K _o, was genuinely evaluated from the constructed model, not imported or adapted from other studies as reported in the literature. All pertinent biokinetic parameters for P. putida have been calculated from continuous system data, which are most appropriate for use in continuous bioprocess applications. Received: 29 July 1996 / Received revision: 18 November 1996 / Accepted: 23 November 1996     

Does the agitation rate and/or oxygen saturation influence exopolysaccharide production by Aureobasidium pullulans in batch culture?

  When Aureobasidium pullulans was grown at a number of agitation rates under batch conditions, exopolysaccharide yields were dramatically reduced at high rates i.e. at least 750 rpm. Investigations with gas blending, which allowed pO₂ manipulation and control independently of the agitation rate, showed that this yield reduction was due solely to the high pO₂ levels that occurred at these agitation rates. Thus, polysaccharide production at 1000 rpm could be elevated by maintaining the pO₂ at a low level during the initial phase of the fermentation. However, both the timing of the pO₂ decrease and the level at which it was maintained were crucial for obtaining yields at 1000 rpm, similar to those observed at low agitation rates. Received: 29 February 1996 / Received revision: 11 July 1996 / Accepted: 15 July 1996     

Dechlorination of polychlorinated biphenyl congeners by an anaerobic microbial consortium

  An anaerobic methanogenic microbial consortium, developed in a granular form, exhibited extensive dechlorination of defined polychlorinated biphenyl (PCB) congeners. A 2,3,4,5,6-pentachlorobiphenyl was dechlorinated to biphenyl via 2,3,4,6-tetrachlorobiphenyl, 2,4,6-trichlorobiphenyl, 2,4-dichlorobi-phenyl and 2-chlorobiphenyl (CB). Removal of chlorine atoms from all three positions of the biphenyl ring, i.e., ortho, meta and para, was observed during this reductive dechlorination process. Biphenyl was identified as one of the end-products of the reductive dechlorination by GC-MS. After 20 weeks, the concentrations of the dechlorination products 2,4,6-CB, 2,4-CB, 2-CB and biphenyl were 8.1, 41.2, 3.0 and 47.8 μM respectively, from an initial 105 μM 2,3,4,5,6-CB. The extent and pattern of the dechlorination were further confirmed by the dechlorination of lightly chlorinated congeners including 2-CB, 3-CB, 4-CB, 2,4-CB and 2,6-CB individually. This study indicates that the dechlorination of 2,3,4,5,6-CB to biphenyl is due to ortho, meta and para dechlorination by this anaerobic microbial consortium. Received: 30 April 1996 / Received revision: 26 July 1996 / Accepted: 5 August 1996     

Biodehalogenation of low concentrations of 1,3-dichloropropanol by mono- and mixed cultures of bacteria

The degradation of low concentrations of 1,3-dichloro-2-propanol (1,3-DCP) and related halohydrins by whole cells and cell-free extracts of soil bacteria has been investigated. Three bacteria (strains A1, A2, A4), isolated from the same soil sample, were distinguished on the basis of cell morphology, growth kinetics and haloalcohol dehalogenase profiles. Strain A1, probably an Agrobacterium sp., dehalogenated 1,3-DCP with the highest specific activity (0.33 U mg protein⁻¹) and also had the highest affinity for 1,3-DCP (K _m, 0.1 mM). Non-growing cells of this bacterium dehalogenated low concentrations of 1,3-DCP with a first-order rate constant (k ₁) of 1.13 h⁻¹ . The presence of a non-dehalogenating bacterium, strain G1 (tentatively identified as Pseudomonas mesophilius), did not enhance the dehalogenation rate of low 1,3-DCP concentrations. However, the mixed-species consortium of strains A1 and G1 had greater stability than the mono-species culture at DCP concentrations above 1.0 gl⁻¹. Received: 30 April 1996 / Received revision: 30 July 1996 / Accepted: 5 August 1996     

Manganese uptake and toxicity in magnesium-supplemented and unsupplemented Saccharomyces cerevisiae

The magnesium content of Saccharomyces cerevisiae was found to vary by up to fivefold at differing␣ stages of batch growth and during growth in the presence of differing magnesium concentrations. Excess Mg was primarily sequestered in vacuoles. Mn²⁺-uptake experiments revealed that Mg-enriched cells had a markedly reduced capacity for Mn²⁺ accumulation. For example, after 6 h incubation in the presence of 50 μM Mn²⁺, Mn levels were approximately twofold higher in cells previously grown in unsupplemented medium than in those from Mg-supplemented medium. These differences were further accentuated at higher Mn²⁺ concentrations and were not attributable to altered cell-surface charge or altered cell-surface Mn²⁺ binding. Cellular Mg status also influenced Mn toxicity towards S. cerevisiae. During exposure to 5 mM Mn²⁺, 50% reductions in the viability of cells with initial Mg contents of approximately 1400 and 2700 nmol (10⁹ cells)⁻¹ occurred after approximately 1.6 h and 3.6 h respectively. In cells containing 3300 nmol Mg (10⁹ cells)⁻¹, more than 75% viability was still maintained after 7 h incubation with 5 mM Mn²⁺. It is concluded that Mn²⁺ uptake and toxicity in S. cerevisiae are strongly influenced by intracellular Mg, possibly through Mg-dependent regulation of divalent-cation transport activity. Received: 15 May 1996 / Received revision: 13 September 1996 / Accepted: 22 September 1996