Purification and biochemical characterization of recombinant alcohol dehydrogenase from the psychrophilic bacterium <Emphasis Type="Italic">Pseudomonas</Emphasis><Emphasis Type="Italic">frederiksbergensis</Emphasis>

A gram-negative psychrophilic bacterium, with potential for biodegradation of long-chain n-alkanes was isolated from ice samples collected in Spitzbergen, Denmark. On the basis of results of biochemical and morphological tests and sequence analysis of 16S rRNA, the strain was identified as Pseudomonas frederiksbergensis. In this work, a short-chain NAD⁺-dependent alcohol dehydrogenase (alcDH) (Accession number: AAR13804) from the P. frederiksbergensis was cloned and transformed in E. coli BL21 (3DE) competent cells. The alcDH activity was highest in the crude extract of cells induced with 1.0 mM IPTG. The recombinant alcDH enzyme was purified to 93.4% homogeneity using three consecutive purification steps including ammonium sulphate, Q-Sepharose Fast Flow column and gel filtration chromatography employing Superdex 200 10/30 HR column. Enzyme enrichment and yield levels of 31.4 folds and 25.5%, respectively, were achieved. While the subunit molecular mass of the enzyme was determined on SDS-PAGE to be ~38 kDa, the aggregated native form of the enzyme had a molecular mass of ~238 kDa by gel filtration analysis. Reaction conditions optima for the recombinant alcDH were determined with propan-1-ol as the substrate. While the optimum pH was 9, the optimum temperature was 35 °C. The alcDH enzyme exhibited moderate thermal stability with half-lives of 150 min at 55 °C, 27 min at 65 °C and 8 min at 75 °C. Results for kinetic parameters indicated that the apparent K _m value for alcDH with propan-1-ol as the substrate was found to be 1.42 mM and the V _max value was 0.63 mmol mg⁻¹ min⁻¹. Experimental evidence revealed that the recombinant alcDH exhibited a wide range of substrate specificity, with higher levels of specific activity for aliphatic alcohols as compared to secondary alcohols. Taken together, the present study highlights the potential of alcDH as a member of cold-adapted enzymes in several key biotechnological applications including environmental bioremediation and biotransformations. It is envisaged that, with the ongoing screening of microorganisms and metagenomes, directed evolution approaches and the subsequent overexpression of recombinant proteins, more enzymes will be found that are suitable for bioremediation purposes.     

Effects of Poly(Ethylene Glycol) on the Production of Poly(β-Hydroxybutyrate) by Azotobacter vinelandii UWD

Azotobacter vinelandii UWD, ATCC 53799, an engineered strain derived from Azotobacter vinelandii UW was used in the poly(ethylene glycol) (PEG)-modulated synthesis of poly(-hydroxybutyrate) (PHB). To the best of our knowledge, this is the first report on modulating the production of PHB by amending the fermentation broth with PEG using A. vinelandii UWD. It was determined that A. vinelandii UWD is prone to back-mutation to the parent strain; hence fermentation experiments require the use of the antibiotic rifampicin. Diethylene glycol (DEG) and PEGs with molecular weights of 400, 2000, and 3400 Da and pentaerythritol ethoxylate (PEE) were used in the modulated fermentation experiments in a concentration of 2% (w/v). The molecular weight of the resulting polymers was reduced by up to 78%. No impact on the productivity of the strain was observed. Spectroscopic evidence showed that PEG-modulated synthesis resulted in the covalent attachment of the ethylene glycol moiety only when a small molecule, DEG, was used. PEGs had the same effects on the polymer formation in terms of molecular weight reduction as DEG, but no spectroscopic evidence was found for the formation of a covalent linkage between PHB and higher molecular weight PEGs.     

Characterization of a poly(3-hydroxybutyrate) depolymerase fromaureobacterium saperdae: Active site and kinetics of hydrolysis studies

An extracellular poly(3-hydroxybutyrate) (PHB) depolymerase was purified fromAureobacterium saperdae cultural medium by using hydrophobic interaction chromatography. The isolated enzyme was composed of a single polypeptide chain with a molecular mass of 42.7 kDa as determined by SDS-PAGE and by native gel filtration on TSK-HW-55S. The enzyme was not a glycoprotein. Its optimum activity occurred at pH 8.0 and it showed a broad pH stability, ranging from pH 3 to pH 11.N-Bromosuccinamide and 2-hydroxy-5-nitrobenzyl bromide completely inactivated the enzyme, suggesting the involvement of tryptophan residues at the active site of the protein. The enzyme was very sensitive to diisopropyl fluorophosphate and diazo-dl-norleucine methyl ester, showing the importance of serine and carboxyl groups. The modification of cysteine residues byp-hydroxy mercuricbenzoate did not cause a loss of activity, whereas dithiothreitol rapidly inactivated the enzyme, revealing the presence of disulfide bonds.A saperdae depolymerase acted on the surface layer of PHB films and the degradation proceeded by surface erosion releasing monomers and dimers of 3-hydroxybutric acid. The degradation of PHB films byA. saperdae depolymerase was partially inhibited in the presence of excess amounts of enzyme. This phenomenon, already observed by Mukaiet al. with poly(hydroxyalkanoates) depolymerases fromAlcaligenes faecalis, Pseudomonas pickettii, andComamonas testosteroni, was analyzed according to the kinetic model proposed by these authors. The experimental data evidenced a general agreement with the kinetic model, although higher initial degradation rates were found withA. saperdae depolymerase.     

Biochemical and Genetic Characterization of an Extracellular Poly(3-Hydroxybutyrate) Depolymerase from Acidovorax Sp. Strain TP4

To determine the properties of enzymes from bacteria that degrade polypropiolactone (PPL), we isolated 13 PPL-degrading bacteria from pond water, river water, and soil. Nine of these strains were identified as Acidovorax sp., three as Variovorax paradoxus, and one as Sphingomonas paucimobilis. All the isolates also degraded poly(3-hydroxybutyrate) (PHB). A PPL-degrading enzyme was purified to electrophoretical homogeneity from one of these bacteria, designated Acidovorax sp. TP4. The purified enzyme also degraded PHB. The molecular weight of the enzyme was estimated as about 50,000. The enzyme activity was inhibited by diisopropylfluorophosphate, dithiothreitol, and Triton X-100. The structural gene of the depolymerase was cloned in Escherichia coli. The nucleotide sequence of the cloned DNA fragment contained an open reading frame (1476 bp) specifying a protein with a deduced molecular weight of 50,961 (491 amino acids). The deduced overall sequence was very similar to that of a PHB depolymerase of Comamonas acidovorans YM1609. From these results it was concluded that the isolated PPL-degrading enzyme belongs to the class of PHB depolymerases. A conserved amino acid sequence, Gly-X₁-Ser-X₂-Gly (lipase box), was found at the N-terminal side of the amino acid sequence. Site-directed mutagenesis of the TP4 enzyme confirmed that ²⁰Ser in the lipase box was essential for the enzyme activity. This is the first report of the isolation a PHB depolymerase from Acidovorax.     

Production and Purification of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) Degrading Enzyme from Streptomyces sp. AF-111

A poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) degrading bacterial strain designated as AF-111 was isolated from sewage sludge sample. The bacterium was identified by 16S rRNA gene sequencing. The results revealed that strain AF-111 showed 99 % similarity with Streptomyces althioticus strain NRRL B-3981 and designated as Streptomyces sp. strain AF-111. An extracellular PHBV depolymerase enzyme was produced under optimized conditions and purified through ammonium sulphate fractionation and column chromatography. The enzyme was purified to homogeneity, indicated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and molecular weight was found to be approximately 51 kDa. Effect of temperature, pH, metal ions and inhibitors on the PHBV depolymerase activity was determined. The enzyme was stable at wide range of temperature (35–55 °C) and pH (6–8). PHBV depolymerase was stable in the presence of different metal ions except iron and zinc which had inhibitory effect on depolymerase activity. Both ethylenediamine teteracetic acid and phenylmethyl sulphonyl fluoride strongly inhibited enzyme activity which indicates that this enzyme belongs to the serine hydrolase family like other polyhydroxyalkanoate depolymerases. The results show that a depolymerase from strain AF-111 can effectively degrade PHBV, therefore, it can be applied in the process of biochemical monomer recycling.     

Degradation of poly(3-hydroxybutyrate), PHB,by bacteria and purification of a novel PHB depolymerase fromComamonas sp.

Bacteria capable of growing on poly(3-hydroxybutyrate), PHB, as the sole source of carbon and energy were isolated from various soils, lake water, activated sludge, and air. Although all bacteria utilized a wide variety of monomeric substrates for growth, most of the strains were restricted to degrade PHB and copolymers of 3-hydroxybutyrate and 3-hydroxyvalerate, P(3HB-co-3HV). Five strains were also able to decompose a homopolymer of 3-hydroxyvalerate, PHV. Poly(3-hydroxyoctanoate), PHO, was not degraded by any of the isolates. One strain, which was identified asComamonas sp., was selected, and the extracellular depolymerase of this strain was purified from the medium by ammonium sulfate precipitation and by chromatography on DEAE-Sephacel and Butyl-Sepharose 4B. The purified PHB depolymerase was not a glycoprotein. The relative molecular masses of the native enzyme and of the subunits were 45,000 or 44,000, respectively. The purified enzyme hydrolyzed PHB, P(3HB-co-3HV), and—at a very low rate—also PHV. Polyhydroxyalkanoates, PHA, with six or more carbon atoms per monomer or characteristic substrates for lipases were not hydrolyzed. In contrast to the PHB depolymerases ofPseudomonas lemoignei andAlcaligenes faecalis T₁, which are sensitive toward phenylmethylsulfonyl fluoride (PMSF) and which hydrolyze PHB mainly to the dimeric and trimeric esters of 3-hydroxybutyrate, the depolymerase ofComamonas sp. was insensitive toward PMSF and hydrolyzed PHB to monomeric 3-hydroxybutyrate indicating a different mechanism of PHB hydrolysis. Furthermore, the pH optimum of the reaction catalyzed by the depolymerase ofComamonas sp. was in the alkaline range at 9.4.     

Biosynthesis and Characterization of Laccase Catalyzed Poly(Catechol)

Enzymatic polymerization of catechol was conducted batch-wise using laccase enzyme produced by the culture Trametes versicolor (ATCC 200801). The polymerization reaction was carried out in 1:1 (v/v) aqueous-acetone solution, buffered at pH 5.0 with sodium acetate (50 mM) in a sealed, temperature-controlled reactor at 25°C. The molecular weight of the produced polymer was determined with GPC. FT-IR, DSC, and TGA were employed to investigate the structure and thermal behavior of synthesized poly(catechol). It was found that catechol units were linked together with ether bonds and thermal stability of the catechol increased in the poly(catechol) polymeric structure effectively. The number average molecular weight of poly(catechol) was found as 813 ± 3 Da with a very narrow polydispersity value of 1.17 showing selective polymerization of catechol by the enzyme.     

Production, Purification and Activity of an Extracellular Depolymerase from Aspergillus fumigatus

A strain of Aspergillus fumigatus, which was observed to rapidly degrade poly-3-hydroxybutyrate (PHB) in a leaf compost, was found to secrete an extracellular hydrolase when grown on PHB as the sole carbon source. Isolation and characterization of the PHB hydrolase (depolymerase) from this fungus revealed that the enzyme had a molecular weight of 57 kDa, an isoelectric point of 7.2, and a PHB hydrolysis activity maxima which occurred at 70°C and pH 8.0. Affinity labeling experiments suggested that this fungal hydrolase is a type of serine esterase. The cyclic trimers of 3-hydroxybutyrate were found to reversibly inhibit the enzymes.     

Gene Cloning and Characterization of a Poly(<Emphasis Type="SmallCaps">l</Emphasis>-Lactic Acid) Depolymerase from <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. Strain DS04-T

A gene encoding a poly(l-lactic acid) (PLA) depolymerase from Pseudomonas sp. strain DS04-T was cloned and overexpressed in Escherichia coli. The recombinant PLA depolymerase with a molecular weight of 19.2 kDa was purified to homogeneity. The optimum pH and temperature of the PLA depolymerase are 8.5 and 60 °C, respectively. K⁺, Ca²⁺ and Ni²⁺ enhance the enzyme activity, while Na⁺, Zn²⁺, Mg²⁺, Cu²⁺, Fe²⁺, Mn²⁺ and Co²⁺ inhibit it. The inhibition of different chemicals on the PLA depolymerase activity were examined, in which EDTA was found to have a significantly inhibitory effect. The main degradation product of the depolymerase is identified as lactic acid monomer by mass spectrometric analysis. Physicochemical properties, substrate specificity and sequence analysis indicated that PME is a new type of PLA depolymerase.     

Assessment of Bacterial Degradation of Aromatic Hydrocarbons in the Environment by Analysis of Stable Carbon Isotope Fractionation

¹³C/¹²C stable carbon isotope fractionation was used to assess biodegradation in contaminated aquifers with toluene as a model compound. Different strains of anaerobic bacteria (Thauera aromatica, Geobacter metallireducens, and the sulfate-reducing strain TRM1) showed consistent ¹³C/¹²C carbon isotope fractionation with fractionation factors between C = 1.0017 and 1.0018. In contrast, three cultures of aerobic organisms, using different mono- and dioxygenase enzyme systems to initiate toluene degradation, showed variable isotope fractionation factors of C = 1.0027 (Pseudomonasputida strain mt-2), C = 1.0011 (Ralstonia picketii), andC = 1.0004 (Pseudomonas putida strain F1). The great variability of isotope fractionation between different aerobic bacterial strains suggests that interpretation of isotope data in oxic habitats can only be qualitative. A soil column was run as a model system for contaminated aquifers with toluene as the carbon source and sulfate as the electron acceptor and samples were taken at different ports along the column. Microbial toluene degradation was calculated based on the ¹³C/¹²C isotope fractionation factors of the batch culture experiments together with the observed ¹³C/¹²C isotope shifts of the residual toluene fractions. The calculated percentage of biodegradation, B, correlated well with the decreasing toluene concentrations at the sampling ports and indicated the increasing extent of biodegradation along the column. The theoretical toluene concentrations as calculated based on the isotope values matched the measured concentrations at the different sampling ports indicating that the Rayleigh equation can be used to calculate biodegradation in quasi closed systems based on measured isotope shifts. A similar attempt was performed to assess toluene degradation in a contaminated, anoxic aquifer. A transect of groundwater wells was monitored along the main direction of the groundwater flow and revealed decreasing concentrations accompanied with an increase in the ¹³C/¹²C stable carbon isotope ratio of the residual toluene. Calculation of the extent of biodegradation based on the isotope values and laboratory derived isotope fractionation factors showed that the residual toluene was degraded to more than 99% by microbial activity. Calculation of the theoretical residual toluene concentrations based on the measured isotope values described the strongly decreasing concentrations along the plume. Other aromatic hydrocarbons like benzene and naphthalene which were analysed in the same course also showed decreasing concentrations along the groundwater flow path accompanied by increasing ¹³C values indicating biodegradation.     

Microbial Poly(L-Lactide)-Degrading Enzyme Induced by Amino Acids, Peptides, and Poly(L-Amino Acids)

Poly(L-lactide)(PLA)-degrading activities of a fungus, Tritirachium album, and two strains of actinomycetes,Lentzea waywayandensis and Amycolatopsis orientalis, were inducible by some proteins (poly-L-amino acid), peptides and amino acids. Extracellular PLA-degrading activity of the culture filtrates was detected when these strains grew in liquid basal medium containing 0.1% (w/v) of (poly-L-amino acids), peptides or amino acids as the enzyme inducer. In addition to PLA-degrading activity, succinyl-(L-alanyl-L-alanyl-L-alanine)-p-nitroanilide (Suc-(Ala)₃-pNA)-degrading activity was observed, implying that the enzymes produced were protease-type. The enzyme activities produced varied between different strains and different inducers. Silk fibroin was the best inducer for A. orientalis and that elastin was the best inducer for L. waywayandensis and T. album.     

Purification of Aspergillus fumigatus (Pdf1) Poly(β-hydroxybutyrate) (PHB) Depolymerase Using a New, Single-Step Substrate Affinity Chromatography Method: Characterization of the PHB Depolymerase Exhibiting Novel Self-Aggregation Behavior

The extracellular poly(-hydroxybutyrate) (PHB) depolymerase of Aspergillus fumigatus Pdf1 was purified by a new, simple, one-step affinity chromatography method using the substrate PHB. The purified enzyme was glycosylated, with the molecular mass of 40 KD, and exhibited a novel self-aggregation behavior by means of hydrophobic interaction that was resolved by Triton X-100 (TX-100) pretreatment of enzyme and also TX-100 incorporation in the native gel. The apparent K _m value of purified enzyme for PHB was 119 g/mL and 3-hydroxybutyrate was detected as the main endproduct of PHB hydrolysis. The depolymerase was insensitive to phenylmethyl sulfonyl fluoride (PMSF), sodium azide, ethylenediaminetetraacetic acid (EDTA), and para-chloromercuric benzoic acid (PCMB), but was inactivated by dithioerythritol (DTT) and showed specificity for short chain-length poly(-hydroxyalkanoates) (PHAs) such as PHB, poly(hydroxyvalerate) (PHV), and copolymers of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV). Medium-chain-length PHA failed to get hydrolyzed. The enzyme, however, exhibited strong cross reactivity with the Comamonas sp. PHB depolymerase antibodies, but not with PHV depolymerase antibodies of Pseudomonas lemoignei. Southern hybridization and dot blot analysis of A. fumigatus Pdf1 genomic DNA with alkaline phosphatase labeled probes of P. lemoignei PHB and PHV depolymerase genes revealed no homology, although the enzyme hydrolyzed both PHB and PHV.     

Purification and Properties of Extracellular Poly(3-hydroxybutyrate) Depolymerase Produced by <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> 202

An extracellular poly(3-hydroxybutyrate) (PHB) depolymerase produced by a thermotolerant fungal soil isolate, Aspergillus fumigatus 202, was purified and characterized. Maximum PHB depolymerase production was obtained at the end of 48 h with initial medium pH 7.0 and 45 °C in Bushnell Haas Minerals medium containing PHB as sole source of carbon. The PHB depolymerase was purified using size exclusion chromatography to a fold purification of 20.62 and 61.62% yield. SDS-PAGE and isoelectric focusing revealed the molecular weight and pI of the purified enzyme as 63,744 Da and 4.2, respectively. N-terminal amino acid sequence of purified enzyme was HAXDAYLVK. This non-glycosylated enzyme was most active at pH 9.0 and 45 °C. Purified enzyme was inactivated by N-bromosuccinimide and dithiothreitol suggesting the involvement of tryptophan residues and disulfide bonds at its active site. Nonionic detergents like Tween 20, Tween 80 and Triton X-100 inhibited the enzyme activity. Ions like Ca⁺² and Mg⁺² (5 mM) increased the enzyme activity 1.5 times. Fe⁺² effectively inhibited the enzyme activity to 88% whereas Hg⁺² completely inhibited the enzyme.     

Biodegradation of Aliphatic and Aromatic Hydrocarbons: Specificity among Bacteria Isolated from Refinery Waste Sludge

Indigenous microorganisms, enriched and isolated from refinery waste sludge, were observed to possess a broad range of metabolic activities for mixtures of several classes of substrates of petroleum hydrocarbons, such as monoaromatic and polycyclic aromatic hydrocarbons (PAHs) and n- and branched alkanes. Three of the best-growing bacterial isolates selectively enriched with these compounds were identified by 16S rDNA sequencing as belonging to the genera Enterobacter and Ochrobactrum. Two of them, Enterobacter sp. strain EK3.1 and Ochrobactrum sp. strain EK6 utilise a hydrocarbon mixture of the branched alkane 2,6,10,14-tetramethylpentadecane and the PAHs acenaphthylene and acenaphthene. Enterobacter sp. strain EK4 can grow with a mixture of 2,6,10,14-tetramethylpentadecane, toluene, acenaphthylene and acenaphthene as carbon sources. Nucleic acid fingerprint analysis, by terminal restriction fragment length polymorphism (T-RFLP) of the PCR-amplified 16S rRNA genes, of the autochthonous bacterial community in contaminated soil samples showed complex and different community structures under different treatments of refinery waste sludge in landfarm areas. The characteristic peaks of the T-RFLP profiles of the individual, isolated degrading bacteria Enterobacter spp. and Ochrobactrum sp. were detected in the T-RFLP fingerprint of the bacterial community of the four months old treated landfarm soil, suggesting the enrichment of bacteria belonging to the same operational taxonomic units, as well as their importance in degrading activity.     

Connectivity of Hybrid Polymer by Tight Binding Approximation: Example of (Si(CH3)2) n (OSiO) p (OH) q

We computed by a tight binding calculation taking into account hybridization, the total electronic energy of different connectivities (amorphous, fractal and linear) of (Si−(CH₃)₂) _n −(OSiO) _p (OH) _q . We found that the total electronic energy of the amorphous connectivity was the smallest as a function of the number of atoms contained by the hybrid polymer. As the total electronic energy is linked to the stability, we may say that the connectivity of such hybrid polymer has the highest stability for the amorphous connectivity.     

Optimization of Poly(<Emphasis Type="SmallCaps">dl</Emphasis>-Lactic Acid) Degradation and Evaluation of Biological Re-polymerization

Poly(dl-lactic acid) or PLA is a biodegradable polymer. It has received much attention since it plays an important role in resolving the global warming problem. The protease produced by Actinomadura keratinilytica strain T16-1 was previously reported as having PLA depolymerase potential and being applicable to PLA biodegradation, which was used in this work. Therefore, this research demonstrates the important basic knowledge on the biological degradation process by the crude PLA-degrading enzyme from strain T16-1. Its re-polymerization was evaluated. The optimization of PLA degradation by statistical methods based on central composite design was determined. Approximately 6700 mg/l PLA powder was degraded by the crude enzyme under optimized conditions: an initial enzyme activity of 200 U/ml, incubated at 60 °C for 24 h released 6843 mg/l lactic acid with 82% conversion, which was similar to the commercial enzyme proteinase K (81%). The degradable products were re-polymerized repeatedly by using commercial lipase as a catalyst under a nitrogen atmosphere for 6 h. A PLA oligomer was achieved with a molecular weight of 378 Da (n = 5). This is the first report to demonstrate the high efficiency of the enzyme to degrade 100% of PLA powder and to show the biological recycling process of PLA, which is promising for the treatment and utilization of biodegradable plastic wastes in the future.     

Influence of Carbon Sources on The Production and Characterization of The Exopolysaccharide (EPS) by Bacillus sphaericus 7055 Strain

In this study, EPS produced by five Bacillus spp. strains was determined. The only one strain (B. sphaericus 7055) was selected due to its high EPS production and it was investigated by growing this strain in LB broth medium containing various carbon sources. The highest EPS production of this strain was found in medium containing fructose. However, the effect of different concentrations of fructose and molasses on EPS production by the strain was studied. The maximum EPS yield of the strain 7055 was recorded with 2.5% (w/v) fructose, also the highest EPS production was found in 2.5% (w/v) molasses. The strain 7055 was found to contain (98.6%) galactose and (1.4%) glucuronic acid in control medium whereas the composition of the strain 7055 (2.5% (w/v) fructose) was found to be (99.9%) neutral sugar and (0.1%) Glucuronic acid while the strain 7055 (2.5% (w/v) molasses) was found to contain (65.9%), neutral sugars and (34.1%) glucuronic acid.     

Purification and Properties of a Poly (β-hydroxybutyrate) Depolymerase From Penicillium sp.

An extracellular poly (β-hydroxybutyrate) (PHB) depolymerase was purified from a Penicillium sp. DS9701-09a by centrifugation, ultrafiltration, precipitation and gel filtration chromatography. The specific activity of the purified enzyme was 37.9-folds higher than that of the culture supernatant and the recovery yield was 11.8%. The PHB deploymerase molecular mass was 44.8 kDa from analysis of both Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Matrix-assisted laser desorption-time-of-flight (MALDI-TOF) mass spectrometer. The isoelectric point of 6.7 for the enzyme was determined by a two-dimensional electrophoresis. The optimum enzyme activity was observed at a temperature of 50 °C and pH 5.0. The apparent K _m of the enzyme was found to be 1.35 mg/mL. The PHB depolymerase consisted of 16 kinds of normal amino acids. The secondary structure of the enzyme was determined by CD spectrum. α-helix and β-turn were found to be 66% and 34% for the enzyme without ammonium sulphite. Chemical inhibition on the PHB depolymerase activity was examined and EDTA was found to have a significantly inhibitory effect.     

Bacteria Degrading PCBs and CBs Isolated from Long-Term PCB Contaminated Soil

Bacteria able to degrade polychlorinated biphenyls (PCBs) and chlorobenzoic acids (CBs) were isolated from soil that had been contaminated with PCBs for 15–30 years. Contaminated soil in which PCB content ranged between 10–470 mg/kg was naturally vegetated with different plants including ash (Fraxinus excelsior), birch (Betula pendula), black locust (Robinia pseudoacacia), Austrian pine (Pinus nigra) and goat willow (Salix caprea) trees as well as a variety of grasses and forbs. Bacteria able to use biphenyl as a sole source of carbon and energy were found in the root zone of all plants, but occurred in the largest numbers beneath pine and black locust. Bacteria able to degrade chlorobenzoic acids were isolated from the same location contaminated with PCBs. Strains that were taxonomically identified by 16S rDNA as Pandoraea were able to use 2-CB, 3-CB, 2,3-CB, 2,5-CB as sole carbon sources, and the strain Arthrobacter utilised 4-CB.     

Isolation of a fungus from denitrifying activated sludge that degrades highly chlorinated dioxins

 This article reports the potential of denitrifying activated sludge to degrade highly chlorinated dioxins, especially from a (landfill) leacheate treatment plant in Japan, and the isolation from this denitrifying activated sludge of a microorganism able to degrade highly chlorinated dioxins. Using a 700-ml bioreactor, denitrifying activated sludge was cultivated under denitrifying conditions by adding 2.0 ng of a mixture of 4- to 8-chlorinated dioxins from fly ash. The dioxin contents of the sample, effluent, and medium before and after cultivation were measured by gas chromatography–mass spectrometry (GC–MS). After 7 days cultivation, about 90% of added dioxins were lost (average percentage of isomer depletion). A dioxin-degrading microorganism was isolated from the activated sludge. Lignin was added to the medium as a color indicator of aromatic compound degradation, and the lignin-decolorizing microorganisms in the denitrifying activated sludge were screened. Some strains were isolated, and one major isolated fungus, strain 622, decolorized lignin effectively. Strain 622 was identified as an Acremonium sp. from its morphological characteristics. It could decolorize lignin by 24% under paraffin-sealed anaerobic conditions. After the cultivation of strain 622 with a 2 ng/ml mixture of 4- to 8-chlorinated dioxins for 1 day, 82% (average for individual isomers) of the added 4- to 8-chlorinated dioxins had been degraded. Added octachlorodibenzo-p-dioxin (OCDD, 100 ng) was degraded under aerobic conditions after 8 h of incubation. During this process, heptachlorodibenzo-p-dioxin was produced and appeared to be a degradation product of OCDD. 1- or 2-hydroxydibenzo-p-dioxin from OCDD was also identified as the degradation product by GC–MS. These results indicated that OCDD was degraded to the nonchlorinated dibenzo-p-dioxins through dechlorination by Acremonium sp. strain 622. Received: October 12, 2001 / Accepted: March 11, 2002