Shu-Yen Liu

Copolymerization of halogenated phenols and syringic acid

Abstract Enzyme-catalyzed copolymerization of phenols with one to five chlorines (4-chlorophenol; 2,4- and 2,6-dichlorophenol; 4-bromo-2-chlorophenol; 2,3,6- and 2,4,5-trichlorophenol; 2,3,5,6-tetrachlorophenol; and pentachlorophenol) and syringic acid was studied with an extracellular laccase of the fungus Rhizoctonia praticola . This reaction is of interest since it presents a model for explaining the incorporation of anthropogenic compounds into humic substances. When the laccase was incubated together with the halogenated phenols and syringic acid, two types of hybrid products were found: (1) phenols covalently bound to an orthoquinone product of syringic acid resulting in the formation of quinonoid oligomers, and (2) phenols covalently bound to decarboxylated products of syringic acid resulting in the formation of phenolic oligomers. Mass spectra of hybrid oligomers gave typical chlorine isotopic patterns which coincided with their respective chlorophenol monomer. It was concluded that all hybrid products contained only one halogenated phenol molecule and that no dehalogenation took place.

Electrophoretic and serological characterization of Porthetria dispar polyhedron protein

Abstract Several modifications of the dissolution method employed for obtaining Porthetria dispar nuclear polyhedrosis virus from polyhedral inclusion bodies (PIBs) were utilized in attempts to obtain undegraded virus particles and polyhedron proteins. Utilization of brief dissolution periods (3–4 min) with an immediate lowering of p H to 8.5–9 yielded predominantly (> 95%) enveloped virus particles. Examination of the polyhedron proteins by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis showed two protein bands: a major component with a molecular weight of 30,350 ± 200 comprising 88–95% of the sample; and a minor component with a molecular weight of 62,750 ± 1800 comprising 5–12% of the sample. Proteolytic activity which led to the degradation of the polyhedron proteins was detected in the alkaline dissolution mixtures. The rate of degradation and the electrophoretic profile of the proteins varied with p H, ionic strength, and length of exposure to these conditions. The degradation could be minimized by addition of a proteolytic inhibitor either to the dissolution mixture or the freshly prepared polyhedron protein solution. Radial immunodiffusion studies conducted with the polyhedron protein fraction and its homologous antiserum showed one precipitin band at its equivalence point and two or three bands in regions of antigen and antiserum excess. There were also two or three faint precipitin bands observed when an excess of virus was assayed against the polyhedron protein antiserum.

Degradation of Sevin by soil microorganisms

Abstract Microorganisms capable of transforming the pesticide 1-naphthyl N-methyl-carbamate (Sevin) were isolated from soil. Three isolates were able to accelerate the hydrolysis of Sevin to 1-naphthol. Several unidentified intermediates were separated by thin-layer chromatography and also by following the decomposition of Sevin-methyl- 14 C. Since 1-naphthol is a biological as well as a chemical decomposition product of Sevin, its transformation by the isolated microbes was also studied. A fungus, identified as Fusarium solani , altered 1-naphthol rapidly. Whereas one strain of bacterium degraded the hydrolysis product gradually, another strain accumulated it under certain conditions. Mixed cultures of the investigated microbes were more effective in transforming Sevin than pure cultures, and this phenomenon was also observed with 1-naphthol as substrate with the exception of one bacterial strain.

Transformation of metolachlor in soil inoculated with a Streptomyces sp.

Microbial detoxication of pesticides may offer a promising alternative to existing physical-chemical treatment methods. We investigated a strain of Streptomyces sp. which can transform metolachlor in a liquid medium for its ability to decontaminate herbicide-treated soil. A cell suspension of Streptomyces sp. was added to a silt loam soil (Hagerstown, pH 6.1) which was amended with 10 μg of metolachlor containing 5 nCi ring-UL-14C metolachlor per gram of soil, and the mixture was incubated at 28°C. Inoculation of the sterile soil resulted in the rapid transformation of metolachlor. Analyses of one-week-old samples indicated that approximately 70% of the added radioactivity was recovered in the ethyl acetate and water fractions as products from the inoculated reaction mixture, whereas in the uninoculated control less than 8% of the 14C was found as products and about 80% was recovered in the form of unchanged metolachlor. In native soil, however, the rate of metolachlor disappearance was not enhanced by Streptomyces inoculation. In inoculated sterile soil the yields of products were affected by inoculum size, inoculation temperature and substrate concentration, but these variables had no effect on product formation in the inoculated native soil. Addition of Na2CO3 (200 μg/g soil) into native soil significantly promoted growth of Streptomyces due to the higher pH (7.8) and also stimulated transformation of metolachlor by 30%. Our results suggest that proliferation of the inoculated organisms under favorable conditions is essential for their function as metolachlor degraders in native soil.

Carbaryl decomposition to 1-naphthyl carbamate by Aspergillus terreus

Abstract The metabolic transformation of the insecticide carbaryl (1-naphthyl N -methyl carbamate) by Aspergillus terreus resulted in the formation of several hydroxylated products. Since approximately 20% of carbaryl was hydroxylated at the carbamate N -methyl group, it was attempted to clarify the pathway of the side chain. 1-Naphthyl N -hydroxymethyl carbamate was chemically identified, and its further biological transformation to 1-naphthyl carbamate could be established. This intermediate was further degraded to 1-naphthol, but it is not known if biological or chemical activity is responsible for it. 1-Naphthol was evidently further metabolized by the fungus.

Bioconversion of alachlor in an anaerobic stream sediment

Abstract We incubated a stream sediment amended with 50 μg per ml of 14C-ring labeled alachlor, 2-chloro-2′,6′-diethyl- N -(methoxymethyl) acetanilide, anaerobically for 10 weeks at 28°C. After 10 weeks, only 50.5% of the 14C-label was extractable into 30% acetonitrile from a nonsterile sediment. Combustion of the soil pellets indicated that increasing amounts of the 14C-label became strongly adsorbed or bound to the sediment. Chromatographic analysis of the hexane extract of the aqueous phase showed that it contained 16% of the unchanged alachlor and 35% of a product which was determined to be the dechlorinated alachlor by mass spectral analysis (C.I. and E.I.). Only alachlor was found in the extract of sterilized samples, indicating that biological activity caused the formation of the dechlorinated product.

Enzymatic binding of the pollutant 2,6-xylenol to a humus constituent

Dimeric and polymeric hybrid oliogmers were obtained by the reaction of 2,6-xylenol and syringic acid in the presence of an enzyme from the fungus Trametes versicolor. Three hybrid dimers were formed in an assay at pH 4.5, while at pH 6.5, the formation of additional higher polymers with a phenyl-ether bond (tail to head linkage) was detected. The major product was identified as 3,5-dimethyl-3′,5′-dimethoxydiphenoquinone (4,4′), and it amounted to approximately 45% of the applied 2,6-xylenol after 3.5 hr incubation at pH 4.5. About 8% of the 2,6-xylenol was transformed to 4,4′-dihydroxy-3,5-dimethyl-3′,5′-dimethoxydiphenol and 22% to 3-methoxy-5-(2′,6′-dimethylphenoxy)-benzoquinone (1,2).

Microbial Transformation of the Herbicide Metolachlor

Abstract Microbial detoxication of pesticides and other xenobiotics is an important alternative to technologies currently being used for pollution control. We studied the possible biotransformation of metolachlor [2-chloro- N -(2-ethyl-6-methylphenyl)- N -(2-methoxy-1-methylethyl)acetamide], a widely used selective herbicide. After screening approximately 100 microorganisms isolated from metolachlor-contaminated soil using various enrichment techniques, we obtained several bacterial and fungal cultures that are capable of transforming metolachlor. From the growth medium of an actinomycete, eight metabolites of metolachlor were isolated and identified by mass and NMR spectral analysis. We only observed benzylic hydroxylation of the aralkyl side chains and demethylation at the N -alkyl substituent during the transformations. All metabolites had a monochlorine isotopic pattern, indicating that no dehalogenation of the chloroacetyl moiety had occurred. In contrast to the results obtained from the pure culture studies, we demonstrated the mineralization of metolachlor in a soil perfusion experiment. If the soil used in a perfusion system had been previously treated with metolachlor for five years, 18.4% of the added 14 C-metolachlor had evolved as 14 CO 2 after 28 days. If the soil had not been previously exposed to metolachlor, only 3.5% of the 14 CO 2 evolved during a similar treatment. The results clearly indicate that a microbial consortium may be more effective in biodegrading a xenobiotic or a recalcitrant chemical such as metolachlor than a single organism.

TRANSFORMATION OF 2,6-DIETHYLANILIE IN SOIL1

We investigated the fate in soil of 2,6-diethylaniline (2,6-DEA), an intermediate in the microbial metabolism of certain acetanilide herbicides. At a concentration of 100 ppm in a soil-water slurry (10g moist soil in citrate-phosphate buffer, pH 5.2), 2,6-DEA was converted to a number of products. These products were extracted with acetone from the soil fraction of the slurry. The two major products isolated with HPLC were identified as a dimer of 2,6-DEA, N-(2,6-diethylphenyl)-2,6-diethyl-p-benzoquinone monoimine (M-1), and a tetramer (M-2). Varying the pH values of the citrate-phosphate buffers used in the preparation of the soil slurries had a significant effect on the quantity of the products. The highest yields of both M-1 and M-2 were obtained at pH 3.8. The yield of the two products decreased with rising pH values. Sterilization of the soil by gamma irradiation did not inhibit the formation of products M-1 and M-2. However, 1-h autoclaving for 3 consecutive days completely destroyed the ability of the soils to catalyze the transformation of 2,6-DEA. Addition of EDTA to the incubation mixture for a final concentration of 0.6% (wt/vol) decreased the formation of M-1 and M-2, and the addition of the same concentration of NaN3 or Na2S2O4 completely inhibited the transformation of 2,6-DEA. Our results support the assumption that physicochemical factors play a significant role in the oligomerization reaction of an aniline in the soil.