Mutagenicity of styrene and styrene oxide

Incubation of S. typhimurium strain TA 1535 with styrene increased the number of his⁺ revertants/plate in presence of a fortified S9 rat-liver fraction. Styrene was also highly cytotoxic for Salmonella cells. Styrene oxide, the presumed first metabolite, had a mutagenic effect towards strains TA 1535 and TA 100 both with and without metabolic activation. Styrene is probably mutagenic because it is metabolized to styrene oxide.     

Bacterial degradation of styrene involving a novel flavin adenine dinucleotide-dependent styrene monooxygenase

By using styrene as the sole source of carbon and energy in concentrations of 10 to 500 microM, 14 strains of aerobic bacteria and two strains of fungi were isolated from various soil and water samples. In cell extracts of 11 of the bacterial isolates, a novel flavin adenine dinucleotide-requiring styrene monooxygenase activity that oxidized styrene to styrene oxide (phenyl oxirane) was detected. In one bacterial strain (S5), styrene metabolism was studied in more detail. In addition to styrene monooxygenase, cell extracts from strain S5 contained styrene oxide isomerase and phenylacetaldehyde dehydrogenase activities. A pathway for styrene degradation via styrene oxide and phenylacetaldehyde to phenylacetic acid is proposed.     

Bacterial degradation of styrene involving a novel flavin adenine dinucleotide-dependent styrene monooxygenase.

By using styrene as the sole source of carbon and energy in concentrations of 10 to 500 microM, 14 strains of aerobic bacteria and two strains of fungi were isolated from various soil and water samples. In cell extracts of 11 of the bacterial isolates, a novel flavin adenine dinucleotide-requiring styrene monooxygenase activity that oxidized styrene to styrene oxide (phenyl oxirane) was detected. In one bacterial strain (S5), styrene metabolism was studied in more detail. In addition to styrene monooxygenase, cell extracts from strain S5 contained styrene oxide isomerase and phenylacetaldehyde dehydrogenase activities. A pathway for styrene degradation via styrene oxide and phenylacetaldehyde to phenylacetic acid is proposed.     

Production of enantiopure styrene oxide by recombinant Escherichia coli synthesizing a two-component styrene monooxygenase

A whole cell biocatalytic process was developed to enable the efficient oxidation of styrene to chiral (S)-styrene oxide with an enantiomeric excess better than 99%. Recombinant Escherichia coli cells were employed to express the genes styAB encoding the styrene monooxygenase of Pseudomonas sp. strain VLB120 from an expression plasmid utilizing the alk regulatory system of P. oleovorans GPo1. The strains reached specific activities of up to 70 U* (g cell dry weight)(-1) in shake-flask experiments with glucose as the carbon source. An efficient two-liquid phase fed-batch process was established for the production of (S)-styrene oxide with hexadecane as an apolar carrier solvent and a nutrient feed consisting of glucose, magnesium sulfate, and yeast extract. Engineering of the phase fraction and the composition of organic phase and feed led to a 2-L scale process with maximal volumetric productivities of 2.2 g (S)-styrene oxide per liter liquid volume per hour. This optimized process was based completely on defined medium and used bis(2-ethylhexyl)phthalate as the apolar carrier solvent, which together with substrate and inducer consisted of 50% of the total liquid volume. Using this system, we were able to produce per liter liquid volume 11 g of enantiopure (S)-styrene oxide in 10 h.     

The metabolism of phenethyl bromide, styrene and styrene oxide in the rabbit and rat

1. The chief sulphur-containing metabolite of styrene and sytrene oxide in the rabbit and rat is chromatographically identical with N-acetyl-S-(beta-hydroxyphenethyl)-l-cysteine and this compound is also formed, together with N-acetyl-S-phenethyl-l-cysteine, as a metabolite of phenethyl bromide. 2. The amounts of the phenethylmercapturic acid and its hydroxy derivative excreted in the urine of animals dosed with phenethyl bromide, styrene, styrene oxide, phenyl glycol, S-phenylethylcysteine and phenethylmercapturic acid have been determined. 3. Liver slices convert phenethylcysteine and phenethylmercapturic acid into N-acetyl-S-(beta-hydroxyphenethyl)-l-cysteine. 4. Methods for the determination by gas-liquid chromatography of mandelic acid and hippuric acid, which are metabolites of some of the compounds studied, are described.     

Microbial transformations of styrene and [14C] styrene in soil and enrichment cultures.

Two different mechanisms were responsible for the disappearance of styrene in enrichment cultures: (i) a mixed population of microorganisms, capable of utilizing styrene as a sole carbon source, oxidized this substrate to phenylethanol and phenylacetic acid; (ii) the culture also mediated polymerization of the monomer to low-molecular-weight styrene oligomers. This chemical reaction probably occurred as the result of microbial degradation of butylcatechol, an antioxidant polymerization inhibitor present in commercial styrene. The resultant polymer material was subsequently metabolized. In soil incubation studies, 14CO2 evolution from applied [8-14C] styrene was used to estimate microbial degradation. Approximately 90 percent of the labeled carbon was evolved from a 0.2 percent addition, and about 75 percent was lost from the 0.5 percent application over a 16-week period.     

Occupational styrene exposure and chromosomal aberrations

Results from a study on the clastogenicity of styrene in vivo are reported. The chromosomes in cultured blood lymphocytes from ten men occupationally exposed to styrene and 5 controls were examined. Styrene-exposed men showed an increase in the rate of chromosomal aberrations. The incidence of aberrant cells ranged from 11 to 26% in the lymphocytes of the exposed subjects and was 3% or less in those of the control group.     

Cell-free styrene biosynthesis at high titers

Styrene is an important petroleum-derived molecule that is polymerized to make versatile plastics, including disposable silverware and foamed packaging materials. Finding more sustainable methods, such as biosynthesis, for producing styrene is essential due to the increasing severity of climate change as well as the limited supply of fossil fuels. Recent metabolic engineering efforts have enabled the biological production of styrene in Escherichia coli, but styrene toxicity and volatility limit biosynthesis in cells. To address these limitations, we have developed a cell-free styrene biosynthesis platform. The cell-free system provides an open reaction environment without cell viability constraints, which allows exquisite control over reaction conditions and greater carbon flux toward product formation rather than cell growth. The two biosynthetic enzymes required for styrene production were generated via cell-free protein synthesis and mixed in defined ratios with supplemented L-phenylalanine and buffer. By altering the time, temperature, pH, and enzyme concentrations in the reaction, this approach increased the cell-free titer of styrene from 5.36 ± 0.63 mM to 40.33 ± 1.03 mM, the highest amount achieved using biosynthesis without process modifications and product removal strategies. Cell-free systems offer a complimentary approach to cellular synthesis of small molecules, which can provide particular benefits for producing toxic molecules.     

Microbial transformations of styrene and [14C] styrene in soil and enrichment cultures.

Two different mechanisms were responsible for the disappearance of styrene in enrichment cultures: (i) a mixed population of microorganisms, capable of utilizing styrene as a sole carbon source, oxidized this substrate to phenylethanol and phenylacetic acid; (ii) the culture also mediated polymerization of the monomer to low-molecular-weight styrene oligomers. This chemical reaction probably occurred as the result of microbial degradation of butylcatechol, an antioxidant polymerization inhibitor present in commercial styrene. The resultant polymer material was subsequently metabolized. In soil incubation studies, 14CO2 evolution from applied [8-14C] styrene was used to estimate microbial degradation. Approximately 90 percent of the labeled carbon was evolved from a 0.2 percent addition, and about 75 percent was lost from the 0.5 percent application over a 16-week period.     

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     

Cytogenetic analysis of human peripheral blood lymphocytes in culture exposed in vitro to styrene and styrene oxide

Styrene and styrene oxide mutagenicity was tested in cultured human lymphocytes treated in vitro with various concentrations of test agents. Styrene alone was found mutagenic at the highest concentration used (5 X 10(-4) mol. l-1, combined with the alkylating agent THIO-TEPA it did not affect the chromosome aberration yield. Exposure to styrene oxide gave a positive result showing a clear-cut dose-effect relationship within the concentration range 5 X 10(-6) to 1 X 10(-3) mol. l-1. In combination with THIO-TEPA its effect on chromosome aberration yields was additive. Styrene oxide proved also to be a very potent inducer of sister chromatid exchanges (SCE) within the concentration range 5 X 10(-6) to 1 X 10(-3) mol. l-1 tested. Combined with THIO-TEPA it exhibited a distinct additive effect in the production of SCEs.     

Bone marrow cell chromosomal aberrations and styrene biotransformation in mice given styrene on a repeated oral schedule

Styrene's capacity to induce chromosomal aberrations was studied in bone marrow cells of CD1 male mice. No mutagenic effect could be detected after either a 4-day treatment course with daily oral doses of 500 mg/kg or a 70-day course with daily oral doses of 200 mg/kg. Urinary elimination of styrene metabolites related to styrene-7,8-oxide formation (i.e. phenylethylene glycol, mandelic acid, benzoic acid, phenylglyoxylic acid and total mercapturic acids) was quantitatively evaluated in the group of mice given the 200 mg/kg dose. In parallel, kinetic studies were made on styrene and styrene-7,8-oxide blood concentrations in the same group of animals. These determinations were carried out on days 1 and 70 of treatment by spectrophotometric, gas chromatographic and mass fragmentographic procedures.Not even nanograms of styrene-7,8-oxide were found in the blood of styrene-treated mice. This suggests that the metabolite does not migrate from the cellular compartment where it is formed being immediately metabolized or irreversibly bound to cellular structures.This observation could well explain the lack of mutagenic effects observed.     

Pilot-scale production of (S)-styrene oxide from styrene by recombinant Escherichia coli synthesizing styrene monooxygenase

Recombinant Escherichia coli JM101(pSPZ10) cells produce the styrene monooxygenase of Pseudomonas sp. strain VLB120, which catalyzes the oxidation of styrene to (S)-styrene oxide at an enantiomeric excess larger than 99%. This biocatalyst was used to produce 388 g of styrene oxide in a two-liquid phase 30-L fed-batch bioconversion. The average overall volumetric activity was 170 U per liter over a period of more than 10 h, equivalent to mass transfer rates of 10.2 mmoles per liter per hour at a phase ratio of 0.5. At this transfer rate, the biotransformation system appeared to be substrate mass-transfer limited. The reactor had an estimated power input in the order of 5 W. L(-1), which is close to values typically obtained with commercially operating units. The product could be easily purified by fractional distillation to a purity in excess of 97%. The process illustrates the feasibility of recombinant whole cell biotransformations in two-liquid phase systems with toxic substrates and products.     

Microbial transformation of styrene by anaerobic consortia

Methanogenic microbial consortia, originally enriched from anaerobic sewage sludge with ferulic acid or styrene (vinylbenzene) as sole organic carbon and energy sources, were used to study transformation of styrene under strictly anaerobic conditions. Styrene, which was added as the substrate in a range of concentrations from 0.1 to 10 mmol/l, was extensively degraded but no methane production was observed during incubation for eight months. The addition of yeast extract during the enrichment stage completely inhibited degradation of styrene. Gas chromatography (GC), gas chromatography/mass spectrometry (GC/MS), high performance liquid chromatography (HPLC) analyses of the culture fluid, and GC analyses of the anaerobic headspace, indicated that the transformation of this arylalkene was initiated through an oxidation-reduction reaction and that the favoured mechanism was most likely the addition of water across the double bond in the alkenyl side-chain. The degradation proceeded through to carbon dioxide, the final product. Benzoic acid and phenol were transient compounds found in highest concentrations in the spent culture fluid and are suggested as the key intermediates of the transformation process. The tentative routes of anaerobic transformation partially overlap with those previously proposed for aromatic hydrocarbons such as toluene. Several pure cultures, which were tentatively identified as Clostridium spp. and Enterobacter spp., were isolated from the styrene-degrading consortia. Two of these cultures were demonstrated to grow on styrene as sole carbon and energy source. Additionally, a pure culture of Enterobacter cloacae DG-6 (ATCC 35929) which had been isolated previously from the ferulate-degrading consortium, was shown to degrade styrene through to carbon dioxide.     

Microbial transformation of styrene by anaerobic consortia

Methanogenic microbial consortia, originally enriched from anaerobic sewage sludge with ferulic acid or styrene (vinylbenzene) as sole organic carbon and energy sources, were used to study transformation of styrene under strictly anaerobic conditions. Styrene, which was added as the substrate in a range of concentrations from 0.1 to 10 mmol/l, was extensively degraded but no methane production was observed during incubation for eight months. The addition of yeast extract during the enrichment stage completely inhibited degradation of styrene. Gas chromatog-raphy (GC), gas chromatography/mass spectrometry (GC/MS), high performance liquid chromatography (HPLC) analyses of the culture fluid, and GC analyses of the anaerobic headspace, indicated that the transformation of this arylalkene was initiated through an oxidation-reduction reaction and that the favoured mechanism was most likely the addition of water across the double bond in the alkenyl side-chain. The degradation proceeded through to carbon dioxide, the final product. Benzoic acid and phenol were transient compounds found in highest concentrations in the spent culture fluid and are suggested as the key intermediates of the transformation process. The tentative routes of anaerobic transformation partially overlap with those previously proposed for aromatic hydrocarbons such as toluene. Several pure cultures, which were tentatively identified as Clostridium spp. and Enterobacter spp., were isolated from the styrene-degrading consortia. Two of these cultures were demonstrated to grow on styrene as sole carbon and energy source. Additionally, a pure culture of Enterobacter cloacae DG-6 (ATCC 35929) which had been isolated previously from the ferulate-degrading consortium, was shown to degrade styrene through to carbon dioxide.