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.     

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.     

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.     

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 degradation of styrene: biochemistry, molecular genetics, and perspectives for biotechnological applications

Large quantities of the potentially toxic compound styrene are produced and used annually by the petrochemical and polymer-processing industries. It is as a direct consequence of this that significant volumes of styrene are released into the environment in both the liquid and the gaseous forms. Styrene and its metabolites are known to have serious negative effects on human health and therefore, strategies to prevent its release, remove it from the environment, and understand its route of degradation were the subject of much research. There are a large number of microbial genera capable of metabolizing styrene as a sole source of carbon and energy and therefore, the possibility of applying these organisms to bioremediation strategies was extensively investigated. From the multitude of biodegradation studies, the application of styrene-degrading organisms or single enzymes for the synthesis of value-added products such as epoxides has emerged.     

The effect of nutrient limitation on styrene metabolism in Pseudomonas putida CA-3.

Styrene degradation in Pseudomonas putida CA-3 has previously been shown to be subject to catabolite repression in batch culture. We report here on the catabolite-repressing effects of succinate and glutamate and the effects of a limiting inorganic-nutrient concentration on the styrene degradation pathway of P. putida CA-3 in a chemostat culture at low growth rates (0.05 h-1). Oxidation of styrene and the presence of styrene oxide isomerase and phenylacetaldehyde dehydrogenase activities were used as a measure of the expression of the styrene degradation pathway. Both glutamate and succinate failed to repress the styrene degradation ability under growth conditions of carbon and energy limitation. Lower levels of enzyme activities of the styrene degradation pathway were seen in cells grown on styrene or phenylacetic acid (PAA) under conditions of both ammonia and sulfate limitation than were seen under carbon and energy limitation. Cells grown on PAA under continuous culture oxidize styrene and styrene oxide and possess styrene oxide isomerase and NAD(+)-dependent phenylacetaldehyde dehydrogenase activities. Catabolite repression of styrene metabolism was observed in cells grown on styrene or PAA in the presence of growth-saturating (nonlimiting) concentrations of succinate or glutamate under sulfate limitation.     

Glutathione depletion in isolated rat hepatocytes caused by styrene and the thermal degradation products of polystyrene

Depletion of reduced glutathione (GSH) was induced in isolated rat hepatocytes incubated with styrene or exposed for 120 min to products from oxidative thermal degradation of polystyrene. The depletion depended on the concentrations of styrene and on the degradation temperature. Styrene (1 mM) or products from degradation of polystyrene at 200°C (concentration of styrene in exposure atmosphere 0.7 ppm) had no detectable effect on glutathione levels in isolated hepatocytes. At higher degradation temperatures (250°C and 300°C, with styrene concentrations of 2.5 and 25 ppm, respectively) a rapid depletion was detected as well as with 3 mM styrene in incubation mixture. The latency of lactate dehydrogenase was affected neither by the polystyrene degradation products nor by the styrene added to the incubation mixture.     

Biochemistry,genetics and physiology of microbial styrene degradation

The last few decades have seen a steady increase in the global production and utilisation of the alkenylbenzene, styrene. The compound is of major importance in the petrochemical and polymer-processing industries, which can contribute to the pollution of natural resources via the release of styrene-contaminated effluents and off-gases. This is a cause for some concern as human over-exposure to styrene, and/or its early catabolic intermediates, can have a range of destructive health effects. These features have prompted researchers to investigate routes of styrene degradation in microorganisms, given the potential application of these organisms in bioremediation/biodegradation strategies. This review aims to examine the recent advances which have been made in elucidating the underlying biochemistry, genetics and physiology of microbial styrene catabolism, identifying areas of interest for the future and highlighting the potential industrial importance of individual catabolic pathway enzymes.     

Bacterial degradation of styrene in waste gases using a peat filter

A biofiltration process was developed for styrene-containing off-gases using peat as filter material. The average styrene reduction ratio after 190 days of operation was 70% (max. 98%) and the mean styrene elimination capacity was 12 g m⁻³ h⁻¹ (max. 30 g m⁻³ h⁻¹). Efficient styrene degradation required addition of nutrients to the peat, adjustment of the pH to a neutral level and efficient control of the humidity. Maintenance of the water balance was easier in a down-flow than in an up-flow process, the former consequently resulting in much better filtration efficiency. The optimum operation temperature was around 23 °C, but the styrene removal was still satisfactory at 12 °C. Seven different bacterial isolates belonging to the genera Tsukamurella, Pseudomonas, Sphingomonas, Xanthomonas and an unidentified genus in the γ group of the Proteobacteria isolated from the microflora of active peat filter material were capable of styrene degradation. The isolates differed in their capacity to decompose styrene to carbon dioxide and assimilate it to biomass. No toxic intermediate degradation products of styrene were detected in the filter outlet gas or in growing cultures of isolated bacteria. The use of these isolates in industrial biofilters is beneficial at low styrene concentrations and is safe from both the environmental and public health points of view. Received: 30 May 1997 / Received revision: 22 August 1997 / Accepted: 25 August 1997     

The start-up period of styrene degrading biofilters

Styrene vapors from contaminated air were eliminated using long-term adapted mixed microbial culture inoculated on four perlite packed biofilters (serial arrangement, up-flow configuration). During start-up the inlet concentration of styrene rose from 175 to 1300 mg/m³ of total carbon. The total actual residence time in the four biofilters was 24 s. Styrene was successfully degraded by the microbial population in the biofilter. An average of 66% of eliminated styrene was transformed to CO₂. The removal efficiency of the pollutant was, after 18 d of start-up, nearly 85% at an organic load of 170g/m³ per h. The concentration profiles along the bed height were linear for various pollutant inlet concentrations. The total amount of microorganisms in analyzed biomass from the biofilters was about 10⁹ per gram of dry packing mass. The moisture content was around 80% in all biofilters.     

Genetic characterization of accumulation of polyhydroxyalkanoate from styrene in Pseudomonas putida CA-3

Pseudomonas putida CA-3 is capable of accumulating medium-chain-length polyhydroxyalkanoates (MCL-PHAs) when growing on the toxic pollutant styrene as the sole source of carbon and energy. In this study, we report on the molecular characterization of the metabolic pathways involved in this novel bioconversion. With a mini-Tn5 random mutagenesis approach, acetyl-coenzyme A (CoA) was identified as the end product of styrene metabolism in P. putida CA-3. Amplified flanking-region PCR was used to clone functionally expressed phenylacetyl-CoA catabolon genes upstream from the sty operon in P. putida CA-3, previously reported to generate acetyl-CoA moieties from the styrene catabolic intermediate, phenylacetyl-CoA. However, the essential involvement of a (non-phenylacetyl-CoA) catabolon-encoded 3-hydroxyacyl-CoA dehydrogenase is also reported. The link between de novo fatty acid synthesis and PHA monomer accumulation was investigated, and a functionally expressed 3-hydroxyacyl-acyl carrier protein-CoA transacylase (phaG) gene in P. putida CA-3 was identified. The deduced PhaG amino acid sequence shared >99% identity with a transacylase from P. putida KT2440, involved in 3-hydroxyacyl-CoA MCL-PHA monomer sequestration from de novo fatty acid synthesis under inorganic nutrient-limited conditions. Similarly, with P. putida CA-3, maximal phaG expression was observed only under nitrogen limitation, with concomitant PHA accumulation. Thus, beta-oxidation and fatty acid de novo synthesis appear to converge in the generation of MCL-PHA monomers from styrene in P. putida CA-3. Cloning and functional characterization of the pha locus, responsible for PHA polymerization/depolymerization is also reported and the significance and future prospects of this novel bioconversion are discussed.     

Growth of the black yeast Exophiala jeanselmei on styrene and styrene-related compounds

The black yeast Exophiala jeanselmei can grow on styrene as the sole source of carbon and energy in concentrations up to 0.36 mm. No growth is observed at higher styrene concentrations. Styrene oxidation is induced by styrene or styrene-related compounds, whereas glucose represses this styrene oxidation. E. jeanselmei shows a broad substrate specificity: various aromatic compounds are used as the sole source of carbon and energy. Styrene-grown cells can oxidize styrene, styrene oxide, phenylacetaldehyde, phenylacetic acid and 2-phenylethanol at a rate of 1.3 to 3.2 g O₂·min^–1·mg^–1 protein. A pathway for the degradation of styrene in E. jeanselmei is suggested.     

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     

Biosynthesis of polyhydroxyalkanoates from styrene by <Emphasis Type="Italic">Enterobacter</Emphasis> spp. isolated from polluted environment

Background

Styrene and its metabolites are known to have serious adverse effects on human health and hence, strategies to prevent its release, eradicate it from the environment, and understand its route of degradation are being considered.

Methods

A total of 18 strains were isolated from 4 samples of diesel contaminated soils. Among them 5 strains were selected for their ability to degrade styrene and use it as a sole carbon source to produce PHA. These strains were identified as Enterobacter spp. on the basis of 16S rRNA gene sequencing. Bacteria were screened for their ability to produce PHA by utilizing glucose and styrene as a carbon sources. Screening for PHA production was done by Nile blue A, Sudan black B, and phase contrast microscopy and the selected 3 strains showed positive results. Growth kinetics along with time profiling of PHA was performed for glucose and styrene as carbon sources.

Results

PHA extraction was done at equal intervals of 12 h by sodium hypochlorite method which showed that these strains accumulate maximum amount of PHA after 48 h in glucose (30.60%). FTIR analysis of PHA was done which revealed homopolymer PHB and copolymer (PHB-co-PHV) production in strains by utilizing glucose and styrene. Gas chromatography mass spectrometry was carried out to identify the metabolites produced by bacterial strains grown on styrene. Metabolites of styrene degradation included propyne and phenylalanine. Genomic DNA isolation was carried out to amplify phaC gene which encodes PHA synthase enzyme.

Conclusions

The conversion of styrene to polyhydroxyalkanoates (PHA) provides a new and unique link between an aromatic environmental pollutant and aliphatic PHA accumulation.

     

Accumulation of polyhydroxyalkanoate from styrene and phenylacetic acid by Pseudomonas putida CA-3

Pseudomonas putida CA-3 is capable of converting the aromatic hydrocarbon styrene, its metabolite phenylacetic acid, and glucose into polyhydroxyalkanoate (PHA) when a limiting concentration of nitrogen (as sodium ammonium phosphate) is supplied to the growth medium. PHA accumulation occurs to a low level when the nitrogen concentration drops below 26.8 mg/liter and increases rapidly once the nitrogen is no longer detectable in the growth medium. The depletion of nitrogen and the onset of PHA accumulation coincided with a decrease in the rate of substrate utilization and biochemical activity of whole cells grown on styrene, phenylacetic acid, and glucose. However, the efficiency of carbon conversion to PHA dramatically increased once the nitrogen concentration dropped below 26.8 mg/liter in the growth medium. When supplied with 67 mg of nitrogen/liter, the carbon-to-nitrogen (C:N) ratios that result in a maximum yield of PHA (grams of PHA per gram of carbon) for styrene, phenylacetic acid, and glucose are 28:1, 21:1, and 18:1, respectively. In cells grown on styrene and phenylacetic acid, decreasing the carbon-to-nitrogen ratio below 28:1 and 21:1, respectively, by increasing the nitrogen concentration and using a fixed carbon concentration leads to lower levels of PHA per cell and lower levels of PHA per batch of cells. Increasing the carbon-to-nitrogen ratio above 28:1 and 21:1 for cells grown on styrene and phenylacetic acid, respectively, by decreasing the nitrogen concentration and using a fixed carbon concentration increases the level of PHA per cell but results in a lower level of PHA per batch of cells. Increasing the carbon and nitrogen concentrations but maintaining the carbon-to-nitrogen ratio of 28:1 and 21:1 for cells grown on styrene and phenylacetic acid, respectively, results in an increase in the total PHA per batch of cells. The maximum yields for PHA from styrene, phenylacetic acid, and glucose are 0.11, 0.17, and 0.22 g of PHA per g of carbon, respectively.     

Microbial degradation of [C14C]polystyrene and 1,3-diphenylbutane.

Microbial degradation of [beta-14C]polystyrene and 1,3-diphenylbutane, a compound structurally representing the smallest repeating unit of styrene (dimer), was investigated in soil and liquid enrichment cultures. Degradation rates in soil, as determined by 14CO2 evolution from applied [14C]polystyrene, varied from 1.5 to 3.0% for a 4-month period. Although relatively low, these percentages were 15 to 30 times greater than values previously reported. Enrichment cultures, containing 1,3-diphenylbutane as the only carbon souce, were used to determine the mechanisms of microbial oxidation of the polymer chain ends. Metabolism of 1,3-diphenylbutane appeared to involve the attack by a monooxygenease to form 2-phenyl-4-hydroxyphenylbutane followed by a further oxidation and subsequent fission of the benzene ring to yield 4-phenylvaleric acid and an unidentified 5-carbon fragment via the classic meta-fission pathway. Phenylacetic acid was probably formed from 4-phenylvaleric acid by subsequent beta-oxidation of the side chain, methyl-oxidation and decarboxylation. An initial examination of the population of microorganisms in the diphenylbutane enrichment cultures indicated that these oxidative reactions are carried out by common soil microorganism of the genera Bacillus, Pseudomonas, Micrococcus, and Nocardia.     

Identification and characterization of epoxide hydrolase activity of polycyclic aromatic hydrocarbon-degrading bacteria for biocatalytic resolution of racemic styrene oxide and styrene oxide derivatives

A novel epoxide hydrolase (EHase) from polycyclic aromatic hydrocarbon (PAH)-degrading bacteria was identified and characterized. EHase activity was identified in four strains of PAH-degrading bacteria isolated from commercial gasoline and oil-contaminated sediment based on their growth on styrene oxide and its derivatives, such as 2,3- and 4-chlorostyrene oxides, as a sole carbon source. Gordonia sp. H37 exhibited high enantioselective hydrolysis activity for 4-chlorostyrene oxide with an enantiomeric ratio of 27. Gordonia sp. H37 preferentially hydrolyzed the (R)-enantiomer of styrene oxide derivatives resulting in the preparation of a (S)-enantiomer with enantiomeric excess greater than 99.9 %. The enantioselective EHase activity was identified and characterized in various PAH-degrading bacteria, and whole cell Gordonia sp. H37 was employed as a biocatalyst for preparing enantiopure (S)-styrene oxide derivatives.