Elsevier

Chemosphere

Volume 61, Issue 7, November 2005, Pages 965-973
Chemosphere

Isolation and characterization of a diverse group of phenylacetic acid degrading microorganisms from pristine soil

https://doi.org/10.1016/j.chemosphere.2005.03.017Get rights and content

Abstract

A diverse range of microorganisms capable of growth on phenylacetic acid as the sole source of carbon and energy were isolated from soil. Sixty six different isolates were identified and grouped according to 16S rRNA gene RFLP analysis. Subsequent sequencing of 16S rDNA from selected strains allowed further characterization of the phenylacetic acid degrading population isolated from soil. Nearly half (30) of the isolates are Bacillus species while the rest of the isolates are strains from a variety of genera namely, Arthrobacter, Pseudomonas, Rhodococcus, Acinetobacter, Enterobacter, Flavobacterium, and Paenibacillus. Sixty-one of the sixty-six strains reproducibly grew in defined minimal liquid culture medium (E2). All strains isolated grew when at least one hydroxylated derivative of phenylacetic acid was supplied as the carbon source, while 59 out of the 61 strains tested, accumulated ortho-hydroxyphenylacetic acid in the assay buffer; when pulsed with phenylacetic acid. Oxygen consumption experiments failed to indicate a clear link between phenylacetic acid and hydroxy-substituted phenylacetic acid in isolates from a broad range of genera.

Introduction

Phenylacetic acid (PA) and hydroxylated derivatives of PA are produced as catabolic intermediates by both bacteria and fungi growing on a wide variety of different naturally occurring aromatic compounds such as aromatic amino acids or lignin as well as various synthetic aromatic compounds. Several bacteria and fungi utilize aromatic amino acids as carbon sources either aerobically (Sariaslani et al., 1974) or anaerobically (Harwood et al., 1999). Aerobic metabolism of PA by fungi such as Exophiala jeanselmei (Cox et al., 1996) and Aspergillus nidulans (Mingot et al., 1999) typically involves the sequential introduction of hydroxyl groups in reactions catalysed by monooxygenases, generating homogentistate as a ring cleavage intermediate. The evidence for direct oxygenation of PA by bacteria is much less clear. It has been proposed that many Pseudomonads convert PA to 4-hydroxyphenylacetate, which is further oxidised to 3,4-dihydroxyphenylacetate (Dagley et al., 1952, Agrawal et al., 1996), while in Flavobacterium sp., PA is believed to be converted to 3-hydroxyphenylacetate and subsequently to 2,5-dihydroxyphenylacetate (Van den Tweel et al., 1988). In Nocardia salmonicolor, 2-hydroxyphenylacetate has been reported as the initial product of PA metabolism (Sariaslani et al., 1974). Recently, a new pathway for aerobic PA metabolism involving the activation of PA to PA-CoA has been reported in different bacterial species including Pseudomonas putida U (Olivera et al., 1998), Escherichia coli (Ferrández et al., 1998) and Azoarcus evansii (Mohamed et al., 2002). Interestingly A. evansii utilizes two different pathways for PA degradation (Mohamed et al., 2002, Rost et al., 2002). Furthermore, microbial genome projects have uncovered homologous pathways in bacteria not previously considered to be PA utilisers, for example, Bordetella sp., Deinococcus radiodurans, Ralstonia metallidurans and Streptomyces coelicolor (Mohamed et al., 2002), expanding our knowledge regarding the diversity of organisms able to degrade PA.

The PA catabolic pathway in P. putida strain U, the so called phenylacetyl-CoA catabolon (Olivera et al., 1998), is believed to be a convergent route responsible for the catabolism of structurally related compounds and much interest has centred on PA metabolism from a biotechnological standpoint (Luengo et al., 2001) particularly in relation to its potential use as a starting material for the biosynthesis of natural penicillins (Luengo et al., 1986, Luengo et al., 2001, Ferrández et al., 1998).

In an attempt to assess the biodiversity, which exists with respect to PA metabolism within a population of soil microorganisms we screened for new isolates, which were capable of growth with PA as the sole source of carbon and energy. In addition we performed preliminary biochemical analysis on these strains in an attempt to determine the pathway for PA metabolism in operation across the various genera which we isolated.

Section snippets

Chemicals

Phenylacetic acid (PA), ortho-hydroxyphenylacetic acid, meta-hydroxyphenylacetic acid, para-hydroxyphenylacetic acid, 3,4-dihydroxyphenylacetic acid, 2,5-dihydroxyphenylacetic acid were purchased at the highest available purity from Fluka Chemie AG, Buchs Switzerland.

Media and buffers

Mineral salts medium (Evans) was made as previously described (Evans et al., 1970). All sterilised growth substrates were added to the medium post-autoclaving to a concentration of 5 mM.

Microbial strains

All PA degrading strains, forming the PA

16S rDNA gene sequence analysis

Phylogenetic analysis of 16S rDNA sequences from isolates capable of utilizing PA as a sole source of carbon and energy is shown in Fig. 1. The 18 representatives from the RFLP analysis did not cluster into a monophyletic group. They were distributed between the Gram-positive and Gram-negative divisions (Table 1). The isolates that clustered in the Gram-positive division were divided between two phyla, the Firmicutes and Actinobacteria (Brosius et al., 1978). The 16S rDNA sequence identity

Discussion

Molecular analysis was performed on a group of soil isolates which were selected based on their ability to utilize PA as a sole source of carbon in an attempt to assess the biodiversity which exists with respect to the metabolism of PA within typical populations of soil microorganisms. 16S rDNA gene analysis reveals a wide diversity of organisms capable of growth on PA (Table 1). Further investigation of the phylogenetic distribution of the isolates shows that these organisms displayed 16S rRNA

References (37)

  • J. Brosius et al.

    Complete nucleotide sequence of a 16S ribosomal RNA gene from Escherichia coli

    Proc. Natl. Acad. Sci. USA

    (1978)
  • H.H.J. Cox et al.

    Styrene metabolism in Exophiala jeanselmei and involvement of a cytochrome P-450-dependent styrene monooxygenase

    Appl. Environ. Microbiol.

    (1996)
  • S. Dagley et al.

    The bacterial oxidation of phenylacetic acid

    J. Bacteriol.

    (1952)
  • W.A. Duetz et al.

    Methods for intense aeration, growth, storage, and replication of bacterial strains in microtitre plates

    Appl. Environ. Microbiol.

    (2000)
  • J. Felsenstein

    Confidence-limits on phylogenies—an approach using the bootstrap

    Evolution

    (1985)
  • T.A. Hall

    BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT

    Nucleic Acids Symp. Ser.

    (1999)
  • C.S. Harwood et al.

    Anaerobic metabolism of aromatic compounds via the benzonyl-CoA pathway

    FEMS Microbiol. Rev.

    (1999)
  • S.I.T. Kennedy et al.

    Metabolism of mandelate and related compounds by bacterium NCIB 8250

    J. Gen. Microbiol.

    (1968)
  • Cited by (9)

    • Toward a high added value compound 3, 4-dihydroxyphenylacetic acid by electrochemical conversion of phenylacetic acid

      2015, Electrochimica Acta
      Citation Excerpt :

      In this context, several methods have been developed to produce 3,4-DHPAA which has the highest antioxidant power. In fact, it is produced by biological method [7,8] and by chemical reaction [9]. Different attempts have been used to evaluate the antioxidant activity of different compounds [10,11] using an accelerated test [12,13], radical species such as ABTS+ (2,2 azinobis (3-ethylbenzthiazoline-6-sulfonic acid) [14], DPPH [15] and ESR (electron spin resonance) spin trapping technique [16].

    • Identification and characterization of a NADH oxidoreductase involved in phenylacetic acid degradation pathway from Streptomyces peucetius

      2010, Microbiological Research
      Citation Excerpt :

      Several microorganisms have been identified from various sources of environment for their ability to degrade phthalate aerobically or anaerobically (Aftring and Taylor 1981; Nozawa and Maruyama 1988). Similarly, a diverse range of microorganisms from a variety of genera were successfully tested for their capability to grow on PA as the sole source of carbon and energy thereby the degrading pathway for PA was explored (O'Connor et al. 2005). PA degradation in bacteria involves an aerobic hybrid pathway, which was initially described in Pseudomonas putida U (Olivera et al. 1998) and Escherichia coli W (Ferrandez et al. 1998), encoded by the PA gene cluster.

    • Microtiter plates as mini-bioreactors: miniaturization of fermentation methods

      2007, Trends in Microbiology
      Citation Excerpt :

      In a similar study, Ferreira-Torres et al.[31] used 96-square deep-well MTPs to compare libraries of recombinant cyclohexanone monooxygenases in E. coli and Acinetobacter calcoaceticus. The use of MTPs for the screening of heterogeneous culture collections for regiospecific hydroxylations was described for a range of educt–product combinations [32–35]. In the past 7 years, MTPs have become a mature alternative to Erlenmeyer flasks for the batch cultivation of microorganisms and have helped to make the handling and screening of large numbers of strains and mutant libraries less time consuming.

    • Synthesis, characterization and comparative studies on the photophysical and photochemical properties of metal-free and zinc(II) phthalocyanines with phenyloxyacetic acid functionalities

      2011, Polyhedron
      Citation Excerpt :

      The synthesis, characterization and spectroscopic behavior as well as photophysical (fluorescence quantum yields and lifetimes) and photochemical (singlet oxygen and photodegradation quantum yields) properties of the new oxygen containing tetra-substituted (peripherally and non-peripherally) metal-free and zinc(II) Pcs with 2-, 3- and 4-phenyloxyacetic acid functionalities (Scheme 1) are reported in this work. Phenylacetic acid (PA) and hydroxylated derivatives of PA are produced as catabolic intermediates by both bacteria and fungi growing on a wide variety of different naturally occurring aromatic compounds such as aromatic amino acids or lignin as well as various synthetic aromatic compounds [20–22]. In this paper we have combined two functional materials (phthalocyanines and phenylacetic acid) into a single compound via a synthetic methodology.

    View all citing articles on Scopus
    View full text