Aerobic biodegradation of 4-methylpyridine and 4-ethylpyridine by newly isolated Pseudonocardia sp. strain M43

Aerobicbiodegradationof4-methylpyridineand4-ethylpyridinebynewly isolatedPseudonocardia sp. strainM43Jay J. Lee1,2, Jung-Hoon Yoon3, Sang-Yong Yang1 & Sung-Taik Lee2

1Nakdong River Environment Research Laboratory, National Institute of Environmental Research, Kyungpook, Korea; 2Department of Biological

Sciences, Korea Advanced Institute of Science and Technology, Taejon, Korea; and 3Korea Research Institute of Bioscience and Biotechnology,

Taejon, Korea

Correspondence: Jay J. Lee, Keum River

Environment Research Laboratory, National

Institute of Environmental Research, 395-1

Dongdae-Ri, Annae, Okchun, Chungbuk,

Republic of Korea. Tel.: 182 43 733 9407;

fax: 182 43 733 9408;

e-mail: [email protected]

Received 20 June 2005; revised 4 October

2005; accepted 10 October 2005.

First published online December 2005.

doi:10.1111/j.1574-6968.2005.00019.x

Editor: Elizabeth Baggs

Keywords

biodegradation; 4-methylpyridine;

4-ethylpyridine; Pseudonocardia strain.

Abstract

A filamentous bacterium capable of utilizing 4-methylpyridine and 4-ethylpyr-

idine as the sole source of carbon, nitrogen and energy was isolated from sludge.

The organism, designated as strain M43, clustered most closely with members of

the genus Pseudonocardia by 16S rRNA gene sequence analysis. During the

degradation of 4-methylpyridine and 4-ethylpyridine, c. 60% of nitrogen in the

pyridine ring was released as ammonia. Metabolite analyses showed that 2-

hydroxy-4-methylpyridine and 2-hydroxy-4-ethylpyridine were transiently accu-

mulated during the degradation of 4-methylpyridine and 4-ethylpyridine, respec-

tively. Strain M43 was also able to degrade pyridine, 3,4-dimethylpyridine, 4-

carboxypyridine and 2-hydroxy-4-methylpyridine. The results indicate that de-

gradation of 4-methylpyridine and 4-ethylpyridine by strain M43 proceeded via

initial hydroxylation.

Introduction

Alkylpyridines are nitrogen-containing aromatic com-

pounds usually found as contaminants in the surface water

and ground water near industries processing synthetic fossil

fuels (Riley et al., 1981; Stuermer et al., 1982). The chemical

properties and toxicities of alkylpyridines are affected dra-

matically by the presence of the alkyl substituents in the

ring. Toxicities of alkylpyridines to the ciliated protozoan

Tetrahymena increases with the size of the alkyl group (Sims

& O’Loughlin, 1989). Despite their importance as a group of

environmental contaminant, relatively little is known on the

biodegradation of alkylpyridines.

Biodegradation of alkylpyridines could be initiated by

one of the three following reactions: (i) reduction of the

aromatic ring, (ii) oxidation of the alkyl group and (iii)

oxidation of the aromatic ring (Sims & O’Loughlin, 1989).

Although the key intermediates have not been found,

numerous reports suggest initial reduction of the pyridine

ring during the degradation of pyridine (Shukla & Kaul,

1974; Watson & Cain, 1975; Rhee et al., 1997) and alkylpyr-

idines (Shukla, 1974; Lee et al., 2001, for reviews, see Kaiser

et al., 1996; Fetzner, 1998). In contrast, oxidation of alkyl

groups in alkylpyridines was reported only with a 3-methyl-

pyridine (3-MP)-degrading Pseudomonas sp. KM3 (Koros-

televa et al., 1981) and microscopic fungi (Modyanova et al.,

1990). On the other hand, there are a few studies presenting

oxidation of the aromatic ring as the initial reaction for the

metabolism of pyridine (Zefirov et al., 1994) and alkylpyr-

idines (Kaiser et al., 1993; Feng et al., 1994).

In the case of the degradation of alkylpyridines, mixed

cultures of bacteria under aerobic and sulfate-reducing

conditions are known to degrade 4-MP and 4-ethylpyridine

(4-EP) via an initial hydroxylation pathway. According to

Kaiser et al. (1993), 2-hydroxy-4-methylpyridine was iden-

tified as a metabolite during the transformation of 4-MP by

a mixed culture under a sulfate-reducing condition. Simi-

larly, when Feng et al. (1994) investigated the transforma-

tion of 2-, 3- and 4-EPs by a mixed culture of aerobic

bacteria, 2-hydroxy-4-ethylpyridine was found during the

degradation of 4-EP. However, to the best of our knowledge,

there has been no report on the metabolic pathways involved

in the degradation of 4-substituted alkylpyridines by pure

culture.

This paper describes the isolation and characterization of

a bacterium that degrades 4-MP and 4-EP. A metabolic

FEMS Microbiol Lett 254 (2006) 95–100 c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

pathway is proposed based on the metabolites observed and

the assimilation of a putative intermediate.

Materials andmethods

Enrichmentand culture

Minimal salts medium (MSM) used for the enrichment

culture of microorganisms was prepared as described pre-

viously (Lee et al., 2001). All cultures were carried out

aerobically at 30 1C on a rotary shaker. Unless otherwise

mentioned, all MSM used for experiments contained the

respective alkylpyridines as the sole source of carbon and

nitrogen, and were incubated in 250 mL Erlenmeyer flasks

on a rotary shaking incubator (170 rpm) at 30 1C.

Enrichment cultures were initiated by mixing 45 mL of

MSM with 5 mL of excess sludge collected from the waste-

water treatment facility in the Taegu dyeing industrial

complex, Korea. Cultures were carried out in 250 mL

Erlenmeyer flasks and appended with 0.5 mM each of 2-

MP, 3-MP, 4-MP, 2-EP, 3-EP, 4-EP, 2,3-dimethylpyridine

(2,3-DMP), 2,4-DMP, 2,5-DMP, 2,6-DMP, 3,4-DMP and

3,5-DMP, respectively, and incubated aerobically at 30 1C

with shaking. Degradation of alkylpyridines was monitored

by scanning absorbances of the culture filtrates at wave-

lengths from 200 to 400 nm using a Beckman (Fullerton,

CA) DU-68 spectrophotometer. All the culture experiments

were duplicated, and medium with a substrate and without

an inoculum was used as control.

Identificationofthe isolate

Some of the physiological characteristics of the isolated

bacterium were tested by the API 20NE test strip (bioMer-

ieux, Marcy L’Etoile, France). For the analysis of cellular

fatty acid composition, strain M43 was cultured on MSM

with sucrose as the carbon source, and fatty acids were

extracted and analyzed according to the instructions of the

Microbial Identification System (MIDI, Microbial ID). Iso-

lation and purification of chromosomal DNA were carried

out according to the method described by Yoon et al. (1996).

The sequencing of 16S rRNA gene of strain M43 was

performed as described by Yoon et al. (1998). A phylogenetic

tree was constructed by the method described previously

(Yoon et al., 2000).

Analyticalmethods

Cell growth was estimated by measuring the cell dry weight

of the total culture filtrate because of the flocculated growth

pattern.

Concentrations of alkylpyridines and metabolites were

analyzed by high-performance liquid chromatography

(HPLC) using a m-Bondapak-C18 column (3.9 mm�

300 mm, Waters, Milford, UK) at a flow rate of 0.8 mL

min,�1 and the mobile phase consisted of 30% methanol

and 0.3% acetic acid in distilled water (volume in

volume). Concentrations of ammonia were measured by

an enzyme-based diagnostic kit (Ammonia kit, Sigma, St

Louis, MO). For the identification of the metabolites, the

culture filtrates were separated by HPLC under the same

conditions as those used for the analysis of alkylpyri-

dines, except that acetic acid was not included in the

mobile phase. 2-Hydroxy-4-methylpyridine, used as the

authentic standard, was purchased from Aldrich (St

Louis, MO). For the mass spectral analyses, fractions of

the metabolites from 4-MP separated by the HPLC were

concentrated 10 times by evaporating the solute at 60 1C,

and subjected to Autospec-Ultima E mass spectrometry

(Micromass, Manchester, UK) with the direct insertion

probe method at 70 eV.

Results anddiscussion

Enrichment cultureand isolationof bacteria

Among the enrichment cultures, 3-MP and 3-EP were

degraded within 4 weeks and 4-MP and 4-EP were degraded

within 8 weeks. After complete disappearance of the appro-

priate alkylpyridines, pure cultures were isolated by trans-

ferring the established enrichment cultures to fresh media,

serial dilution and spread plating. Isolation and character-

ization of bacterium that degrades 3-MP and 3-EP have

been described previously (Yoon et al., 2000; Lee et al.,

2001). Isolation of pure cultures degrading 4-MP and 4-EP

was initially unsuccessful. Although pure colonies were

isolated by serial dilution and spread plating of the enrich-

ment culture suspension, none of the isolates was able to

degrade 4-MP or 4-EP in the liquid MSM. However, careful

observation of the 4-MP and 4-EP degrading enrichment

cultures indicated that successive transfer yielded white flocs

on the surface of the cultures. Therefore, isolation of

microorganisms was attempted by spread plating the flocs

on MSM agar supplemented with 1 mM of 4-MP. After 7

days, microbial colonies with an appearance similar to the

flocculation in the enrichment cultures developed. The

ability of the bacteria to degrade 4-MP was confirmed by

liquid culture. One of the 4-MP-degrading isolates was

designated as strain M43 and deposited at the Korean

collection for type cultures (KCTC) under the accession

number KCTC 9914.

Identificationofthebacterium

When cultured on complex media such as Luria broth,

Nutrient broth, Trypticase soy broth and Potato dextrose

broth, strain M43 required more than 14 days for the

visualization of colonies. However, when cultured on MSM

FEMS Microbiol Lett 254 (2006) 95–100c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

96 J.J. Lee et al.

supplemented with 4-MP, fructose or sucrose as the carbon

source, visible colonies developed within 7 days. Therefore,

MSM agar plate supplemented with sucrose was used for the

culture and maintenance of the bacterium. Strain M43 was

maintained on MSM agar appended with sucrose (5 g L�1)

for 6 months without losing the ability to degrade 4-MP. In

the liquid culture using MSM, strain M43 grew as surface or

submerged flocculation, leaving the culture broth clear.

Suspended growth was not achieved by cultivation in any

of the media and culture conditions tested. Accordingly,

difficulties were encountered in the growth measurement,

cell collection and other tests for taxonomic identification.

When cultured on MSM agar plates supplemented with

4-MP or sucrose, the strain M43 showed leathery white

colonies. Old colonies sometimes exhibited thick brownish

yellow aggregates. Light microscopic examination of the

cells showed clumps of filaments about 1 mm in width. The

basic taxonomic characteristics of strain M43 are described

in Table 1, along with the physiological characteristics tested

by the API 20NE test strip and the major cellular fatty acid

composition.

The 16S rRNA gene sequence determined for strain M43

was 1442 nucleotides long. The highest similarity of 99.1%

was found with the corresponding sequence of Pseudono-

cardia sulfidooxidans DSM 44248 (Reichert et al., 1998). The

16S rRNA gene sequence similarity values for strain M43

and the type strains of other validly described Pseudonocar-

dia species were in the range of 95.3–99.1%, indicating

that strain M43 belongs to the genus Pseudonocardia.

A phylogenetic tree showing the position of strain M43

among the members of the genus Pseudonocardia and

related taxa is shown in Fig. 1. Filamentous morphology

and the presence of Iso-, 10-methyl-branched saturated, iso-

branched monounsaturated, straight-chain saturated and

monounsaturated components as major fatty acids of strain

M43 also support the idea that strain M43 belongs to the

genus Pseudonocardia (Holt et al., 1994). The 16S rRNA

gene sequence obtained from the strain M43 was deposited

at the GenBank database under the accession number

AF 378364.

Degradationof4-MPand4-EP

Strain M43 degraded 4-MP and 4-EP without additional

nitrogen sources. 1.88 mM of 4-MP was completely de-

graded in 121 h and 1.63 mM of 4-EP was degraded in

209 h in batch flask cultures. Ammonia concentrations were

increased in the broth during the course of 4-MP and 4-EP

degradation (Fig. 2). About 60% of ring nitrogen for each

compound was released as ammonia. As there was no

additional nitrogen source, detection of ammonia indicates

that the aromatic ring was cleaved and the nitrogen was

released. A total of 86 mg L�1 of cell mass was produced by

inoculating 2.1 mM of 4-MP. Up to 6.4 mM of 4-MP was

degraded within 240 h of incubation in the aerobic batch

culture. Incubation with 4-MP concentrations higher than

6.4 mM and 4-EP concentrations higher than 3.7 mM re-

sulted in more than 60 h of lag time and reduced the rate of

degradation. The optimum initial pH for the degradation of

4-MP was 7.5, but a pH range of 6.5–8.0 was tolerated.

Identificationofmetabolites

During the degradation of 4-MP, changes in the UV absorp-

tion pattern were observed. The alkylpyridine peak at

254 nm decreased and new peaks emerged at wavelengths

around 289 and 224 nm. The new peaks eventually dimin-

ished, indicating that the metabolites were further de-

graded. A similar spectral change was observed during the

degradation of 4-EP. The metabolite from 4-MP was sepa-

rated by the HPLC with a retention time of 2.8 min. When

fractions of the separated metabolites were subjected to UV

spectral analyses, an absorption pattern resembling the

Table 1. Taxonomic characteristics of strain M43

Characteristic Property Compound Assimilation Major fatty acid %

Morphology Filament Glucose 1 Iso-C16 : 0 19.8

Width 1 mm Sucrose 1 10-methyl-C16 : 0 13.7

Motility � Arabinose � C17 : 0o6c 13.1

Gram stain 1 Mannose � C16 : 0 9.0

Oxidase � Mannitol 1 C15 : 0 7.9

Catalase 1 N-Acetylglucosamine � Iso-C16 : 1H 6.7

Reduction of NO3� 1 Maltose 1 Iso-C15 : 0 6.5

Indole from tryptophan � Gluconate 1 10-methyl-C17 : 0 4.3

Glucose acidification � Caprate � C17 : 0 3.7

Arginine dehydrogenase � Adipate 1

Urease � Malate 1

b-galactosidase � Citrate 1

Hydrolysis of esculin � Phenyl acetate 1

Hydrolysis of gelatin � Glycerol 1


97Biodegradation of alkylpyridines by Pseudonocardia sp. strain M43

crude culture filtrate was observed (absorption peaks at

around 289 and 224 nm). The UV absorption spectrum of

commercially available 2-hydroxy-4-methylpyridine was

identical to that of the metabolite and when both com-

pounds were coinjected for the HPLC analysis, a single

symmetric peak appeared. Similarly, the metabolite from

4-EP was separated on the HPLC with a retention time of

4.4 min and showed an identical UV absorption pattern.

Mass analysis of the metabolite produced from 4-MP gave

a molecular ion [M1] of m/z 109 (Fig. 3b). The molecular

ion corresponds to the molecular formula C6H7NO. The

metabolite and commercial 2-hydroxy-4-methylpyridine

(Fig. 3a) showed similar fragmentation patterns. The meta-

bolite from 4-EP was extracted and analyzed by the same

method as the one used for the 4-MP intermediate. Mass

analysis of the metabolite gave a molecular ion [M1] of m/z

123 (Fig. 3c), corresponding to the molecular formula

C6H7NO.

On the basis of UV absorption spectra, the mass spectra

and the data obtained for the 2-hydroxy-4-methylpyridine

intermediate, the metabolites produced from 4-MP and

4-EP were identified as 2-hydroxy-4-methylpyridine and 2-

hydroxy-4-ethylpyridine, respectively. In summary, during

the degradation of 4-MP (1.88 mM) and 4-EP (1.63 mM), 2-

hydroxy-4-MP and 2-hydroxy-4-EP corresponding to 37%

of 4-MP and 41% of 4-EP, respectively, were transiently

accumulated in the culture media (Fig. 2). Identification of

2-hydroxy-4-MP as an intermediate of 4-MP degradation by

an anaerobic sulfate-reducing consortia has been described

previously (Kaiser et al., 1993). Similarly, Feng et al. (1994)

reported accumulation of 2-hydroxy-4-ethylpyridine during

the degradation of 4-EP by an aerobic mixed culture.

0.01

Actinobispora xinjiangensis CCTCC AA97020T (AF056709)

Actinobispora aurantiaca CCTCC AA97002T (AF056707)

Actinobispora alaniniphila CCTCC AA 97001T (AF056708)

Actinobispora yunnanensis IMSNU 22019T (AJ252822)

Pseudonocardia petroleophila IMSNU 22072T (AJ252828)

Pseudonocardia saturnea IMSNU 20052T (AJ252829)

Pseudonocardia thermophila IMSNU 20112T (AJ252830)

Pseudonocardia asaccharolytica DSM 44247T (Y08536)

Pseudonocardia sulfidoxydans DSM 44248T (Y08537)

Pseudonocardia hydrocarbonoxydans IMSNU 22140T (AJ252826)

Strain M43Pseudonocardia halophobica IMSNU 21327T (AJ252827)

Pseudonocardia compacta IMSNU 20111T (AJ252825)

Pseudonocardia autotrophica IMSNU 20050T (AJ252824)

Pseudonocardia alni IMSNU 20049T (AJ252823)

Actinosynnema mirum DSM 43827T(X84447)

Lentzea albidocapillata DSM 44073T

Saccharothrix australiensis ATCC 31947T (X53193)

Kutzneria viridogrisea JCM 3282T (U58530)

Actinokineospora riparia IFO 14541T (X76953)

Streptoalloteichus hindustanus IFO 15115T (D85497)

Kibdelosporangium aridum ATCC 39323T (X53191)

Amycolatopsis orientalis DSM 44040T (X76958)

Saccharopolyspora hirsuta ATCC 27875T (U93341)

Saccharomonospora viridis NCIMB 9602T (Z38007)

Prauserella rugosa DSM 43194T (AF051342)

Thermocrispum agreste DSM 44070T (X79183)

Actinopolyspora halophila ATCC 27976T (X54287)

Fig. 1. Phylogenetic tree based on 16S rRNA gene sequences showing the position of strain M43 within the family Pseudonocardiaceae. Scale bar

represents 0.01 substitutions per nucleotide position.


98 J.J. Lee et al.

However, to the best of our knowledge, this is the first report

on the 2-hydroxylated metabolites produced from 4-MP and

4-EP by pure culture.

Degradationof pyridineand related compounds

Degradation of pyridine and pyridine derivatives was tested

by monitoring changes in the UV absorbance spectrum at

wavelengths of 200–400 nm after 7 and 14 days of incuba-

tion. Among the tested compounds, pyridine, 4-MP, 4-EP

and 2-hydroxy-4-methylpyridine were completely degraded

within 7 days. 3,4-DMP and 4-carboxypyridine were de-

graded within 14 days (Table 2).

In the present study, strain M43 displayed a relatively

narrow substrate range among many pyridine derivatives as

is the case with many microorganisms degrading alkylpyr-

idines (Kaiser et al., 1996). Interestingly, although 2-hydro-

xy-4-methylpyridine, the putative intermediate of 4-MP

degradation, was easily used as a growth substrate, none of

the hydroxylated pyridines were degraded. The inability to

utilize hydroxylated pyridines was confirmed by cells grown

on 4-MP, 4-EP, pyridine and sucrose. Therefore, degrada-

tion of pyridine by strain M43 seems to follow a pathway

different from the one used for the degradation of 4-MP and

4-EP. Although there are numerous reports on the pyridine-

degrading microorganisms, evidence supporting pyridine

degradation via an initial hydroxylation step is scarce (Kaiser

et al., 1996; Fetzner, 1998). In this context, in the case of

strain M43, although degradation of 4-MP and 4-EP pro-

ceeded via 2-hydroxylated intermediates, degradation of

pyridine by this bacterium may follow a pathway different

from the one involving an initial hydroxylation step.

Con

cent

ratio

n (m

M)

0.0

0.5

1.0

1.5

2.0

Time (h)

0 40 80 120 160 200 240

Con

cent

ratio

n (m

M)

0.0

0.5

1.0

1.5

2.0

Time (h)

0 40 80 120 160 200 240

(a)

(b)

Fig. 2. Degradation of 4-methylpyridine (a) and 4-ethylpyridine (b) by

strain M43. Symbols: circle, 4-methylpyridine (a) and 4-ethylpyridine (b);

triangle, 2-hydroxy-4-methylpyridine (a) and 2-hydroxy-4-ethylpyridine

(b); and square, ammonia. The error bars indicate the distribution of

duplicates.

50 75 100 1250

50

100

80

109

5771

109

80

7157

80 123

67

94

53

Rel

ativ

e in

tens

ity (

%)

m/z

50 75 100 1250

50

100

Rel

ativ

e in

tens

ity (

%)

m/z

50 75 100 1250

50

100

Rel

ativ

e in

tens

ity (

%)

m/z

(b)

(c)

(a)

Fig. 3. Electron ionization mass spectra of 2-hydroxy-4-methylpyridine

(a, standard compound), metabolite from 4-methylpyridine (b) and

metabolite from 4-ethylpyridine (c).


99Biodegradation of alkylpyridines by Pseudonocardia sp. strain M43

Acknowledgement

This study was supported by the Eco-Technopia-21 project,

Ministry of Environment, Republic of Korea.

References

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Table 2. Degradation of pyridine and pyridine derivatives by strain M43

Compound

Concentration

(mM)

Degradation

within 7

days

Degradation

within 14

days

Pyridine 1.26 1 1

2-methylpyridine 1.07 � �3-methylpyridine 1.07 � �4-methylpyridine 1.07 1 1

2,3-dimethylpyridine 0.93 � �2,4-dimethylpyridine 0.93 � �2,5-dimethylpyridine 0.93 � �2,6-dimethylpyridine 0.93 � �3,4-dimethylpyridine 0.93 � 1

3,5-dimethylpyridine 0.93 � �2-ethylpyridine 0.93 � �3-ethylpyridine 0.93 � �4-ethylpyridine 0.93 1 1

2-carboxypyridine 0.81 � �3-carboxypyridine 0.81 � �4-carboxypyridine 0.81 � 1

2-hydroxypyridine 1.05 � �3-hydroxypyridine 1.05 � �4-hydroxypyridine 1.05 � �Pyridine-N-oxide 1.05 � �2,3-dihydroxypyridine 0.90 � �2,4-dihydroxypyridine 0.90 � �2,6-dihydroxypyridine 0.90 � �2-hydroxy-4-

methylpyridine

0.92 1 1


100 J.J. Lee et al.

Documents

Aerobic biodegradation of 4-methylpyridine and 4-ethylpyridine by newly isolated Pseudonocardia sp. strain M43