importance of gram-positive naphthalene-degrading bacteria in oil-contaminated tropical marine...

Importance of Gram-positive naphthalene-degrading bacteriain oil-contaminated tropical marine sediments

W.-Q. Zhuang1, J.-H. Tay1, A.M. Maszenan1, L.R. Krumholz2 and S.T.-L. Tay1

1Environmental Engineering Research Centre, School of Civil and Environmental Engineering, Nanyang Technological University,

Singapore 639798, and 2Department of Botany and Microbiology, 770 Van Vleet Oval, University of Oklahoma, Norman, OK 73019,

USA

2002/314: received 17 October 2002, revised 24 January 2003 and accepted 31 January 2003

ABSTRACT

W.-Q. ZHUANG, J . -H . TAY, A .M. MASZENAN, L .R . KRUMHOLZ AND S.T . -L . TAY. 2003.

Aims: The aim of this study was to isolate, characterize and evaluate the importance of naphthalene-degrading

bacterial strains from oil-contaminated tropical marine sediments.

Methods and Results: Three Gram-positive naphthalene-degrading bacteria were isolated from oil-contaminated

tropical intertidal marine sediments by direct isolation or enrichment using naphthalene as the sole source of carbon

and energy. Bacillus naphthovorans strain MN-003 can also grow on benzene, toluene, xylene and diesel fuel while

Micrococcus sp. str. MN-006 can also grow on benzene. Staphylococcus sp. str. MN-005 can only degrade

naphthalene and was not able to use the other aromatic hydrocarbons tested. Strain MN-003 possessed the highest

maximal specific growth rate with naphthalene as sole carbon source. An enrichment culture fed with naphthalene as

sole carbon source exhibited a significant increase in the relative abundances of the three isolates after 21 days of

incubation. The three isolates constituted greater than 69% of the culturable naphthalene-degrading microbial

community. Strain MN-003 outcompeted and dominated the other two isolates in competition studies involving

batch cultures inoculated with equal cell densities of the three isolates and incubated with between 1 and 10 mg l)1

of naphthalene.

Conclusions: Three Gram-positive naphthalene-degrading bacteria were successfully isolated from oil-

contaminated tropical marine sediments. Gram-positive bacteria might play an important role in naphthalene

degradation in the highly variable environment of oil-contaminated tropical intertidal marine sediments. Among the

three isolates, strain MN-003 has the highest maximal specific growth rate when grown on naphthalene, and

outgrew the other two isolates in competition experiments.

Significance and Impact of the Study: This research will aid in the development of bioremediation schemes for

oil-contaminated marine environments. Strain MN-003 could potentially be exploited in such schemes.

Keywords: Bacillus, biodegradation, Gram-positive bacteria, Micrococcus, naphthalene, Staphylococcus.

INTRODUCTION

Polycyclic aromatic hydrocarbons (PAHs) are widely distri-

buted contaminants in diverse environments because of their

common association with many anthropogenic activities,

such as petroleum refining and incomplete combustion of

fossil fuels (Berardesco et al. 1998). PAH bioremediation is

considered an effective and environmentally benign cleanup

technology as it involves the partial or complete bioconver-

sion of these pollutants to microbial biomass, carbon dioxide

and water (Head and Swannell 1999). A successful biore-

mediation strategy will require an in-depth understanding of

Correspondence to: Stephen T.-L. Tay, Environmental Engineering Research Centre,

School of Civil and Environmental Engineering, Nanyang Technological University,

50 Nanyang Avenue, Singapore 639798 (e-mail: [email protected]).

ª 2003 The Society for Applied Microbiology

Letters in Applied Microbiology 2003, 36, 251–257

the factors that influence the biodegradation process and the

ecology of pollutant-degrading bacteria (Langworthy et al.

1998).

Naphthalene, the simplest PAH, has long been used as a

model compound in PAH bioremediation studies. Common

naphthalene-degrading bacteria include Pseudomonas spp.,

Vibrio spp., Mycobacterium spp., Marinobacter spp., and

Sphingomonas spp. (Hedlund et al. 1999). Although many

naphthalene-degrading bacteria have been isolated, these

bacteria may thrive in one environment but may not be able

to compete with other micro-organisms in another environ-

ment as environmental conditions will impose a selection

pressure on specific types of bacteria. Furthermore, indi-

genous bacteria have been shown to outcompete artificially

introduced strains in several bioremediation investigations

(Iwabuchi et al. 1997). Therefore, implementation of a

successful bioremediation strategy should necessitate a

detailed evaluation of the roles of the indigenous bacteria

(Piehler et al. 1999).

This study describes the isolation and characterization of

several strains of naphthalene-degrading bacteria obtained

from oil-contaminated tropical marine sediments. Their

ability to compete for naphthalene in mixed culture was also

examined.

MATERIALS AND METHODS

Isolation procedure

Tropical marine sediments contaminated with marine fuel

oil were aseptically collected from a beach in south

Singapore and stored at –20�C for several months before

use. ONR 7a media (Dyksterhouse et al. 1995) was used for

isolating naphthalene-degrading bacteria. The direct isola-

tion method and the enrichment isolation method were

performed as previously described (Zhuang et al. 2002) with

incubations at 25�C. Isolates were screened to select for

bacteria that can grow rapidly on ONR 7a agar plate with

naphthalene as sole carbon source. Three of the isolates

exhibited relatively faster growth rates than the rest and

were picked and chosen for further study. One isolate,

Bacillus naphthovorans strain MN-003, was obtained using

the direct isolation method and was described previously

(Zhuang et al. 2002). Colonies of strain MN-003 were also

obtained in the enrichment isolation experiments as con-

firmed by partial 16S rDNA partial sequencing (500 bp) of

different colonies. Staphylococcus sp. str. MN-005 and

Micrococcus sp. str. MN-006 were obtained using the

enrichment isolation method. Bacterial counts were per-

formed to monitor the relative abundances of different

bacterial groups in the enrichment culture from which the

isolates were derived. Counts of total culturable heterotro-

phic bacteria were determined using the plate-counting

method and marine agar 2216 (BBL, Difco). Counts of total

culturable naphthalene-degrading bacteria and of strains

MN-003, MN-005 and MN-006 were determined based on

the appearance of colonies on naphthalene-incubated ONR

7a plates inoculated with the enrichment culture. Colony

identity was confirmed by partial 16S rDNA sequencing of

randomly selected colonies with the same morphotype as

strains MN-003, MN-005 and MN-006.

Morphological, phenotypic and phylogeneticcharacterizations

Growth of isolates at different temperatures and salinities

were monitored as previously described (Zhuang et al. 2002).

Enzyme profiles and carbon substrate utilization character-

istics were determined using the API ZYM and API 20E

assays according to the manufacturer’s instructions (Bio-

Merieux, Marcy–l’Etoile, France). Gram-stain and the

Voges-Proskauer test were performed as previously des-

cribed (Smibert and Krieg 1994). A non-staining Gram-

stain method (Buck 1982) was also performed to validate the

Gram-stain result. Isolates were also tested for growth on

benzene, toluene, xylene and diesel oil. Growth was

confirmed by colony formation on agar plates containing

the target substrate as sole carbon source and compared with

control plates without the target substrate.

A whole cell direct lysis PCR amplification method was

used to amplify 16S rDNA, as described previously

(Maszenan et al. 1999). The nearly full-length 16S rRNA

gene was amplified by PCR with forward primer Eubac27F

and reverse primer Universal 1492R1 (Lane 1991). The 16S

rDNA sequence and phylogenetic analysis were performed

as previously described (Tay et al. 1998).

Monod growth kinetics

The Monod growth kinetics were determined for the three

isolates as described previously (Zhuang et al. 2002). Total

cells numbers were counted using DAPI staining

(4¢,6-diamidino-2-phenylindole) and naphthalene disappear-

ance was monitored using gas chromatography (Dykster-

house et al. 1995). Naphthalene concentrations were

determined by reference to calibrated standards. Starting

naphthalene concentrations ranged from 0Æ5 to 10 mg l)1

which is less than the solubility of naphthalene in water

(approx. 30 mg l)1 at 20�C).

Competition in mixed culture

In the competition studies, batch cultures were prepared

with ONR 7a media and inoculated with equal numbers of

each of the three isolates. The mixed cultures were prepared

in triplicate in acid-washed 100 ml serum bottles which

252 W.-Q. ZHUANG ET AL.

ª 2003 The Society for Applied Microbiology, Letters in Applied Microbiology, 36, 251–257

served as batch reactors. Each bottle contained 20 ml ONR

7a media and approx. 0Æ33 · 106 cells ml)1 each of strains

MN-003, MN-005 and MN-006 in exponential growth

phase. The inocula were previously grown on naphthalene

as sole carbon source. The batch cultures were incubated at

three different naphthalene concentrations (1, 3, and

10 mg l)1) and placed on a shaker at 150 rev min)1 at

25�C. Naphthalene was delivered to the bottles in methy-

lene chloride and the methylene chloride was removed by

evaporation in a flow cabinet (Hedlund et al. 1999). The

densities of each isolate were monitored over a period of

21 days, while naphthalene concentrations were measured

every 2 days and replenished where necessary.

Strains MN-003, MN-005 and MN-006 formed orange,

white and yellow colonies, respectively, on Marine 2216 agar

plates. The different colony morphologies allowed the direct

plate counting method to be used to determine the bacterial

densities of individual strains in enrichment cultures that

contained all three strains. For each assay, 1 ml of cell

suspension was sampled and serially diluted (10)3 to 10)6).

A quantity of 100 ll of each dilution was spread on a marine

2216 agar plate. The plates were incubated for 3 days at

25�C and then counted. All experiments were performed in

triplicate.

RESULTS

Isolations and enrichments

A total of six naphthalene-degrading bacterial strains were

isolated from oil-contaminated tropical marine sediments.

Three of them were designated as strains MN-003, MN-005

and MN-006 and chosen for further study, because they

exhibited relatively fast growth rates on ONR 7a agar plates

fed with naphthalene as sole carbon source. Strains

MN-003, MN-005 and MN-006 formed visible colonies

on ONR 7a agar plate within 7 days, while the slower-

growing isolates required at least 10 days. Strain MN-003

was successfully isolated using the direct isolation method

and was also detected with the enrichment isolation method.

Strains MN-005 and MN-006 could not be isolated with

direct isolation, but were obtained with enrichment isolation.

The enrichment culture incubated with naphthalene as

sole carbon source exhibited a gradual and significant

increase in counts of heterotrophic bacteria, from 4Æ4 ± 1Æ8 ·105 cells ml)1 initially to 1Æ4 ± 0Æ4 · 108 cells ml)1 after

21 days of incubation (Fig. 1). An enrichment culture

incubated without naphthalene served as a negative control

and did not show any significant increase in counts of

heterotrophic bacteria. Three types of colonies were detec-

ted on ONR 7a agar plates incubated with inoculum from

the starting enrichment culture, while six types of colonies

were detected from the enrichment culture after incubation

for 21 days. Three colony types were directly associated

with strains MN-003, MN-005 and MN-006. Colonies of

strain MN-003 could be observed from the beginning of the

enrichment experiment, but colonies of strains MN-005 and

MN-006 were only detected after a sufficient period of

incubation. The relative abundances of these three strains

were calculated based on the appearance of the colonies on

the ONR 7a agar plates (Fig. 1). Strains MN-003, MN-005

and MN-006 were present in the enrichment culture

incubated for 21 days at concentrations of 5Æ5 · 107,

0Æ44 · 107 and 2Æ9 · 107 cells ml)1, respectively. These cell

densities are equivalent to relative abundances of 39Æ5, 3Æ2and 20Æ6% of total culturable heterotrophic bacteria, and

43Æ4, 3Æ5 and 22Æ7% of total culturable naphthalene degra-

ding bacteria, respectively.

Characterization of naphthalene-degradingbacteria

Characterization of B. naphthovorans strain MN-003 has

been previously described (Zhuang et al. 2002). Strain MN-

005 is spherical in shape, Gram-positive, catalase-positive

and oxidase-negative. Cells ranged between 0Æ6 and 1Æ3 lm

010203040

6050

708090

100

0 210 3 6 9 12 15 18 21

9

8

7

6

5

Log

CF

U (

ml–1

)

Rel

ativ

ely

abun

danc

e (%

)

Time (days) Time (days)

(a) (b)

Fig. 1 Bacterial enumerations in enrichment

culture. (a) Counts of heterotrophic bacteria

in enrichment culture with 10 mg l)1 naph-

thalene as sole carbon source (j) and without

naphthalene (d). (b) Relative abundance of

naphthalene-degrading bacteria to heterotro-

phic bacteria. (j) Total naphthalene-

degrading bacteria, (j) strain MN-003, (h)

strain MN-005, ( ) strain MN-006 and ( )

other strains

GRAM-POSITIVE NAPHTHALENE DEGRADERS 253


in diameter when grown in marine broth 2216 media at

25�C. Strain MN-005 grew at salinities ranging from 0Æ28 to

5% and temperatures ranging from 15 to 37�C. Strain MN-

006 is spherical in shape, Gram-positive, catalase-positive

and oxidase-positive and occurs in pairs, in triplets and as

tetrads. Cells ranged between 0Æ4 and 1 lm in diameter

when grown in marine broth 2216 media at 25�C. Strain

MN-006 grew at salinities ranging from 0Æ28 to 7% and

temperatures ranging from 15 to 41�C. API ZYM and API

20E results of the three isolates are summarized in Table 1.

Strain MN-003 can use naphthalene, benzene, toluene,

xylene and diesel oil as sole carbon source. Strain MN-005

can only use naphthalene as sole carbon source. Strain MN-

006 can use naphthalene as well as benzene.

Phylogenetic analysis based on the 16S rDNA sequence

showed that strain MN-005 belonged to the Staphylococcus

Strains

API ZYM MN-003 MN-005 MN-006

Alkaline phosphatase + + +

Esterase (C 4) + + +

Esterase Lipase (C 8) + + +

Lipase (C 14) ) ) )Leucine arylamidase + + +

Valine arylamidase + ) +

Cystine arylamidase + ) +

Trypsin + ) +

a-Chymotrypsin + ) +

Acid phosphatase ) + +

Naphthol-AS-BI-phosphohydrolase ) + +

a-Galactosidase ) ) )b-Galactosidase + + )b-Glucuronidase ) ) )a-Glucosidase + + +

b-Glucosidase ) ) )N-acetyl-b-glucosaminidase ) ) )a-Mannosidase ) ) )a-Fucosidase ) ) )

API 20E MN-003 MN-005 MN-006

Beta-galactosidase + + )Arginine dihydrolase + ) )Lysine decarboxylase ) ) )Ornithine decarboxylase ) ) )Citrate utilization ) ) )H2S production ) ) )Urease ) + )Tryptophane deaminase ) ) )Indole production ) ) )Acetoin production + + +

Gelatinase + ) +

Glucose + + +

Mannitiol + + +

Inositol + ) )Sorbitol ± ) )Rhamnose ) ± )Sucrose + + )Melibiose ) ) )Amygdalin + + ±

Arabinose + ) )NO3 -NO2 ) ) )

Table 1 API ZYM and API 20E tests for

strains MN-003, MN-005 and MN-006



genus and was most closely related to Staphylococcus xylosus;

the sequence identity was 99Æ9%. Strain MN-006 belonged

to the Micrococcus genus and was most closely related to

Micrococcus luteus; the sequence identity was 99Æ8%.

Monod growth kinetics

Strains MN-003, MN-005 and MN-006 had maximal

specific growth rates (lmax) of 0Æ32 ± 0Æ03, 0Æ082 ± 0Æ008

and 0Æ30 ± 0Æ02 h)1, respectively, and half-saturation con-

stants (Ks) of 2Æ85 ± 0Æ54, 0Æ79 ± 0Æ10 and 2Æ52 ± 0Æ32 mg l)1,

respectively, when grown with naphthalene as sole carbon

source.

Competition in mixed culture

Results of batch competition experiments incubated with

naphthalene concentrations of 1, 3, and 10 mg l)1 are shown

in Fig. 2. At a naphthalene concentration of 1 mg l)1, cell

concentrations of strains MN-003, MN-005 and MN-006

peaked at 20Æ3 ± 1Æ0 · 106, 2Æ72 ± 0Æ1 · 106 and

11Æ0 ± 2Æ0 · 106 cells ml)1, respectively. At a naphthalene

concentration of 3 mg l)1, cell concentration of strains MN-

003, MN-005 and MN-006 peaked at 25Æ1 ± 0Æ3 · 106,

3Æ64 ± 0Æ1 · 106, and 15Æ4 ± 1Æ2 · 106 cells ml)1, respect-

ively. At a naphthalene concentration of 10 mg l)1, cell

concentrations of strains MN-003, MN-005 and MN-006

peaked at 26Æ6 ± 0Æ6 · 106, 3Æ93 ± 0Æ2 · 106, and 21Æ1 ± 0Æ8· 106 cells ml)1, respectively. Strain MN-003 was most

abundant at the three naphthalene concentrations tested

and grew faster than strains MN-005 and MN-006 under

these mixed culture conditions.

DISCUSSION

Screening for relatively fast-growing naphthalene-degrading

bacteria from oil-contaminated tropical marine sediments

resulted in the recovery of three candidate isolates

B. naphthovorans strain MN-003, Staphylococcus sp. strain

MN-005 and Micrococcus sp. strain MN-006. Although the

isolation methods were unbiased and could select for both

Gram-positive and Gram-negative bacteria, all three candi-

date strains were Gram-positive. This dominance of Gram-

positive bacteria is demonstrated in the high relative

abundances of B. naphthovorans strain MN-003, Staphylo-

coccus sp. strain MN-005 and Micrococcus sp. strain MN-006

in the enrichment culture. The three strains constituted

more than 63% of the total culturable heterotrophic bacteria

and more than 69% of the culturable naphthalene-degrading

bacteria in the enrichment culture. The dominance of

Gram-positive bacteria should not be surprising. Gram-

positive bacteria have a stronger cell envelope than Gram-

negative bacteria and this allows them to thrive in the highly

variable intertidal sediment environment, where sediment

0·00

5·00

10·00

15·00

20·00

25·00

30·00

0 6 12 15 18 21

0·00

5·00

10·00

15·00

20·00

25·00

30·00Time (days)

CF

U (

×106

cells

per

ml)

MN-003

MN-006

MN-005

MN-003

MN-006

MN-005

MN-003

MN-006

MN-005

3 9

0 6 12 15 18 21

Time (days)

3 9

CF

U (

×106

cells

per

ml)

(a)(b)

(c)

Fig. 2 Direct plate counting results of com-

petition among Bacillus naphthovorans strain

MN-003 (r), Staphylococus sp. strain MN-

005 (j) and Micrococcus sp. strain MN-006

(m), at (a) 1 mg l)1, (b) 3 mg l)1, and (c) 10

mg l)1 naphthalene concentration and with

ONR 7a media. Initial cell concentration of

each strain was approx 0Æ33 · 106 cells ml)1



temperatures are high in the day and osmotic pressures and

nutrient supply may change periodically over a daily cycle.

Many different species of bacteria with the ability to

degrade naphthalene and other PAHs have been isolated,

mostly from soil environments. The majority of the PAH-

degrading bacteria were previously found to belong to the

genus Pseudomonas, and the PAH-degradative gene clusters

in these bacteria were highly homologous to the naphthalene

gene (nah gene) cluster from the NAH7 plasmid in

Pseudomonas putida strain G7 (Cerniglia 1993). However,

recent investigations of contaminated soils have uncovered

naphthalene-degrading bacteria that did not hybridize with

NAH7-derived gene probes (Ahn et al. 1999; Lloyd-Jones

et al. 1999), and indicate that there are still many uniden-

tified bacteria with diverse PAH biodegradation pathways

that involve hitherto undiscovered genes and gene clusters.

The microbial communities in marine environments have

generally been reported to be dominated by Gram-negative

bacteria (Dyksterhouse et al. 1995; Berardesco et al. 1998;

Hedlund et al. 1999; Macnaughton et al. 1999; Ravenschlag

et al. 2001). Melcher et al. (2002) reported characterization

of phenanthrene-degrading bacteria from San Diego Bay

sediments that belonged to the genera Vibrio, Marinobacter

or Cycloclasticus, Pseudoalteromonas, Marinomonas, and

Halomonas. However, there is little information on

Gram-positive naphthalene-degrading bacteria in marine

environments, although PAH-degrading bacteria belonging

to the Gram-positive nocardioforms and spore-forming

Paenibacillus groups have recently been isolated from the

rhizosphere of salt marsh plants (Daane et al. 2001). The

three isolates reported in the current study extend our

knowledge of the range of naphthalene-degrading bacteria

found in marine environments. This work suggests that

Gram-positive bacteria may play a key role in PAH

degradation on contaminated tropical beaches.

The relative abundance of strains in the naphthalene-fed

enrichment culture experiment and in the batch culture

competition studies was MN-003 > MN-006 > MN-005.

This trend in relative abundance follows the trend in the

maximal specific growth rates exhibited by the three strains

when grown on naphthalene. Strain MN-003 had the fastest

maximal specific growth rate, while the growth rates of

strains MN-005 and MN-006 were slower than that of strain

MN-003 by 74 and 6%, respectively. Strain MN-005 had a

maximal specific growth rate that was significantly smaller

than the other two strains. Although strain MN-005 had the

smallest half-saturation constant of 0Æ79 mg l)1 and, conse-

quently, the largest substrate affinity for naphthalene, it was

outcompeted in all competition experiments by strains MN-

003 and MN-006, even at the lowest tested naphthalene

concentration of 1 mg l)1.

Of the three strains, the dominant position of strain MN-

003 appears to be related to its superior ability to grow on

naphthalene as there are no specific reasons why the bacilli

would be expected to outcompete cocci in culture. Gram-

positive bacteria isolated from marine sponges are known to

show antimicrobial activities against other Gram-positive

bacteria (Hentschel et al. 2001), but there is no evidence for

release of antimicrobial agents in these experiments. The

ability to produce endospores is an adaptation mechanism

found in Bacillus sp. that may allow strain MN-003 to

survive in the highly variable environmental conditions of

the intertidal marine sediments from which it was isolated.

Endospores are highly resistant to environmental insults

(e.g. heat, dessication, radiation, oxidants and proteases) and

allow the bacilli to persist and be ubiquitous in the

environment without losing the capacity for germination

and outgrowth (Francis and Tebo 2002).

Strain MN-003 was successfully isolated using both the

direct isolation method and the enrichment isolation

method. Strains MN-005 and MN-006 were isolated by

the enrichment isolation method and could not be obtained

by direct isolation. The direct isolation method involves

growing isolates directly from environmental samples and is

often used to isolate the dominant members in a microbial

community. On the other hand, the enrichment isolation

method entails a period of acclimatization in the laboratory

to produce an enrichment culture from which the micro-

organisms of interest are subsequently isolated. The isolates

that are obtained from the enrichment isolation method may

not necessarily be the dominant bacterial members in the

original environmental sample. Strain MN-003 is likely a

dominant member of the microbial community in the oil-

contaminated tropical marine sediment and has potential for

application in bioremediation schemes. This is based on its

high maximal specific growth rate and its high relative

abundance in enrichment culture and in the competition

batch cultures. These results vindicate the use of the direct

isolation method to discover environmentally important

strains.

ACKNOWLEDGEMENTS

This work was supported by Nanyang Technological

University’s Academic Research Fund Project RG52/98 to

S.T.-L. Tay.

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