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Mercury-Resistant Bacteria and Petroleum Degradation

Article in Applied Microbiology · February 1974

DOI: 10.1128/AEM.27.1.285-287.1974 · Source: PubMed

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APPLIED MICROBIOLOGY, Jan. 1974, p. 285-287 Vol. 27, No. 1
Copyright 0 1974 American Society for Microbiology Printed in U.SA.

Mercury-Resistant Bacteria and Petroleum Degradation

J. D. WALKER AND R. R. COLWELL
Department of Microbiology, Univ'ersity of Maryland, College Park, Maryland 20742
Received for publication 18 July 1973

The concentration of mercury in water and sediment and in the oil extracted
from water and sediment was determined for samples collected in Colgate Creek,
located in Baltimore Harbor of the Chesapeake Bay. The concentration of
mercury in the oil was 4,000 times higher than in sediment and 300,000 times
higher than in water samples. The mercury-resistant bacterial populations of the
samples studied have been shown to degrade oil, suggesting these bacteria to be a
significant factor in the degradation of oil in Colgate Creek.

The full extent of the effects of petroleum on Hatch and Ott (4), with modifications according
biological communities is not yet fully known to Nelson et al. (8). Additional surface sedi-
(2), although the fact that petroleum persists in ment samples were collected from Baltimore
the natural environment is universally accepted Harbor by using a gravity corer, and these sam-
(1). Recently, Matsumura et al. (7) reported ples were extracted by using a Soxlet method,
that alkaline pH, organic content of sediments, with hexane as the solvent. Preliminary experi-
and microorganisms affect release of mercury ments demonstrated that hexane and benzene
into water. Our own studies in the Chesapeake extracted similar hydrocarbons, quantitatively
Bay have shown that greater populations of and qualitatively. The hexane extracts were
petroleum-utilizing and mercury-resistant bac- analyzed for lead, zinc, copper, nickel, and
teria occur in areas where high concentrations of manganese, in addition to mercury, by atomic
petroleum and mercury are found (9, 14). We absorption spectrophotometry. Cadmium and
were aware that high concentrations of pesti- copper content were measured by atomic ab-
cides have been shown to be associated with oil sorption spectrophotometry, utilizing a graphite
slicks (10). The fortuitous discovery in our atomizer. Benzene extracts of the sediment
laboratory that the concentration of mercury in samples were loaded into a 0.75-inch (3.39-cm)
benzene-extractable material of Chesapeake capillary tube closed at one end. Samples were
Bay sediment was greater than in unextracted tared prior to and after the analysis. Low-res-
Chesapeake Bay sediment led us to examine the olution mass spectrometry scanting to about
mercury and oil relationship with respect to mi- m/e 500 was employed. A CEC 21-103C mass
crobial degradation of petroleum. One aspect of spectrometer was used for the analysis.
the research underway in our laboratory is the From the results shown in Table 1, the
problem of petroleum degradation carried out concentration of mercury in the extracted oil
by the autochthonous microbial populations of was 4,000 times that found in the sediment and
the natural environment (3, 14). Mobilization 300,000 times that of the water column. Labora-
of mercury and the cycling of this heavy metal in tory studies have demonstrated microbial con-
the environment are also being investigated (8, version of mercuric chloride to methyl mercury
9). This paper reports the degradation of oil by (11) and to inorganic mercury (11-13), as well as
mercury-resistant bacteria. conversion of phenylmercuric acetate to di-
Methods for isolating, culturing, and iden-
tifying mercury-resistant bacteria from Colgate TABLE 1. Concentration of mercury in water,
Creek in Baltimore Harbor have been described sediment, and benzene extracts of water and
elsewhere (9, 14). The surface sediment and sediment from Colgate Creek
water samples examined in this study were ~~Mercury concn
collected in Colgate Creek by using a ponar grab Sample
Sample (Mg/gm)
and Niskin sampler, respectively. The concen-
tration of mercury in sediment, water, and ben- Sediment ............... ....... 0.67
zene-extracted water and sediment samples Benzene extract of sediment ............ 2,795.02
was measured by using flameless atomic ab- Water ........... ........... <0.01
sorption spectrophotometry, as described by Benzene extract of water ............... 2,960.56
285
286 NOTES APPL. MICROBIOL.
phenyl mercury (6) and inorganic mercury (8). degrading bacteria were resistant to concentra-
However, the nature of the mercury compounds tions of mercuric chloride ranging from 2 to 30
associated with oil is not known and is presently mg/liter with a Pseudomonas sp. being most
under investigation in our laboratory and also resistant. The fact that these bacteria were
as part of a cooperative study with the Inorganic resistant to such high concentrations of mercury
Chemistry Section, National Bureau of Stan- is of ecological importance, since most bacteria
dards, Washington, D.C. Evidence obtained by require adaptation to mercury resistance. Since
mass spectrometric analysis of the extracted oil these petroleum-degrading bacteria were iso-
suggests that the oil is a complex mixture of lated from the mercury- and oil-rich water and
hydrocarbons (Table 2). Measured concentra- sediment of Colgate Creek, the observed mer-
tions of mercury probably represent the amount cury resistance may be due to the continuous
present in the oil in Colgate Creek rather than exposure to mercury concentrated in the oil
that which is preferentially extracted with ben- found in this environment.
zene. The concentrations of mercury detected To determine whether a high concentration of
in benzene extracts of Colgate Creek sediment heavy metals, including metals other than mer-
and water are significantly higher than the levels cury, was associated with increased oil content
normally present in crude oil. Preliminary in the sediments of Chesapeake Bay, sediment
results indicate that the mercury is associated samples collected in Baltimore Harbor were
with specific organic complexes in the oil. examined for oil and heavy metal content (Fig.
To determine whether mercury-resistant bac- 1). The results obtained indicated that with an
teria are capable of degrading petroleum, repre-
sentative strains of several genera of bacteria TABLE 3. Mercury-resistant strains of bacteria
were examined for ability to utilize petroleum isolated from samples collected in the Chesapeake
(Table 3). Of the six representative strains Bay and tested for ability to degrade petroleum
examined, only an Enterobacter sp. (strain 85) Maximum Petroleum
was unable to utilize petroleum as a sole source
of carbon. Utilization of petroleum by Genus Strain HgCl
no. tolerance utilization
Pseudomonas spp. may prove important since (mg/liter) 7' 14 21
most of the mercury-resistant bacteria isolated
from the Chesapeake Bay have been identified Pseudomonas sp ...... 94 24-40 +
as Pseudomonas spp. (9). In addition to screen- Arthrobacter sp ..... 72 . 4-5 +
ing mercury-resistant bacteria for petroleum- Flavobacterium sp. 119 20-24 +
Vibrio sp ............. 639 12-16 +
degrading ability, a number of petroleum- Pseudomonas sp ...... 244 50-60 +
degrading bacteria were screened for mercury Pseudomonas sp ...... 127 20-24 +
(HgCl) resistance. Since mercury resistance is Citrobacter sp .........132 40-50 +
dependent upon the strain, species, and genus Pseudomonas sp ...... 187 16-20 +
of bacteria examined, a wide range of tolerances Enterobacter sp. ..... 1_12-14
85 -
to mercury was expected. In fact, petroleum- a
Data from Nelson et al. (8).
b Days.
TABLE 2. Composition of benzene-extractable
material from Colgate Creek sediment
Compound type
Paraffins .................................
Percent
20.0
0600,~ - _

I _
CHIOWJM

Monocycloparaffins ........................ 26.0

Dicycloparaffins ........................... 8.5 . bm -j

Tricycloparaffins .......................... 7.2 Ri ---kly i -- . -

Tetracycloparaffins ........................ 2.4 MCKEL

Pentacycloparaffins ........................ 0.5 * coo"

Hexacycloparaffins . 0

Alkylbenzenes ............................. 28.8

Benzcycloparaffins .............. .......... 0.6
Benzdicycloparaffins ....................... 0.2
Alkylnaphthalenes ......................... 3.1
Alkylacenaphthenes ............. .......... 1.0 PCEMT a.

Fluorenes ................................. 1.0 FIG. 1. Relationship of oil and heavy metal con-
Phenanthrenes ............................ 0.5 centrations in sediment samples collected in Balti-
Cyclopentaphenalenes ........... .......... 0.4 more Harbor.
 VOL. 27, 1974 NOTES 287
increased oil content, i.e., > 1%, an increase in Microbial ecology and the problem of petroleum degra-
zinc, chromium, lead, copper, nickel, cadmium, dation in Chesapeake Bay, p. 185-197. In D. G. Ahearn
and S. P. Meyers (ed.), The microbial degradation of
and mercury, but not manganese, can be ex- oil pollutants. Louisiana State Univ. publ. no.
pected. SG-73-01, Center for Wetlands Research, Louisiana
In summary, we suggest that in a heavy State University Baton Rouge, La.
metal-enriched environment, mercury and those 4. Hatch, W. R., and W. L. Ott. 1968. Determination of
heavy metals soluble in oil may concentrate in sub-microgram quantities of mercury by atomic ab-
sorption spectrophotometry. Anal. Chem.
the oil and that the mercury-resistant microor- 40:2085-2087.
ganisms present in the mercury-rich, oil-laden 5. Mast, R. F., R. R. Ruch. 1973. Survey of Illinois crude
samples are capable of utilizing oil. This phe- oils for trace concentrations of mercury and selenium.
nomenon may prove important in the biological Environ. Geol. Notes 65:1-9.
6. Matsumura, F. Y., Y. Gotoh, and G. M. Boush. 1971.
transfer of both mercury and oil in the food Phenylmercuric acetate; metabolic conversion by mi-
chain. croorganisms. Science 173:49-51.
7. Matsumura, F., Y. Gotoh, and G. M. Boush. 1972.
The assistance of W. R. Taylor, Chesapeake Bay Institute, Factors influencing translocation and transformation
Johns Hopkins University, in obtaining facilities for field of mercury in river sediment. Bull. Environ. Contam.
work, is gratefully acknowledged. Ship time on the R/V Toxicol. 8:267-272.
Ridgely Warfield was provided through NSF Grant 8. Nelson, J. D., Jr., W. Blair, F. E. Brinckman, R. R.
GD-31707. Petroleum-utilizing bacteria were screened by J. Colwell, and W. P. Iverson. 1973. Biodegradation of
D. Nelson, Jr., for mercury resistance. W. Blair and F. E. phenylmercuric acetate by mercury-resistant bacteria.
Brinckman graciously provided assistance in the mercury Appl. Microbiol. 26:321-326.
analysis of sediment and petroleum. We are also grateful to 9. Nelson, J. D., Jr., H. L. McClam, and R. R. Colwell.
0. Villa and J. Marks, EPA field laboratory, Annapolis, Md., 1972. The ecology of mercury-resistant bacteria in
for making available to us their data on sediment analyses for Chesapeake Bay, p. 303-312. Proc. Marine Technol.
Baltimore Harbor in the Chesapeake Bay. Raymond C. Soc. Washington, D.C.
Allred, Harrell T. Ford, Mynard C. Hamming, and Gerald 10. Seba, D. B., and E. F. Corcoran. 1969. Surface slicks as
Perkins of Continental Oil Co., Ponca City, Okla., provided concentrators of pesticides in the marine environment.
the mass spectrometry analyses. Pestic. Monit. J. 3:190-193.
This work was supported by Office of Naval Research 11. Spangler, W. J., J. L. Spigarelli, J. M. Rose, and H. M.
Contract no. N 00014-69-A-0220-0006. J.D.W. acknowledges Miller. 1973. Methylmercury: bacterial degradation in
partial support from an Environmental Conservation Post- lake sediments. Science 180:192-193.
doctoral Fellowship granted by the National Wildlife Federa- 12. Summers, A. O., and E. Lewis. 1973. Volatilization of
tion and American Petroleum Institute. mercuric chloride by mercury-resistant plasmid-bear-
ing strains of Escherichia coli, Staphylococcus aureus,
LITERATURE CITED and Pseudomonas aeruginosa. J. Bacteriol.
113:1070-1072.
1. Blumer, M., and J. Sass. 1972. Oil pollution, persistence 13. Summers, A. O., and S. Silver. 1972. Mercury resistance
and degradation of spilled fuel oil. Science in a plasmid-bearing strain of Escherichia coli. J.
176:1120-1122. Bacteriol. 112:1228-1236.
2. Button, D. K. 1971. Petroleum: biological effects in the 14. Walker, J. D., and R. R. Colwell. 1973. Microbial ecology
marine environment, p. 421-429. In D. W. Hood (ed.), of petroleum utilization in Chesapeake Bay, p.
Impingement of man on the oceans. John Wiley and 685-691. API/EPA/USCG conference on prevention
Sons, New York. and control of oil spills. American Petroleum Institute,
3. Colwell, R. R., J. D. Walker, and J. D. Nelson, Jr. 1973. Washington, D.C.

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