To define the natural habitats of meroury-resistant bacteria, water and
sediment samples were separated into filterable, planktonic, and inter-
stitial fractions, respectively (Table 5). Results showed that a non-
uniform distribution of both mercury-resistant bacteria and total mer-
cury concentrations existed among water, plankton, and sediment samples.
The relative enrichment, i.e., greater concentrations, of mercury in
sediments and living organisms, in comparison to water, is consistent
with other published data (3, 4) and agrees with the hypothesis that
planktonic forms of life may be influential in the transport of mercury,
as well as introduction of mercury, into food chains. A definite trend
in the data was that mercury-resistant bacterial populations found asso-
ciated with plankton were relatively larger than those for water or sed-
inent. Observations of water and sediments indicated that in most
cases, mercury-resistant bacteria were also distributed non-wniformly
between different particle size fractions. This suggests that an exam
ination of mercury levels and bacterial populations of individual micro-
enviroments would be the most logical approach to the problem of de-
fining the relationship of mercury-resistant bacterial population size
to environmental mercury levels.

A variety of bacterial cultures were isolated and characterized during
the initial 2 years of the investigation. Fig. 1 shows the average
comparative population distributions of HgClg resistant and total popu-
lations of bacteria. These distributions were obtained by sampling and
testing cultures randomly selected from count plates with and without
added HgCla. The relatively greater diversity of the total population,
with respect to the generic categories used, and the relative enrichment
for Pseudomonas species among the resistant population is evident.

—
MERCURY RESISTANT I I TOTAL
GENUS PERCENT
20 40 60 20
VIBRIO
maACTE |
ACINETOBACTER

CORYNEBACTERIUM

CYTOPHAGA/FLAVOBACTERIUM

BACILLUS

ENTERICS

— — |

Figure 1, Average distribution of genera in total and HgCl2 resistant
populations. (Reproduced with the permission of the Marine

Technélogy Society. )


Te

Table 14. HgClg METABOLIZING BACTERIA

Percent change in (203Hg)4& Incubation

mg Cells Decrease Cellular® time

Source? Culture (dry weight) Live Killed Uninoculated _ uptake (hn)
B2 1 0.99 12.6 5.4 4.6 22.7 48
Be2 3 2.36 38.1 8.5 4.5 51.8 72
Be2 4 1.17 1.1 5.5 4.6 29.6 48
Be 5 1,08 83.4 3.2 - 4.8 1
B2 7 0.84 5.6 4.1 8.0 26.6 24
B2 8 1.17 15.0 5.9 4.6 21.0 48
Be2 10 0.42 26.1 1.1 4.5 31.8 72
Be2 1 0.84 33.4 7.6 - 20.0 1
B-2 12 1,02 9.0 2.5 4.6 20.6 48
Be2 13 0.17 1.7 1.2 45 8.8 72
Be v4 1.26 21.9 7.3 4.6 36.5 48
Be2 15 0.72 14.7 2.7 - 23.8 1
Be2 16 1.41 81.6 6.5 -- 16.0 1
Be2 17 0.72 11.8 ~_ 8.0 _ 24
B-2 19 0.81 31.4 3.0 4.5 30.2 72
Be2 20 0.51 30.4 7.5 4.5 20.2 72
EB-1 21 0.84 14.2 2.0 4.5 13.5 72
EB-1 wa 0.84 33.3 3.2 4.5 11.8 72
EB-1 26 1.48 21,0 5.9 _ 38.2 1
EBe1 28 1.04 37.8 4.8 4.5 30.0 72

ay suspension of washed cells, maintained at 25 C in 4.9 ml of PES and aerated via a
capillary tube, was incubated for 5 min prior to the addition of 0.1 ml of 203pg..
labeled HgCl2 (final concentration of 6 ppm HgClg; approx. 100,000 CPM/ml). 0.1 ml
samples were removed at zero time and at regular intervals to 10.0 ml scintillation
cocktail for counting. Percentage decrease or uptake was based upon counts corrected
for quenching and decay relative to radioactivity in the killed control at t = 0,

selected from 6 ppm HgCl2 count plates of water and sediment from Colgate Creek and
sediment from Eastern Bay. Of the 30 original colonies suboultured from the coun:
plates to broth containing 6 ppm HgClg, 22 grew, with 17 actively metabolizing .

°0.1 m2 aliquots were filtered through .45 Pp Millipore filters and washed three times
with 1.0 ml volumes of PES (25 C).


Asssy for Mercury Evolution

Cultures were incubated with shaking in Erlenmeyer flasks of broth con-
taining 6 ppm HgCl2 for 16 h at 25 C and subcultured (20% inoculum size)
in fresh medium, with subsequent incubation for 1 to 6h, After incuba-
tion, the cells were centrifuged from the medium and washed twice in
0.01 M potassium phosphate-buffered (pH 7.0) estuarine salts (PES).
After resuspension, the cells were divided into three portions: one was
assayed directly, the second was autoclaved at 121 C for 15 min to serve
as a control, and the third was centrifuged, resuspended in distilled
water, and dried at 80 C for dry weight determination. Suspensions of
cells in PES were added to PES in a test tube to 4.9 ml final volume.
The suspensions were aerated with moist air to keep the cells evenly
dispersed and to enhance ohhe evolution of Hg° from solution. After 5
min aeration, 100 ul of 203Hg-labeled HgClg (Amersham Searle Corp.,
Arlington Heights, Illinois) was added. Samples (100 ul) were removed
at intervals and mixed with 10.0 ml of 3a70 preblended liquid scintilla-
tion vials. Duplicate samples were filtered through 0.45 micron Milli-
pore filters (Millipore Corp., Bedford, Massachusetts) and washed thrice
with 1.0 ml volumes of PES at 25 C. The filters were immersed in 10.0
ml scintillation cocktail. Samples were counted in an Intertechnique
model SL-40 liquid scintillation spectrophotometer (Teledyne Isotopes,
Westwood, New Jersey). Radioactivity, expressed in disintegrations per
min, was calculated automatically with a channels ratio program. Cor-
rections for decay (47 day half life) were made when necessary.

Statistical Analyses

Data expressed as percent were converted by arc sine transformation (29)
to approximate a normal distribution. Multiple stepwise linear regres-
sions were performed by an IBM 1108 computer using biomedical program
BMDO3R from the University of Maryland Computer Center Library.

Bacteria and seston were recovered from water samples by centrifugation
at 13,200 x g for 15 min, The greenish-brown material was resuspended
in sterile PES before use. Bacteria were extracted from sediments by a
modification of the methods of Balkwill and Casida (30) developed by
those authors for soils. One hundred grams (wet weight) of sediment was
homogenized with 200 ml of sterile 0.1% sodium pyrophosphate in salts
solution (PES) in a Waring blender for two 30 sec.. blending intervals.
The mixture was centrifuged at 365 x g for 5 min and the supernatant so-
lution was decanted and recentrifuged at 365 x g. The supernatant solu-
tion was again decanted and centrifuged at 13,200 x g for 10 min. The
straw-colored supernatant solution was discarded, and the pellet was re-
suspended in sterile PES and centrifuged at 475 x g for 10 min, The
dark brown pellet was discarded, and the greenish brown supernatant so-
lution was retained for use.

26


Nelson, J.D., L.W. Wan, Z. Vaituzis, and R.R. Colwell. Effects of mer-
curic chloride on the morphology of selected bacterial strains. Present-
ed at the Annual Meeting of the American Society of Microbiologists,

May 6-11, Miami Beach, Florida. (1973)

Nelson, J.D. and R.R. Colwell. Effects of tropical storm Agnes upon the

bacterial flora of Chesapeake Bay. Chesapeake Research Consortium,
Publication #27. (1974)

82


g 3

% of TVC RESISTANT to, 6ppm HgCle
io]

Figure 9.

30

2

20

% of TVC RESISTANT to Gppm HgCl

Figure 10.

MONTH

Seasonal variation in percent of HgClg resistant bacteria in

water. The percent of total viable, aerobic, heterotrophic
bacterial count (TVC) capable of growth on a solid medium con-
taining 6 ppm of HgCl2 was determined. Water les from Station
B-1, 1972 (a); Station B-2, 1972 and 1973 (A,®); Station 4-1,
1972 (@); Station A-2, 1972 and 1973 (TM,0); and Station EB-1,
1972 and 1973 (@,O), were plated and incubated at 25 C for 1 week.

SN \_
—_— ss 2 °.
=

ee
M A M

J F J J

MONTH

Seasonal variation in percent of HgClo resistant bacteria in
sediment. The percent of total, viable, aerobic heterotrophic
bacterial count (TVC) capable of growth on a solid medium con-
taining 6 ppm of eg was determined. Sediment samples from
Station B-1, 1973 (&); Station B-2, 1972 and 1973 (A ©);
Station A-2, 1972 and 1973 (M,€); and Station EB-1, 1972 and
1973 (@,0) were plated and incubated at 25 C for 1 week.

36


SECTION III
INTRODUCTION

Mercury and its compounds (organic and inorganic) are known to undergo a
number of chemical and biological transformations. Microorganisms, in
particular bacteria, have been documented as agents in the formation of
methyl mercury and elemental mercury from inorganic mercury. Similarly,
bacteria have been shown to be capable of degrading PMA, MeHg and other
organomercurials to Hg° and simple carbon compounds.

Inorganic mercury in complexation with organic material is felt to be
the predominant form of mercury in the estuarine environment. We and
other investigators have observed the evolution of Hg° from samples of
water and sediment. Based upon these observations and the known meta~
bolic capabilities of bacteria, we hypothesized a role for bacteria in
the transformations and mobilization of mercury in Chesapeake Bay. This
report describes our investigation of the physiological and ecological
properties of mercury-resistant bacteria. It also presents evidence for
the relationship of bacterial mercury resistance and metabolism.


SECTION VII
THE ROLE OF BACTERIA IN THE MOVEMENT OF MERCURY

THROUGH A SIMPLIFIED FOOD CHAIN

INTRODUCTION

Whether mercury levels in the oceans have risen significantly is a high-
ly controversial issue (65, 66, 67). Undisputable, however, is the fact
that marine animals are capable of concentrating mercury and other heavy
metals to levels several orders of magnitude above that found in sea
water (4), particularly in the proximity of kmown sources of pollution
(3, 68). Regardless of the source, the consequences of the sequestra-
tion of mercury are serious because the highly toxic and persistent
alkylated forms of mercury comprise most of the mercury in fish tissue
(22, 69, 70). The mechanism(s) by which mercury is accumulated in higher
organisms are not completely known. Barber et al. found no apparent re-
lationship between mercury concentrations in marine fish and ambient sea
water levels of mercury (66). Similarly, Windom et al, (71) found no
significant differences in mercury concentrations in inshore and offshore
catches of 35 species of North Atlantic fin fish. However, the amount
of mercury accumulated in fish appears to be related both to the size
(66, 70) and the species of fish involved (66). It is not known, as yet,
whether given species of fish, such as tuna or swordfish, acquire rela-
tively large amounts of mercury by unique mechanisms, which are species
specific, or from participation in, or association with, a particular
mercury-accumulating food web (72). Evidence both for (73, 74) and
against (4, 69, 75) the magnification of mercury concentrations through
trophic levels has been presented. The question has not been satisfac-
torily answered since none of the investigations were specifically
undertaken to trace mercury flow through a specific food chain, under
controlled conditions. Mercury uptake has been studied in individual
marine animals such as the oyster (69, 76) and the fiddler crab (77),

but in neither case were the microbiological parameters of the experiment
measured or controlled.

We have investigated the ecology of mercury-resistant bacteria in Chesa-
peake Bay and have concluded that bacteria, by virtue of their ability
to accumulate mercury and reduce Hg? to Hg®, are influential in the
mobilization and transformation of mercury in the Chesapeake Bay habitat.
Consequently, an investigation of the role of bacteria in the

64


non-glucose-utilizing type III organisms correlated with average season-
al resistance (0.76 correlation). Interestingly, the two mst metabol-
lically active, reducing strains assayed in a survey of cultures
(Table 14) were both type III Pseudomonas spp. In contrast, glucose
oxidizing Pgeudomonas spp. types I and II declined in abundance from
winter through the spring, summer and fall months.

Mercury mercaptide formation, via metabollically-generated sulfhydryl
compounds has been shown to be a mechanism for mercury resistance in
some microorganisms (50, 52). However, our data show that H2S produc-
tion is not an exclusive property of mercury-resistant bacteria, hence
could not account for the level of mercury resistance in the bacterial
populations studied. This particular point was carefully examined in
order to establish the validity of a plate count index for potential
mercury metabolism. Our conclusion was further supported by the fact
that the seasonal distribution of Hg9S-producing bacteria did not coin-
cide with that of average mercury resistance in the viable, aerobic,
heterotrophic populations in Chesapeake Bay water and sediment.

PMA was found to be considerably more toxic for the bacteria in Chesa-
peake Bay samples than HgClo. At 3 ppm, it was also more selective for
spp. When the PMA concentration was reduced to 0.3 ppm,

inhibition of the total population was found to be comparable to that
induced by the presence of 6 ppm of HgCl2 in the plating medium. The
relatively greater toxicity of organomercurials for microorganisms is a
general phenomenon (34, 51, 52) and is very likely related to the lipo-
philic character of these compounds.

In summary, an ecological analysis of the distribution and function of
mercury-resistant bacteria in Chesapeake Bay has been accomplished. The
seasonal fluctuations in the numbers of these bacteria has been demon-
strated and some of the influencing environmental factors elucidated.
The cyclic oscillation observed in that physiological group of micro-
organisms comprising the mercury-resistant bacteria may be only a single
component of a species succession. Work underway on the species compo-
sition of the total aerobic, heterotrophic bacterial populations in
Chesapeake Bay should provide some further insight into this fascinating
and heretofore undescribed aspect of bacterial population dynamics in
nature,

SUMMARY

Total ambient mercury concentrations and numbers of mercury-resistant,
aerobic, heterotrophic bacteria at six locations in Chesapeake Bay were
monitored over a 17 month period. Mercury resistance expressed as the
proportion of the total, viable, aerobic, heterotrophic bacterial popu-
lation, reached a reproducible maximum in spring and was positively
correlated with dissolved oxygen concentration and sediment mercury con-
centration and negatively correlated with water turbidity.

A relationship between mercury resistance and metabolic capability for

46


to be expected in the natural environment, since temperatures and cell
concentrations used in vitro were much higher than measured in gitu.
However, the in vitro results clearly indicate a mechanism for the rapid
mobilization of mercury in an estuary. The generation of Hg° may effect
a net loss trom the environment through volatilization, since Holm and
Cox (38) have shown Hg° added to aqueous systems to be stable for
periods up to 2 weeks.

Analysis of Seasonal Data

We have observed a seasonal fluctuation in numbers of mercury-resistant
bacteria, and presumably mercury metabolism, and have attempted to re-
late it to various physical parameters. There are precedents for sea-
sonal variations of marine bacteria, suggesting successions of (39, 40),
but none more apropos than Jernelov's (41) observation of the seasonal
changes in numbers of mercury-methylating bacteria in fish slime. Mul-
tiple regression analysis (Table 18) indicated that numbers of mercury-
resistant bacteria are determined by different factors at each sampling
area, A similar approach was employed by Brasfield (42) in her investi-
gation of the incidence of pollution indicator bacteria.

Percentage resistance and mercury concentrations in sediments were posi-
tively correlated at the Annapolis and Eastern Bay stations, although
not significantly at Annapolis. In contrast, a negative correlation was
observed for samples collected in Baltimore Harbor, where Colgate Creek
sediment contained higher levels of both Hg and petroleum, If mercury
concentration is sufficient to specify bacterial population structure,
then differences in the mercury-resistant populations of similar locales

might reflect the proportion of total mercury which is biologically
available.

Stations for comparative analysis were selected on the basis of similar-
ities in sediment type and differences in mercury content. On this
basis, data for Stations A-1 and EB-2 (sandy sediments) and B-1 were de-
leted from the regression equation. Percentage of mercury-resistant
bacteria in the water colum was found to be related to percentage of
mercury-resistant bacteria in the sediment, suggesting a common deter-
minant, most likely the mercury occurring in the sediments. The per-
centage of mercury-resistant bacteria in sediments was found also to be
correlated with water transparency and dissolved oxygen.

A peak in sesten levels in the upper Chesapeake Bay coincides with the
spring run-off from the Susquehanna River (32, 43). Smith has reported
that most of the mercury in estuarine water is associated with the par-
ticulates (44). We have observed a negative correlation between water
turbidity and presumptive in situ bacterial mercury metabolism, which
may correspond to a decreased availability of mercury to the bacterial
population. This process may be related to the adsorption of Hg to par-
ticulates. In fact, the binding of mercury and other heavy metals to
organic suspended particulates, is the basis for their removal from
water during sewage treatment (45, 46). Thus, the occurrence of a peak

Wy


Assay for He° Production

The method of assay using 203g-labeled HgC12 was as previously described.
The assays were carried out at 25 C, using 1 ppm of HgCl2.

RESULTS AND DISCUSSION
Survey of Morphological Effects

Representative cultures of bacteria isolated from colonies on agar media
containing HgClo or PMA were grown in broth containing sublethal concen-
trations of HgCl2 in shaker flasks incubated at 25 C, The experimental
cultures and controls without HgClg were grown to early log phase and
harvested, Thin sections were prepared from fixed and stained specimens,
and the preparations were examined by transmission electron microscopy.
Growth in the presence of mercury commenced after varying lengths of
time, with an array of morphological effects which did not appear in con-
trol cultures of the same physiological age (Table 20). The most fre-
quently observed defect involved the cell wall and cell wall synthesis.
This was suggested by the formation of projections from the cell wall,
elongated pleomorphic cells of the Gram-negative bacteria examined, and
irregular cross-wall formation in the Gram-positive bacterial strains.

A number of cultures also showed plasmolysis, indicating interference

by the mercury with cytoplasmic membrane transport phenomena. These
effects may be related to the finding that most of the mercury bound to
bacterial cells has been found either on the cell wall or cytoplasmic
membrane (this report, 10, 62).

Growth and Morphology of Representative Gram-positive and Gram-negative
Species

Figure 15 shows growth of Bacillus strain 394 in the presence and absence
of 5 ppm HgCl . After a one day delay ("lag") in the onset of growth,
the cells were able to undergo logarithmic growth at a normal rate. A
similar lag phase has been observed in the pattern of growth of other
microorganisms in the presence of mercury (5, 9, 12, 53, 54, 63).

50


by the slightly shorter lag phase in the unlabeled mercury-containing
culture. During the initial phase of incubation, radioactivity was
rapidly lost from both the inoculated broth and from the sterile control,

10 r
Lo .
3
3
8
o
rr) v deo 2
s
5
°
g
~
>
E60 4.0
= =)
3 wy
2
2st 2 a ww
z Ns
a 2 th
e © 40] any ans)
z 5
z
+ é
G 20h 2 by
2°T &
=
a vA
Fe
S 1s * A?
2
Lf
a —
PS nt 17
°
{ +.

2 4 624 2 2 30

INCUBATION TIME (hr)

Figure 18, Growth and uptake and metabolism of mercuric ion by Entero-
bacter, 85. Shaker flasks containing broth with and
without 203Hg- and un-labeled HgClg (2 ppm) were inoculated
with an 18 h culture and incubated at 34.C. Turbidity of
the culture without mercury (@) and the culture with labeled
mercury (4) were measured, Total (A) and non-filterable
(@) activity of the latter were also measured, An uninocu-
lated, | Hg-labeled control flask (D0) was also assayed for
total radioactivity. Aliquots were removed from flasks
without and with unlabeled mercury at intervals for examina-
tion by electron microscopy (arrows). Total viable counts
of the unlabeled mercury culture (©) were obtained by
spreading suitable dilutions upon basal nutrient agar.

Other investigators have shown that reducing agents such as gincosg, (64)
and yeast extract (63) in growth media promote the reduction of Hg“".
The loss of radioactivity from the control broth represented the vola~
tilization of Hg as Hg® and did not arise from adsorption to the glass

5h


Figure 13.

Figure 14.

PERCENT

80: | |
60)
12345676
FALL 1972
“e a

12345678
20
12348678
SPRING 1972

SUMMER 1972
12346678

WINTER 1972

SEASON

Seasonal population distribution of HgClz resistant aerobic,
heterotrophic bacteria. Five hundred and thirty-nine pure
cultures found to be resistant to 6 ppm of HgCl were collected
from Stations B-1, B-2, A-1, 4-2, and EB-1, and EB-2 during
January 1972 through December 1972. "Winter" = 1/13/72 - 1/31/72,
“Spring” = 3/29/72 - 6/1/72, "Sumer" = 7/25/72 - 8/1/72, and
"Fall" = 10/6/72 ~ 12/5/72. For generic key, see Fig. 12.

80)

70

60

50) D water
@ sepiMent

PERCENT

GENUS

Mercury resistant bacterial population distributions in water and
sediment in Chesapeake Bay. Three hundred and fity-six pure cul-
tures found to be resistant to 6 ppm HgCl2 were isolated during
October 1971 through December 1973, The cultures were classified
into genera or generic groups as listed in Fig. 12.

ho


activity may be particularly relevant to the short term response of
natural bacterial populations to mercury pollution, for the induction
time frame may be in terms of only minutes or hours, under the proper

conditions.
Table 7. HEAVY METAL RESISTANCE OF MERCURY-RESISTANT BACTERIA

Culture Maximm concentration (ppm) -- Metal fon?

No. mot pet ag ast cot*  cu2t ame? cae? ark? Hg?
72 167.09 167.0 16.7 501.0 334.0 334.0 55.1 100.2 668.0 4.0
119 167.0 167.0 16.7 501.0 55.1 334.0 167.0 167.0 1.7 20.0
187 16.7 167.0 1.7 501.0 55.1 334.0 110.2 50.1 668.0 16.0
132 16.7 167.0 1.7 501.0 167.0 334.0 167.0 167.0 16.7 40.0
85 167.0 167.0 1.7 501.0 167.0 334.0 55.1 50.1 100.2 12.0
639 «16.7 167.0 1.7 501.0 110.2 334.0 55.1 100.2 50.1 12.0

9% 16.7 167.0 1.7 501.0 55.1° 334.0 55.1 16.7 334.0 24.0
244 16.7 167.0 1.7 501.0 55.1 334.0 55.1 50.1 100.2 50,0
127 16.7 167.0 1.7 501.0 18.4 334.0 18.4 16.7 16.7 24.0

“one drop of culture was added to 3 ml of basal broth containing dilutions
of filter-sterilized solution of heavy metal, and the tubes were incubated,

without agitation, at 25 C for 7 or more days.
were scored positive.

out metals were included in the assay.

Metal ion salts x vere: A1CLy
Tigo, Cole 30,
(nap otro ae oe OO

2

6H20, Pb (CoH
» ZnSO, “7H,

"05 cabo, an,

“Broth with salt added was adjusted to pH 7 with 1 N NaOH and filter-

sterilized.

Sypoocdmmam concentrations tested were:

Al - 167.0,

Tubes showing turbidity
Uninoculated and inoculated controls with and with-

Pb - 167.0, Ag - 167.0,

As - 501.0, Co ~ 668.0, Cu - 334.0, Zn ~ 334.0, Cd - 334.0, Cr - 668.0,

Hg - 100.0.

13


An examination of one of the most active isolates (culture #5) further
suggested a correlation of resistance and metabolism, The organism was
grown in the absence of HgC12, with concomitant diminution of its toler-
ance for, and ability to metabolize, HgClg (Table 15), Experiments uti-
lizing a closed system with a HgBro-KBr mercury trap (18), in which the
radioactive volatile product of the reaction was quantitatively collected,
suggested that Hg° evolution accounted for the loss of Hg from the sus-
pension (Table 16). Radioactivity in the acidified trapping solution

was not benzene-extractable, nor were methyl or other alkyl mercury com-
pounds detected in thin layer chromatograms of the dithizone derivative.

Table 15. MERCURY RESISTANCE AND METABOLISM OF BACTERIAL STRAIN NO. 5
GROWN IN THE PRESENCE AND ABSENCE OF HgCl,

A, Resistance
Growth®
HgCl concentration (ppm)
Culture? i 3 72 16 20 2h
Grown with HgCly + + + + + ~
Grown without HgCl, + + +

*tubes were inoculated with 1 drop of culture, incubated, with shaking,
at 25 C, and observed daily for up to 21 days.

Pourtures were serially transferred twice with or without 6 ppm HgClg

in the growth medium, A 24 hour old culture was used to inoculate the
test medium.

B,_ Metabolism
ng Percent change in (293Hg)/30 min®
Culture? Dry _ weight Decrease in suspension Uptake by cells
Grown with
oO. 28 13.7
HgCly 39 38.
Grown with-
out HgCl, 0.43 501 15.6

24 suspension of washed cells, maintained at 25 C in 4.9 ml of PES and
aerated via a capillary tube, was incubated for 5 min prior to the ad-
dition of 0.1 ml of HgCl2 (final concentration of 6 ppm HgClg; approx.
300,000 CPM/m1). 0.1 ml samples were removed at zero time and at
regular intervals to 10.0 of scintillation cocktail. Percentage de-
crease in the suspension or increase in the cellular (non-filterable)
radioactivity was based upon counts corrected for quenching.

me cultures were grown for 2 h at 25 C, with shaking, in broth in the
presence and absence of 6 ppm HgCl2, Inocula were cells grown for 24

h with and without 6 ppm of HgCl2 in the growth medium. Equal turbidi-
ties were attained in each culture at the end of the incubation period.

32


The in vitro experiments carried out in this study may be related to con-
ditions in the natural environment. The positive correlation observed
between mercury concentration and number of mercury-resistant bacteria
in sediment suggests that selection does occur jn gity. It is logical
to propose that despite adverse effects of mercury or other heavy metals,
microorganisms will prevail, in some form, because of their remarkable
versatility. However, it is not yet possible to predict, quantitatively,
the effects such a perturbation would have upon population structure in

a natural microbial commmity or at the higher ecological level.

63


ABSTRACT

This report describes the physiology and ecology of mercury-resistant,
mercury-metabolizing bacteria from Chesapeake Bay. Evidence is present-
ed which establishes a role for bacteria in the cycling of meroury in
the estuarine environment.

Mercury-resistant aerobic, heterotrophic bacteria were isolated from
water and sediment at six stations, representing various levels of en-
vironmental quality, in upper Chesapeake Bay. These organisms were
found to be resistant to an array of inorganic and organic mercury com
pounds and heavy metal ions. It was also observed that in individual
cultures demonstrating resistance to HgCl2 and phenylmercuric acetate,
the resistance was adaptive and was also related to metabolic capability
for degrading the compounds to elemental mercury (Hg°). However, cul-
tures in the process of adaptation evidenced delayed growth and cell di-
vision, which were dependent upon mercury concentration. The cultures
also developed a variety of morphological irregularities associated with
cell well and cytoplasmic membrane synthesis and function.

From the results of a survey of Hg®° production among a group of randomly
selected, HgClo-resistant bacteria and mixed natural microbial popula-

tions, it was established that the enumeration of mercury resistant bac-
teria by plate counting is a valid index of potential Hg~" metaboliem ip

The population of mercury-resistant bacteria, primarily Pseudomonas spp.,
varied quantitatively in time and from station to station. The distri-
bution of mercury-resistant bacteria was significantly different in vater
and » from station to station, and seasonally. Yet, the propor-—
tion of Hg“" resistant bacteria among the total, viable, heterotrophic
bacterial population reached a reproducible maximm in spring and was
positively correlated with water turbidity, dissolved oxygen concentra-
tion, and mercury concentration in the sediment.

These findings and the observation of the evolution of Hg° from freshly
collected water and sediment suggest that bacteria may contribute sub-
stantially to the mobilization and transformation of mercury from exist-
ing deposits in Chesapeake Bay, specifically, and in the aquatic environ-
ment, in general.

This report was submitted in fulfillment of Grant #602529 by the Univer-

sity of Maryland under the partial sponsorship of the Environmental Pro-
tection Agency. Work was completed as of November 1, 1974.

iii


Figure 27. Sterilizable oyster aquarium. Sampling port with rubber
septum (1); air inlet with sterile fiber glass filter (2);
air outlet (3); water effluent (4); magnetic stirring bar
(5); gas dispersion tube (6).

of aquarium water were determined periodically by spreading suitable di-
lutions of the water on solid basal medium and incubating the plates at
room temperature for 2 weeks.

Experimental Procedures

Six oysters of uniform size were selected for each experiment. Two were
shucked and freeze dried for total mercury analysis. The remaining four
were placed in the sterilized aquarium. Bacterial suspensions were

added through a port in the lid, followed by 1.0 ml of sea water containing

66


introduction of mercury into a simple estuarine food chain was initiated.
Based upon a comparative survey of benthic and pelagic animals in Hawai-
ian waters, Klemmer and Iuoma (73) concluded that mercury is probably
most readily transported through short food chains directly linked to
benthic organisms. After considering the available information and the
fact that mollusks are known to accumulate mercury and other heavy
metals (78), we selected for our study a food chain incorporating bac-
teria and a benthic filter feeder, the American oyster, Crassostrea
virginica, A series of experiments were conducted to elucidate the
routes and mechanisms of mercury introduction into aquatic food chains
at the lowest trophic level.

MATERIALS AND METHODS

Aquarium System

The apparatus used in each of the three experiments which were carried
out consisted of a flanged, 14 liter pyrex fermenter jar equipped with a
stainless steel lid which was sealed with a rubber O-ring and fastened
with bolts (Fig. 27). The lid had threaded nylon hose fittings wrapped
with teflon tape to prevent leaks, A rubber serum bottle cap was placed
on the central fitting to permit sampling of the closed system witha .
sterile 12 inch canula attached to a sterile syringe. A rack constructed
from a glass rod was used to suspend the oysters off the bottom of the
jar, above the teflon-coated magnetic stirring bar. A gas dispersion
tube was connected to an outside air pump via Tygon tubing and a nylon
hose fitting in the lid. The entire aquarium, complete with fiber glass
air filter, was sterilized by autoclaving at 121 C for 15 min. Ten
liters of 0.45 micron filter-sterilized, dilute, artificial sea water
(Seven Seas Marine Mix, Utility Chemical Co., Paterson, N. J., 33% of
sea water strength) were pumped in, and the aquarium was immersed in a
15 C constant temperature water bath. Circulation was provided by an
external, submersible magnetic stirrer powered by water, and the water
in the jar was oxygenated by air pumped through the fiber glass filter
to the submerged gas dispersion tube. Effluent air from the aquarium
was transported through Tygon tubing to two scrubbers in series, each
containing 100 ml of HgBro - KBr solution (see Section V) to trap vola-
tilized mercury (Fig. 28).

Oysters and Bacteria

Oysters were dredged from Tolly Bar outside of Annapolis Harbor, Maryland,
in March and April 1974, and maintained in raw bay water at in situ tem
perature and salinity until used in the experiments. The animals were
serubbed and placed in artificial sea water (approximately 12.0 ©/oo
salinity) at 6 C for approximately 1 week, The temperature of the water
in which the oysters were held was brought up to 15 C gradually and was
held for 3-4 days at 15 C prior to placing them in the sterile aquarium
system. Bacterial cultures were grown in liquid basal medium (see pre-
vious sections) containing 6 ppm of HgCl9. Fresh transfers of overnight
cultures were grown for 3-5 h, centrifuged and resuspended in sterile
salts solution, and added to the aquarium. Total viable bacterial counts

65


10-20 micro C of 203 Hg Clo to give a final concentration of 10.2 ppb.
After an equilibration period of 10-15 min, duplicate 1 ml samples of
aquarium water and scrubber solution were removed for scintillation
counting. A second 1 ml sample of aquarium water was filtered through a
0.45 micron membrane filter, which was rinsed with three 1 ml volumes of
artificial sea water. The filters were saved for scintillation counting.
The above sampling procedure was repeated on four successive days. At
the conclusion of the experiment, the oysters were removed from the
aquarium, shucked, and dissected (Fig. 29). Tissues were rinsed in arti-
ficial sea water and placed in tubes for scintillation counting, Fecal
material which had settled to the bottom was aspirated from the aquarium
and collected on 8 micron membrane filters and rinsed in artificial sea
water prior to scintillation counting. Radioactivity was measured in a
Packard TRICARB Spectrometer equipped with an Auto-Gamma Spectrometer.
The standard deviation was 1% of total counts for all oyster tissues.

Figure 29. Oyster dissection. Oysters were shucked, and the mantle fluid
was drained and retained. The oysters were dissected, and each
of the tissues selected for analysis was rinsed in artificial
sea water prior to measurement of radioactivity. Mantle,
gills + labial palps, visceral mass, and adductor muscle (left
to right).

68


SECTION V
MICROBIAL ECOLOGY OF MERCURY-RESISTANT BACTERIA
IN CHESAPEAKE BAY

INTRODUCTION

Comparatively little is known about chemical and biological processes
involved in the availability and mobility of inorganic mercury, the pre-
dominant pollutant form of mercury in the environment and a precursor of
methyl mercury.

Microbial transformations of mercury, other than those involved in the
formation of methyl mercury, have been described by several investiga-
tors. The reductive decomposition of orten) and inorganic mercurials,
with the formation of elemental mercury (Hg°), have been reported for
several genera of mercury resistant bacteria (1, 2, 8, 9, 13, 15, 16).
This phenomenon may account for the low ambient levels of methyl mercury
(17, 18) and, possibly, for the observed loss of Hg from contaminated
sediments and soil (19, 20, 21, 22). However, the process of microbial
Bg° generation should be emphasized as a potential mechanism for the mo-
bilization of mercury and for the generation of substrate for methyla-
tion. The latter ans ees is supported by evidence that Hg® is
readily oxidized to aT situ under the influence of dissolved oxygen
and organic matter (23, 24).

Although direct pollution of the aquatic environment with mereury has
been drastically curtailed during the early 70's, extensive sediment
deposits exist which constitute a continuing methyl mercury hazard (25).
In view of this Benet a understanding of microbial processes in the

generation of Hg® and is essential, particularly for evaluating en-
vironmental impact.

This report describes a study of the geographical and seasonal distribu-
tions of aerobic, heterotrophic, mercury-resistant bacteria involved in
the formation of Hg° in Chesapeake Bay.

2h


OT

Table 5. DISTRIBUTION OF BACTERIAL HgCl, RESISTANCE® AND TOTAL MERCURY CONCENTRATIONS AMONG WATER AND SEDIMENT FRACTIONS

Source
Falt Interstitial
Surface water water Surface sediment water® Plankton?

% Resistent (/fig/ (ppb) % Resistant % Resistant (ppm) % Resistant % Resistant /Bg7 (ppm)
Sample® HgCl, PMA HgC12 HgCla PMA HgCl2 HgCla PMA
42 - 3/29/72 6.5 =~ <0.2 + .02 3.1 7.0 -- 0.27 + .03 33.6 W4 — 0.77 + .04
B2- 4/03/7200 -- =e ~ 1.5 29.8 -- 0,824.02 22.5 31.0 == 0.68 + .03
ee 48 —~ <0.2 + .02 7.3 23.8 — 040+ .0 5.9 - - -
Bl - 5/15/72 4.2 — <0.2 + .02 _ 8.2 — 0.17 +.01 ~ 10.9 -- -
42 - 6/01/72, 0.3 - - <0.4 1.5 -- 0.37 4 .02 1.7 2.9 —- 3.0 +.90
B2 ~10/05/72 13.2 -- 0,09 + .01 35.0 6.3 — 0.97 + .04 35.7 - - -_
EBI-10/06/72 - -- - _- 4.8 -- 0.04 + .00 31.0 -- - _
a2 -12/05/72 6.0 -- 0.04 + .01 8.1 1.5 -- 0.20 + .08 1.8 -- - -~
B2 = 1/04/73 8.9 22.4 -_ - 33 5.5 0.67+.01 - 18.8 -- 0,09 + .01
B2 - 5/24/73 8.3 19.6 - ~ 6.0 8.2 ~ - 6.1 31.8 0,05 + .00
EBI- 5/25/73 0.08 «(0.14 - - 0.31 0 = - - 0.23 53.5 0,06 + .05

“Dilutions of material were apread on basal medium, with and without 6 ppm of HgCl2 or 0.3 ppm of PMA added. The percent
of the total, viable, aerobic, heterotrophic bacterial population resistant to meroury was calculated.

Yuater waa filtered through sterile 8 um pore size Millipore filters.

°supernatant solution resulting from centrifugation of sediment at 1,610 x G for 20 min.
4coriected with #20 nylon mesh plankton net.

see Fig. 7, Section V.


reductive decomposition of PMA, methyl mercury (Me Hg) and ethyl mercury
has also been reported by others (2, 10, 13).

22

oa

2.0

b
@

ol

a
GROWTH (LOG KLETT UNITS)

20349 - PMA (cpmx 1074)

y
é
t
|
1
a
\
°
rs

3 4
TIME (HOURS)

Figure 4, Uptake and metabolism of PMA by Pseudomonas 244. Cells were
added to broth containing 6.08 ppm of radiolabeled PMA after
5 min, preincubation. Radioactivity in 0.1 ml of cell sus-
pension (triangles) and in 1.0 ml of cel] suspension collect-
ed on a membrane tilter (open symbols) were determined. 0,1
ml samples of uninoculated broth (squares) were also analyzed.
Growth was measured turbidimetrically (closed symbols).

Washed, non-proliferating cells of Pseudomonas 244 retained the ability
to metabolize PMA for extended periods of time and were used to study
Li Ran ieeet in detail. Aerated suspensions of the cells co:

labeled PMA in PES were used to assay decomposition through the
loss of radioactivity. vity was dependent upon cell density and
aeration driving off the ~ as formed. Cultures previously grown and
harvested from media with and without 6 ppm PMA supplementation were
assayed (Fig. 6). As in the case of the production of Hg? and Hg@*t, the
induced culture was much more active. Cells of both cultures accumlated
label rapidly, but the non-induced, non-PMA-metabolizing culture accum-—
lated label significantly more than the induced culture. Thus, for both

+ and PMA, resistance and metabolism were related and inducible

phenomena.

16


SEDIMENT

PERCENT RESISTANT
40 60 80 20. 49 60 8©

24 ppm PMA
1S ppm PMA
6 ppm PMA
3 ppm PMA
Sppm MeHgCl

100 ppm HgCl,

SOppm Hecl,

Figure 2. Comparative percentages of mercury-resistant isolates from
sediment and water. (Reproduced with the permission of the
Marine Technology Society. )

Mercury resistance in bacteria is often accompanied by resistance to
other heavy metals and to drugs (5, 6, 7, 8). Resistance is believed to
be conferred by factors borne by extra-chromosomal fragments ("plasmids")
which are transferrable. A group of representative mercury~resistant
bacteria were screened for resistance to a set of heavy metal ions uging
a broth pedium, | Variable patterns f resistance, particularly to >
Co**, Zn", Cd“*, and Cr®®, and were observed (Table 7), suggesting
that resistance was not generalised, i.e., that determinants of resis-
tance are independent. The possibility of plasmid-mediated resistance
among these bacteria is currently being investigated in our laboratory.

Bacterial Adaptation to Mercury Resistance

It wes observed that, after serial-passages of bacteria through growth
medium in the absence of mercury, resistance to mercury decreased sig-
nificantly. When 30 cultures were grown in the absence and presence of
inorganic and/or organic mercury, followed by replicate plating onto
wercury-containing media of various types, it was found that growth of
bacteria in the presence of mercury clearly decreased sensitivity to
this metal (Table 8). "Adaptation" for growth in the presence of mer-
cury compounds had been observed earlier by others (5, 9, 10). This
phenomenon has been found to be a consequence of the "induction" of
ensymes for the detoxification of mercury compounds via reductive de-
composition of mercury compounds to volatile Hg° (8, 11, 12). This

12


MATERIALS AND METHODS

Sampling

Water samples for bacterial plate counts were collected in sterile dilu-
tion bottles or sterile Niskin bag samplers (General Oceanics, Miami,
Florida) 10 cm below the surface. Sediment samples for bacterial plate
counts and mercury analysis were taken from the upper 5 - 10 om layer
using an Ekman dredge or Petite Ponar grab (Wildlife Supply Co., Saginaw,
Michigan), Water samples for mercury analysis were collected in 0.5 N
HNO3-washed bottles and acidified promptly with 5 ml of concn. HNO3 per
liter. Horizontal plankton tows for 10 - 30 min, using plankton nets
equipped with 20 mesh nylon cloth, provided ample material for analysis.
The collected plankton were centrifuged, resuspended in sterile salts
solution (see below) and either homogenized for bacterial plate counts
or recentrifuged and lyophilized for mercury analysis. Appropriate con-
trols and experiments eliminating the possibility that lyophilization
affected the mercury analyses were made.

Mercury Analysis

Sediment samples were allowed to settle, the water layer, if any, was
removed, and the sediments were air dried. Each sample of water or sed-
iment was wet ashed according to Environmental Protection Agency proce-
dures and analyzed by flameless atomic absorption spectrophotometry
(26). Analyses were performed at the National Environmental Research
Center, Cincinnati, Ohio and at the National Bureau of Standards, Wash-
inton, D.C.

Isolation, Culture, and Identification of Bacteria

The medium employed for determination of the total viable count (TVC)
and enumeration of mercury-resistant bacteria consisted of glucose, 2.0
gm; Casamino acids (Difco Laboratories, Detroit, Michigan), 5.0 gm;
Yeast extract (Difco), 1.0 gm; and Bacto agar (Difco), 20 gm; per liter
of artificial estuarine salts solution, i.e., 10.0 gm of NaCl, 2.3 gm of
MgClo°6H20, and 0.3 gm of KCl per liter. The salts mixture was adjusted
to pH 7.2 and autoclaved at 121 C for 15 min. Mercury-containing media
were prepared by adding freshly prepared solutions of HgClo in sterile
salts solution or PMA in 95% ethanol to sterile, molten agar medium.
Suitable dilutions of material were prepared in sterile salts solution
and spread on agar medium with and without added mercury within 5 hours
of sampling. The inoculated plates were incubated for 7 days at 25 C,
after which the colonies were counted. Colonies appearing on mercury-
containing media were selected on a random basis and purified. The or-
ganiams were identified according to a taxonomic scheme originated by
Shewan (27) which was modified by incorporation of the methods and proce-
dures described by Colwell and Wiebe (28).

25


with dilutions of water and sediment and incubated aerobically and an-
aerobically, for comparison. The percent of meroury-resistant bacteria
was always lower for samples taken from three different locations when
plates were incubated anaerobically (Table 3). These findings are not
consistent with properties of methyl mercury-metabolizing bacteria as
published by others, but they do agree with those of mercuric ion-
reducing bacteria reported later in this section.

Table 2, EFFECT OF INCUBATION TEMPERATURE ON BACTERIAL VIABLE COUNT AND
RESISTANCE TO HgCl,

Incubation Incubation
temperature time Total le Percent
(c) (days) count (x 10°/gram) resistant?
37 7 1.45 16.7
25 7 2.60 7.8
15 14 18.80 1.4
2 14 11.70 14

"a sample of sediment (B2-5/24/73) was diluted and plated on basal
medium, with and without 6 ppm of FigCl,. Surface water temperature was
16.9 C when the sample wes taken.

Peorcentage of the IVC resistant to 6 ppm of HgCla.

Table 3. COMPARATIVE EFFECTS OF AEROBIC AND ANAEROBIC INCUBA-
TION ON BACTERIAL RESISTANCE TO HgCl,

Percent resistant®

Sample Aerobic inoubation ‘Anaerobic incubation®
B2 - 5/24/73 7.0 1.0
A2 ~ 5/24/73 1.3 0.3
EB1 - 5/25/73 1.8 0.3

8samples of sediment were diluted and spread on basal medium
agar, with and without 6 ppm HgCl,, and incubated for 7 days
at 25 C.

DiIncubated in BicQuest (Cockeysville, Maryland) anaerobic jars
containing CO2-enriched anaerobic atmosphere produced by the
Gas Pak (BioQuest).


15.

76,

77.

78.

719.

80,

Jernelov, A. and H. lann, Mercury accumulation in food chains,
OIKOS 22:403-406. (1971)

Cunningham, P.A. and M.R. Tripp. Accumulation and depuration of mer-

cury in the American oyster, Crassostrea virginica. Marine Biol,
20:14-19. (1973)

Vernberg, W.B, and J, O'Hara, Temperature-salinity stress and mer-
cury uptake in the fiddler crab, Uca pugilator. J. Fish. Res. Bd.
Canada 29:1491-1494. (1972)

Glatsoff, P, The American oyster, Crassostrea virginica (Gmelin).
U.S. Fish, Wildlife Service Fishery Bulletin 64:1-480. (1964)

Pentreath, R.J. The accumulation from water of 652n, Shan , 58c0,

and 27Fe by the mussel, Mytilus edulis. J. Mar. Biol. Ass. U.K.
53:138-153. (1973)

Kopfler, F.C. The accumulation of organic and inorganic mercury com-

pounds by the Eastern oyster ( virginica). Bull. Environ.
Contam. Toxicol. 141:275-280. (1974.

80


Figure 28. Oyster aquarium in operation. HgBr2 - KBr traps (1); immer-
sible, water propelled magnetic stirrer (2).

67


0.94

Cx)

0.7]

06]

2
rs

OPTICAL DENSITY (6700m)

10 20 30
INCUBATION TIME (hr)

Figure 15. Growth of Bacillus, species 394, in the presence and absence
of 5 ppm of HgClg. Broth in shaker flasks with (A) and
without (@) HgCl2 was inoculated with overnight culture
(0.1 m1/5 ml fresh broth). Growth was measured turbidimetri-
eally during h at 25 C. Samples were removed from each
flask (arrows) for the morphological analysis of stained
thin sections (see Figs. 15 and 16).

These cells showed irregular cross-wall formation and grossly deformed
outer cell walls (Fig. 16). The effects were noticeably increased at a
concentration of 15 ppm HgCl, (Fig. 17).

A Gram-negative species was selected for a more detailed investigation
since Gram-negative organisms constitute 90% of the mercury-resistant
bacteria isolated from Chesapeake Bay in the course of the study. The
objective, in this instance, was to clarify events occurring while bac-
teria were apparently quiescent in mercury-containing broth and to relate
these events to cytological changes associated with adaptation. Entero-
bacter, strain 85, was serially transferred several times in the absence
of mercury, prior to inoculation into shaker flasks with and without 2
ppm of 203Hg-lebeled HgClg. After a 24h lag phase, the mercury-contain-
ing culture grew at a rate comparable to that of the control culture
(Fig. 18). Although the turbidity of the culture remained constant, a
nearly 3-log drop in viable count occurred. Viable count and turbidity
increased at the end of the lag phase. An isotope effect was suggested

52


16

17

18
19

8

25

CHARACTERIZATION OF THE VOLATILE Hg METABOLITE PRODUCED
BY PSEUDOMONAS, STRAIN 5

Hg® EVOLUTION BY NATURAL POPULATIONS OF BACTERIA ISOLATED
FROM WATER AND SEDIMENT

MULTIPIE REGRESSION ANALYSIS OF SEASONAL DATA

PHYSIOLOGICAL CHARACTERISTICS OF HgCl2 AND PMA RESISTANT
BACTERIA

SURVEY OF MORPHOLOGICAL EFFECTS IN SELECTED CULTURES OF
BACTERIA GROWN IN THE PRESENCE OF Hg0l,

MERCURY RESISTANCE AND MERCURY METABOLISM OF STRAIN 85
BEFORE AND AFTER GROWTH IN THE PRESENCE OF HgCl

EXPERIMENTAL OUTLINE
AVERAGE TOTAL MERCURY CONCENTRATION IN UNTREATED OYSTERS

RELATIVE DISTRIBUTION OF MERCURY FOLLOWING EXPOSURE OF
OYSTERS

TISSUE DISTRIBUTION OF MERCURY AMONG TREATED OYSTERS

ix

33

35

70

vA)
72


Table 12. DISTRIBUTION OF RADIOACTIVITY IN 7O3pg-PMA-LABEIED CELLS OF
PSEUDOMONAS STRAIN 244°

Percent radio-

b Total Percent activity recovered
Cell fraction! counts/min® of total in fractions
Cell free extract 1.59 x 109 100.0 _
3,000 x G pellet 2.28 x 104 14.3 21.0
35,000 x G pellet 5.85 x 10% 3A 53.7
126,000 x G pellet 8.06 x 107 5.1 7.3
126,000 x G supernatant 1.96 x 104 12.3 18.0

35 ml of a 24 h culture growing in broth containing 6 ppm of PMA was
added to 100 ml of fresh broth containing 6 ppm of PMA. The culture
¥B incubated for 2 h, with shaking, at 20 C, and 20 microcuries of

labeled PMA were added. The culture was incubated an additional
hour, and labeled cells were harvested by centrifugation.

Othe labeled cells, suspended in chilled 0.01 M TRIS-buffered (pH 7.0)
"three salts", were fragmented by two passages through a French Pres-
sure Cell. The resulting cell-free extract was fractionated by dif-
ferential centrifugation.

Caliquots of each fraction were collected, and radioactivity was mea-
sured by liquid scintillation counting.

Cells of induced and non-induced cultures of Pseudomonas strain 244 were
compared to identify a cell component responsible for mercury resistance,
Preliminary investigations have shown no differences in gross lipid com-
position or in the morphology, by inspection of thin sections of cells
using electron microscopy. However, striking cytological effects were
observed in cultures which were less resistant to mercury and which were
grown in the presence of HgClg (Section VI). Cells of several cultures
grown in the presence of HgCl2 showed morphological similarities, sug-
gesting that mercury impairs normal cell well and membrane synthesis and
function. This observation most likely is related to the propensity of
mercury to bind to the cell envelope.

SUMMARY

This section described the sources of mercury-resistant bacteria and
techniques used in this investigation, The cultural characteristics of
mercury-resistant bacteria and the physiology of aerobic mercury metabo-
lism were discussed.

22


Figure 23.

Thin section of culture #85 cells after 6 h incubation in

broth containing 2 ppm of HgCl2, The most prominent cyto-
logical features are plasmolysis and lysis in a large por-
tion of the cells, Arrows indicate electron dense groups

of ribosomes.

59


Figure 26.

Thin section of culture #85 cells after 28 h incubation in
broth containing 2 ppm of HgClz. The cells show reversion
to rod-shaped morphology, although cell wall contours are
still somewhat irregular. Electron dense clusters of ribo-
somes are present in fewer numbers.

61


Table 21. MERCURY RESISTANCE AND MERCURY METABOLISM OF STRAIN 85 BEFORE
AND AFTER GROWTH IN THE PRESENCE OF Hgc1,*

A. Mercury resistance?
HgClp concentration (ppm)

Culture 2 4 6 8 10
85 + + - - -
85-L + + + - -
85-S + + + + +

8see the first experiment (Fig. 18).

Pone cultures were serially subcultured in the
absence of mercury. Each tube of mercury-
containing broth was inoculated with 3 drops of
@ log phase culture and incubated, with shaking,
at 25 C for 4 days.

“culture 85 was the original inoculum, strain
numbers 85-L and 85-S refer to small and large
colonial types isolated during incubation of the
HgClg containing broth. (See Fig. 19.)

a
B. Formation of Hg®

Percent_decrease in 203 np radioactivity/60 min
Dry weight liv Heat killed! Net biological

Culture (mg) suspension suspension reduction®
85 2.05 9.8 16.1 0
85-L 1.50 7.41 3.6 3.5
85-S 1.90 11.3 3.0 8.3

“The assay was carried out at 25 C, with aeration, as described in Materi-
als and Methods. The assay contained 1 ppm of 12 (approx. 120,000

dpm/m1).
Osterile inocula were prepared by autoclaving at 121 C for 15 min.
°the difference in percent decrease between live and sterile inocula.

62


10 AA ame Ae — —_—s

“NON INDUCED
8 \ oe CELLS

4a 44 TOTAL

INDUCED \

rN

N

A ee ood

NON INDUCED TM—_—"”

rs

n

203149 (cpm x10-*)
+

i

INDUCED
}

10 20 30 40
TIME (MIN)

Figure 6. Uptake and metabolism of PMA by induced and non-induced rest-
ing cells of Pseudomonas 244. The reaction was begun when
cells were added to the buffered PMA-salts mixture (arrow).
0.1/m2 samples of cell suspension were taken at the indicated
times.

The effect of salinity upon PMA metabolism by Pseudomonas strain 244 was
tested by varying the total concentration of salts in the three salts
solution, which is comprised of NaCl, MgClo, and KCl. Cells placed in
three salts (33% sea water strength) buffered with 0.01 M tris (hydroxy-
methyl) aminomethane (TRIS)-hydrochloride (pH 7.0) were as active as
those in a salts solution at 82% of the strength of sea water (Table 10).
However, when the salt content was reduced to &.2%, the cells were in-
active. Inhibition was partially due to decreased salt concentration and
to TRIS buffer, since the same concentration of salts in the absence of
TRIS gave partial activity. When 0.01 M phosphate-buffered solutions of
NaCl, 12, and KCl of equal ionic strength were tested, it was found
that don, alone, satisfied the ionic requirement for activity. In
phosphate-buffered suspensions, a concentration of Ng2t of from 5 - 10 mTM
was optimm for PMA d ‘tion. A similar requirement for the
cell-free reduction of to Hg° by a mercury-resistant strain of
Escherichia coli has also been reported (14). Magnesium ion concentra-
tion also effects resistance of the organism to PMA, as shown by the

data given in Table 11. The optimum concentration for resistance was
found to be in the. range 1.2 - 5.6 mM *. This observation further
established the certainty of the relationship of PMA resistance and PMA

19



el

Table 24. RELATIVE DISTRIBUTION OF MERCURY FOLLOWING EXPOSURE OF OYSTERS

Percentage of total added mercury® after 4 days

Oyster
Fecal Adherent Unfiltered Suspended
Experiment Uptake material? organisms® water particulates? Volatile® Recovery!
I 22.00 -- - 4.20 2.8 9.25 35.5
I 39.30 2.07 0.80 7230 5.9 8.70 58.3
III 36.90 2.10 0.55 5.60 4e1 8.00 53-1

"the initial mercury concentration was 10.2 ppb of HgClg.
Yoollected on 8 micron membrane filter after settling of aquarium contents.
*Vorms and crustaceans adhering to oyster shells,

Sveroury retained by filtration through 0.45 micron membrane filters.

“Mercury accumulated in first HgBrg - KBr trap. Mercury accwmlated in the secc.d traps ranged

from 0,03-0,09% of the total added mercury.

Experiment I did not include fecal material or adhering organisms, Mercury adsorbed to aquarium

and shell surfaces was not measured in any of the experiments.


+ &

10

1

12

13

14

FIGURES

Average distribution of genera in total and HgClg resistant
populations

Comparative percentages of mercury-resistant isolates from
sediment and water

Induction of resistance to PMA by Pseudomonas 244
Uptake and metabolism of PMA by Pseudomonas 244

Change in mercury contained in bioreactor atmosphere as a
function of time

Uptake and metabolism of PMA by induced and non-induced
resting cells of Pseudomonas 244

Experimental sampling sites in upper Chesapeake Bay

Elemental mercury evolution by bacteria grown on agar
plates containing HgCl,

Seasonal variation in percent of HgClg resistant bacteria
in water

Seasonal variation in percent of HgClg resistant bacteria
in sediment

Seasonal variation in salinity, temperature, and dissolved
oxygen concentration in surface waters from three locations
in Chesapeake Bay

Distribution of HgClo resistant, aerobic, heterotrophic
bacteria isolated from water and sediment of Chesapeake
Bay

Seasonal population distribution of HgCl2 resistant,
aerobic, heterotrophic bacteria

Mercury resistant bacterial population distributions in
water and sediment in Chesapeake Bay

vi

12
15
16

18

19

30

36

36

4


II
III

VII

VIII

CONTENTS

CONCLUSIONS
RECOMMENDATIONS
INTRODUCTION

PHYSIOLOGICAL AND CULTURAL CHARACTERISTICS OF
MERCURY-RESISTANT BACTERIA

MICROBIAL ECOLOGY OF MERCURY-RESISTANT BACTERIA
IN CHESAPEAKE BAY

EFFECTS OF MERCURIC CHLORIDE UPON GROWTH AND MORPHOL-
OGY OF SELECTED STRAINS OF MERCURY-RESISTANT BACTERIA

THE ROLE OF BACTERIA IN THE MOVEMENT OF MERCURY
THROUGH A SIMPLIFIED FOOD CHAIN

REFERENCES
LIST OF PUBLICATIONS
GLOSSARY OF TERMS

Ww ND

~

F & R


Since an estuary is subjected to extensive changes in salinity, among
other parameters, the effects of the salt content of the growth medium
used for routine isolation of mercury-resistant bacteria was evaluated
for samples taken from sites encompassing a wide range of salinities
(Table 4). Media of three salinities, including the routine isolation
medium were compared. Media of the highest (26.59 °/oo) and lowest
(2.66 °/oo) salinities consistently yielded higher proportions of mer-
cury-resistant bacteria than the medium in general use in our studies
(11,38 °/oo). The effects of salt concentration may be related to a
salt requirement for meroury metabolism (this Section), or to selective
effects on the bacterial populations in the samples tested. The extreme
effects of salt observed in the case of samples of lowest salinity (B2-
5/15/72) suggested that the latter argument should also be considered.

Table 4. EFFECT OF SALT CONCENTRATION ON BACTERIAL RESISTANCE TO HgClp

Salinity of Salinity of

eurface growth Percent, resistant?
Sample _____vater (°/o0) mediua® (°/oo) __Water Sediment
BY - 1/18/72 5.94 2.66 7.60 22.90
11.38° 7.90 16,40

26.59 17.00 19.50

EB1 - 1/31/72 10.56 2.66 - 0.79
11.38 - 0.08

26.59 = 0.74,

Bi = 5/15/72 1.56 2.66 12.90 11,50
11.38 4.20 8,20

26.59 50.00 29.20

York R, mouth 16.40 2.66 5.30 6.30
11.38 0.50 3.50

26.59 0.16 5.90

B2 = 5/24/73 5.60 2.66 - 9.25
11,38 - 7.80

26.59 _ 11.10

* samples of water and sediment were spread on basal medium containing one
of three concentrations of artificial sea water (NaCl : MgCl5-6H,0 3
KCl = 100 : 23 : 3) with and without 6 ppm of HgCl, added.

Percent of total viable, heterotrophic bacterial population capable of
growth in medium containing 6 ppm of HgCl, after 7 days at 25 C.

°concentration of salts routinely used in the medium for assay of mer-
cury resistance.


oOo an nro

10

1

12

13

1
15

TABLES

COMPARISON OF RESISTANCE OF SEDIMENT AND WATER POPULATIONS
TO DIFFERENT LEVELS OF HgCl2 AND PMA

EFFECT OF INCUBATION TEMPERATURE ON BACTERIAL VIABLE COUNT
AND RESISTANCE TO HgCl>

COMPARATIVE EFFECTS OF AEROBIC AND ANAEROBIC INCUBATION ON
BACTERIAL RESISTANCE 10 HgCl,

EFFECT OF SALT CONCENTRATION ON BACTERIAL RESISTANCE TO
HgCly

DISTRIBUTION OF BACTERIAL HgClo RESISTANCE AND TOTAL MER-
CURY CONCENTRATIONS AMONG WATER AND SEDIMENT FRACTIONS

CROSS RESISTANCE OF BACTERIAL CULTURES TO MERCURY COMPOUNDS
HEAVY METAL RESISTANCE OF MERCURY-RESISTANT BACTERIA
ADAPTATION TO MERCURY RESISTANCE

METABOLISM OF PHENYIMERCURIC ACETATE BY MERCURY-RESISTANT
BACTERIA

EFFECTS OF SALTS AND INHIBITORS ON PMA METABOLISM BY
PSEUDOMONAS, STRAIN 244,

EFFECT OF MAGNESIUM ION CONCENTRATION ON PMA RESISTANCE
IN PSEUDOMONAS, STRAIN 244

DISTRIBUTION OF RADIOACTIVITY IN 203hg-pMa-LABELED CELLS
OF PSEUDOMONAS, STRAIN 244

TOTAL MERCURY CONCENTRATIONS AND PERCENTAGE OF THE TOTAL
VIABLE BACTERIAL COUNT RESISTANT TO 6 PPM OF HgClo

HgCl, METABOLIZING BACTERIA

MERCURY RESISTANCE AND METABOLISM OF BACTERIAL STRAIN
NO. 5 GROWN IN THE PRESENCE AND ABSENCE OF HgCl,

viii

Page

10
11
13
14

17

20


SECTION IX
LIST OF PUBLICATIONS

Articles

Nelson, J.D., H.L. McClam, and R.R. Colwell. The ecology of mercury-
resistant bacteria in Chesapeake Bay. In: Preprints. Proceedings of the
Eighth Annual Conference of the Marine Technology Society, Washington,
D.C. pp. 303-312. (1972)

Nelson, J.D. and R.R. Colwell. Metabolism of mercury compounds by bac-
teria in Chesapeake Bay. In: Third International Congress on Marine Cor~
rosion and Fouling. Northwestern University Press, Evanston, Illinois.
pp. 767-777. (1973)

Nelson, J.D., F.E. Brinckman, R.R. Colwell, and W.P. Iverson. Biodegra-
dation of phenylmercuric acetate by mercury-resistant bacteria. Appl.
Microbiol. 26:321-326. (1973)

Walker, J.D. and R.R. Colwell. Mercury-resistant bacteria and petroleum
degradation. Appl. Microbiol. 27:285-287. (1974)

Nelson, J.D. and R.R. Colwell. Ecology of mercury-resistant bacteria in
Chesapeake Bay. J. Microbial Ecol. In Press.

Papers Presented

Nelson, J.D., T.B. Elliot, and R.R. Colwell. Metabolism of mercury com- |
pounds by bacteria in Chesapeake Bay. Presented at the 35th Annual Meet-
ing of the American Society of Limnology and Oceanography, March 19-22,
Tallahassee, Florida. (1972)

Nelson, J.D., H.L. MeClam, and R.R, Colwell. The ecology of mercury-
resistant bacteria in Chesapeake Bay. Presented at the Eighth Annual Con-
Gon) of the Marine Technology Society, Sept. 11-13, Washington, D.C.
1972

Nelson, J.D. and R.R. Colwell. Metabolism of mercury compounds by bac-
teria in Chesapeake Bay. Presented at the Third International Congress
on Marine Corrosion and Fouling, Oct. 2-6, Gaithersburg, Maryland. (1972)

81


2.

3.

4

5.

9.

10.

SECTION VIII
REFERENCES

Nelson, J.D., W. Blair, F.E. Brinckman, R.R, Colwell, and W.P. Iver-
son. Biodegradation of phenylmercuric acetate by mercury-resistant
bacteria. Appl. Microbiol. 26:321-326. (1973)

Spangler, W.J., J.L. Spigarelli, J.M. Rose, R.S. Flippin, and H.H,
Miller, Degradation of methyl mercury by bacteria isolated from en-
vironmental samples. Appl. Microbiol. 25:488-493. (1973)

Wein, D. and E. Goldberg. Mercury in the marine environment.
Environ. Sei, Technol. 4:765-768. (1970)

Williams, P.M. and H.V. Weiss. Mercury in the marine environment:
Concentration in seawater and in a pelagic food chain. J. Fish.
Res, Board Can, 30:293-295. (1973)

Kondo, I., T. Ishikawa, and H. Nakahara. Mercury and cadmium resis-
tances mediated by the penicillinase plasmid in Staphylococcus
aureus. J, Bacteriol. 117:1-7. (1974)

Novick, R. and C, Roth. Plasmid-linked resistance to inorganic
salts in Staphylococcus aureus. J. Bacteriol. 95:1335-1342. (1968)

Smith, D. R factors mediate resistance to mercury, nickel, cobalt.
Science 156:1114-1115. (1967)

Summers, A.O. and E. Lewis. Volatilization of mercuric chloride by
mercury-resistant plasmid-bearing strains of Escherichia

Staphylococcus ai and Pseudomonas aeruginosa. J. Bacteriol.
143:1070-1072, (1973

Komura, I, and K, Izaki. Mechanism of mercuric chloride resistance
in microorganisms. I. Vaporization of a mercury compound from mer-
curic chloride by multiple drug resistant strains of Escherichia
goli., J. Biochem. (Japan) 70:885-893. (1971)

Tonomura, K., K, Maeda, F, Futai, T, Nakagami, and M, Yamada, Stim-

ulative vaporization of phenyl mercuric acetate by mercury-resistant
bacteria, Nature (London) 217:644-646. (1968)

Th


36,

wT.

38.

39.

40.

A

46.

47,

Iandner, L, Biochemical model for the biological methylation of mer-
cury suggested from methylation studies in v: with Neurospora
crassa. Nature (London) 230:452-454. (1971

Bongers, L.H. and M.N. Khattak. Sand and gravel overlay for control
of mercury in sediments. EPA-16080HVA01/72, U.S. Environmental Pro-
tua Agency. U.S. Government Printing Office, Washington, D.C.
1972

Holm, H.W. and M.F. Cox. Mercury transformations in aquatic sedi-
ments. In: Abstracts. Ann. Meeting Amer. Soc. Microbiol., Chicago,
Tllinois. p. 25. (1974)

Kaneko, T. and R.R. Colwell, Ecology of tpae Jerahacmolriious in
Chesapeake Bay. J. Bacteriol. 113:24-32. (1973

Murchelano, R.A. and C. Brown. Heterotrophic bacteria in Long Island
Sound. Marine Biology 7:1-6. (1970)

Jernelov, A. Mercury and food chains. In: Environmental Mercury
Contamination (R. Hartung and B.D. Dinan, eds.). Ann Arbor Science
Publishers, Inc., Ann Arbor, Michigan. pp. 174-177. (1972)

Brasfield, H. Environmental factors correlated with size of bacterial
populations in a polluted stream. Appl. Microbiol. 24:349-352. (1972)

Biggs, R.B. Geology and hydrography. In: Gross Physical and Bio-
logical Effects of Overboard Spoil Disposal in Upper Chesapeake Bay.
Contribution No. 397, Natural Resources Institute of the University

of Maryland, Solomons, Maryland: pp. 7-15. (1970)

Smith, J.D., R.A. Nicholson, and P.J. Moore. Mercury in water of
the tidal Thames. Nature (London) 232:393-394. (1971)

Brown, H.G., C.P. Hensley, G.L. McKinney, and J.L. Robinson. Effi-
ciency of heavy metals removal in municipal sewage treatment plants.
Environ, Letters 5:103-114. (1973)

Logsdon, G.S. and J.M. Symons. Mercury removal by conventional water
treatment methods. In: Proceedings 92nd Annual Conference American
Water Works Association, Chicago, Illinois. (1972)

Flemer, D.A. Phytoplankton. Im: Gross Physical and Biological Ef-
fects of Overboard Spoil Disposal in Upper Chesapeake Bay. Contribu-
tion No. 397, Natural Resources Institute of the University of
Maryland, Solomons, Maryland. pp. 16-25. (1970)

Goodwyn, F. Zooplankton. In: Gross Physical and Biological Effects
of Overboard+Spoil Disposal in Upper Chesapeake Bay. Contribution
No. 397, Natural Resources Institute of the University of Maryland,
Solomons, Maryland. pp. 39-41. (1970)

TT


Table 8, ADAPTATION 10 MERCURY RESISTANCE®

Before hin After growth in Hg

Test medium Medium % Cultures Medium % Cultures
grown in resistant grown in resistant
3 ppm Me HgCl Control 20 100 ppm HgCl 63
24 ppm PMA Control 3 100 ppm HgCl 1
24 ppm PMA Control 7 50 ppm HgCl Tl
24 ppm PMA Control 7 3 ppm PMA 13

“total mmber of cultures tested = 30.

Paeudomonas culture #5, when grown in the absence of HgCl2, showed de-
creased resistance to HgCl2. The same culture also evidenced a relative-
ly decreased ability to volatilig radiolabeled Hg from a buffered
suspension of cells containing Sig-labeled HgClg (Table 15, Section V).
These results indicated the capability to metabolize inorganic Hg in
this bacterial strain is inducible and related to mercury resistance.

The volatile radioactive product of the reaction was isolated and char-
acterized as Hg° (Section II), in agreement with the findings of others
(9, 12). A simplified system, consisting of non-proliferating cells in
a phosphate-buffered (pH 7.0), artificial, estuarine salts solution
(PES), was used to establish kinetic parameters. When the suspension was
bubbled with nitrogen, instead of air, Hg° formation was diminished by as
much as 45%. This observation is possibly related to effect of anaero-
biosis upon mercury resistance of mixed bacterial populations described
above. The reaction was dependent upon cell concentration and exhibited
saturation kinetics, characteristics which are attributable to enzyme-
catalyzed reactions. At 25 C, the half saturation constant (Km) was 20
ppm of HgClo and the maximm velocity of reduction was 35.4 pe/ng ary
weight of cells/min.

Similarly, bacterial adaptation to an organic mercury compound, PMA, was
investigated using Pseudomonas strain 244, which was originally isolated
from PMA-containing medium. Figure 3 shows the change in resistance of
the culture to a test concentration of 24 ppm of PMA, when grown with and
without 6 ppm of PMA "inducer." In contrast to the non-induced culture,
the induced culture, when plated on 24 ppm PMA medium, was able to grow
and maintain resistance for a long period of time. The number of PMA
resistant colony forming units (CFU) in the induced culture rose 200-fold,
whereas CFU of the non~induced culture decreased 30-fold. In either case,
these changes were preceded by a transient, but reproducible, increase in
resistance prior to the onset of growth. This rapid response is consis-
tent with the induction of an enzyme(s). The experiment was repeated
using 6 ppm of HgC1 as inducer. In contrast to the previous experiment,

14


Ecological Research Series



49.

50.

51.

52.

53.

5h.

55.

56.

57.

58.

59.

60.

61.

lovelace, 1.E., H. Tubiash, and R.R. Colwell. Quantitative and

Qualitative commensal bacterial flora of Crassostrea v: in
Chesapeake Bay, Proc. Natl. Shellfish Assoc. 58:82-87. (see)

Ross, I.S. and K.M. Old. Thiol compounds and resistance of
Prrenenbors avenae to mercury. Trans. Br. Mycol. Soc. 60:301-310.

Stutzenberger, F.J. and E.0, Bennett. Sensitivity of mixed popula-

tions of Staphylococcus aureus and Goli to mercurials.
Appl. Microbiol. 133570-574. (1965

Nuzzi, R. Toxicity of mercury to phytoplankton. Nature (London)
227:38-40. (1972)

Ben-Bassat, D., G.-Shelef, N. Gruner, and H. Shuval. Growth of
in a medium containing mercury. Nature (London) 240:
43-44. (1972)

Nelson, J.D. and R.R. Colwell. Metabolism of mercury compounds by
bacteria in Chesapeake Bay. Jn: Third International Congress on
Marine Corrosion and Fouling. Northwestern University Press,
Evanston, Illinois. pp. 767-777. (1973)

Furukawa, K., T. Suzuki, and K. Tonomura. Decomposition of organic
mercurial compounds by meroury-resistant bacteria. Agr. Biol. Chem.
(Japan) 33:128-130. (1969)

Tingle, L.E., W.A. Pavlat, and I.L, Cameron. Sublethal cytotoxic
effects of mercuric chloride on the ciliate Tetrahymena pyriformis.
J. Protozool. 20:301-304. (1973)

Bernheim, F. The effect of cyanogen iodide and mercuric chloride
on the permeability of cells of Pseudomonas aeruginosa and the
antagonistic action of sulfhydryl compounds. Proc. Soc. Expt. Biol.
Med. 138:444~447. (1971)

Kellenberger, E, and R. Ryter. Cell wall and cytoplasmic membrane
of Escherichia coli. J. Biophys. Biochem. Cytol. 4:323-326. (1958)

Inft, J.H. Improvement in epoxy resin embedding material. J. Bio-
phys. Biochem. Cytol. 9:409-414. (1961)

Watson, M.L. Staining of tissue sections for electron microscopy
with heavy metals. J. Biophys. Biochem, Cytol. 4:475-478. (1958)

Venalle, J.H. and R. Coggeshall. A simplified lead citrate stain
for use in electron microscopy. J. Cell Biol. 25:407-408. (1965)

78


Table 18. MULTIPLE REGRESSION ANALYSIS OF SEASONAL DATA

Partial standard

a Correlation coefficients regression Significance

Y xX Simple Partial Multiple coefficient level af Sample

& vater 3 +0,08557  -0.90433 0.9871> 9.14 0.05 10 1-81, a2

% vater 5 0.76012 -0.84457 0.9871 1.29 0.05 10 "

% sediment 6  +0.50568 +40.82607 0.9232 40.42 0.05 10 "

% vater 7 ~+0.79797 +0.81540 0.9640 40.85 0.05 11 2-B1, B2

% vater 9 0.42607 -0,72052 0.9640 -0.57 0.10 1 "

% water 2 40.79752 +0.94433 0.9731 1.96 0,05 9 3-EBI, EB2

% veter 4  +0.26347  -0.89642 0.9731 0.95 0.10 9 "

% sediment 1 +40.79752 +40.94434 0.9950 +0.45 0.05 9 "

% sediment 3 +0.68272 +0.94348 0.9950 0.64 0.05 9 "

% sediment 4 +0.45798 +0.95714 0.9950 40.49 0.01 9 "

% sediment 5  +0.01878 -0.96038 0.9950 8.30 0.01 9 "

% sediment 6 -0.14795 -0.92919 0.9950 1.09 0.05 9 "

% sediment 8 -0.00870 -0.96131 0.9950 -7.30 0.01 9 "

% sediment 9 -0.15035 +0.94953 0.9950 0.67 0.05 9 "

% water 3  +0.67893 +0.60978 0.7358¢ 0.65 0.001 32 aa, 2
EBI, ER2

% water 2 +0.66167 40.5777 0.8171 0.71 0.05 17 5rAa, 2

% sediment 1 +0.66167 +0.5777 0.88344 0.47 0.05 17 "

% sediment 7 -0.47979 +0.50305 0.8834 0.59 0.10 17 "

% sediment 8 +0.14737 +0.53308 0.8834 1.55 0.10 17 "

% sediment 9 40.66394 +0.59526 0.8834 0.83 0.05 17 "

ay = % water; X2 = % sediment; X3 = TVC water; X, = TVC sediment; X5 = surface water tem-
perature; 16 = salinity; 7 = turbidity; Xg = dissolved oxygen; Xg = total mercury concen-
tration in sediment. Only those variables whose partial regressien coefficients were
significantly >O are included (see "Significance level" colum).

b,c, a

F value for analysis of variance for multiple linear regression is significant (0.10,
0.01, and 0.05 levels of significance, respectively).

38


SECTION I
CONCLUSIONS

The results of this investigation prove that aerobic, heterotrophic bac-
teria can be influential in the mobilization of mercury from existing
resources in Chesapeake Bay.

Mercury-resistant bacteria are numerous throughout Chesapeake Bay in
water and sediment, but are most prevalent in those areas where high
total mercury levels are found in sediment. Bacteria "adapt" to mer-
cury, in terms of total numbers and generic distribution in a given lo-
cation and in the ability to resist and reduce mercury, presented in the
form of mercuric and phenylmercuric ions, to the elemental state. The
former adaptation probably represents a long-term, selective effect, and
the latter a more rapid, or transient, microbial response to mercury
contamination, Although bacteria adapt successfully, in vitro studies
suggest that bacteria can undergo cell damage in the presence of exces-
sive levels of mercury contamination. Bacterial growth occurs at a
threshold inhibitory concentration of mercury, with the appearance of
numerous morphological effects, which indicate cell wall and cytoplasmic
membrane damage.

The proportion of mercury-resistant bacteria in the total viable, aero-
bic, heterotrophic bacterial population serves as a valid index of po-
tential mercury mobilization in Chesapeake Bay water and sediment.

Using this criterion, it can be concluded that bacterial mobilization of
mercury, through the formation of Hg®, varies seasonally and is related
to water turbidity, water dissolved oxygen, and total mercury concentra-
tions in sediment. A reproducible peak in activity occurs in Spring.
This phenomenon can have serious consequences, in terms of effects on
life cycles of higher marine organisms, and the rates of accumulation of
mercury through marine and estuarine food webs.


ACKNOWLEDGMENTS

Shiptime aboard the R/V RIDGELY WARFIELD was made possible by Grant
#GD-31707 from the National Science Foundation to the Chesapeake Bay
Institute, Johns Hopkins University. We are indebted to Dr. E. Taylor
and H. Whaley of the Chesapeake Bay Institute and the crew of the R/V
RIDGELY WARFIELD for their excellent cooperation and assistance during

the field studies. Shiptime was also provided by the State of Maryland
Department of Natural Resources.

We are also grateful for heavy metal analyses performed by 0. Villa and
J. Marks, Annapolis Field Laboratory, Environmental Protection Agency;
J. F. Kopp, Environmental Monitoring and Support Laboratory, Cincinnati,
Ohio; and W. Blair, National Bureau of Standards, Washington, D.C.
Analytical support was provided via the National Bureau of Standards
(Environmental Protection Agency Grant #R 801002).

We also acknowledge electron microscopists, Z. Vaituzis and L. W. Wan,
for their considerable efforts.


Table 20, SURVEY OF MORPHOLOGICAL EFFECTS IN SELECTED CULTURES OF BAC-
TERIA GROWN IN THE PRESENCE OF HgC1,°

b HgC1g lag
Culture concentration phase

Genus Strain (ppm) (h) Morphological effects®

Pseudomonas % 10 2 Cell wall outgrowth

Citrobacter 132 20 70 Plasmolysis, damaged
membrane

Flavobacterium 119 10 40 Cell wall outgrowth,
plasmolysis, elongated
cells, irregular meso-
somes

Vibrio 639 5 72 Giant, pleomorphic and
rod-shaped cells,
elongated cells

Bagillus 394, 15 Al Irregular septum forma-
tion

Arthrobacter 72 5 24 Irregular septum forma-
tion

Enterobacter 85 1 ie} Pleomorphic cells, out-
growth of cell wall,
plasmolysis

Pseudomonas = B-164 15 6 Small spherical cells

days bounded by common

outer membrane

Pseudomonas 244 25 ) None

>selected cultures were inoculated into shaker flasks of broth with and
without HgClo added and were incubated at 25 C until early log phase.

b
Cultures were isolated and purified from colonies picked from mereury-
containing nutrient medium inoculated with water and sediment samples
collected in Chesapeake Bay and the Potomac River.

“Fixed and stained thin sections of cells grown, with and without HgClo,
to the same cell density were compared.

4a plied by R. A, Macleod.

51


The microbial populations of water and sediment, collected by differen-
tial centrifugation, also carried out the reductive process. Recoveries
of bacteria isolated by this method were essentially quantitative for
water and approximately 1% for sediments, based upon total, viable,
aerobic, heterotrophic bacterial counts. Assays for total and non-
filterable radioactive mercury in suspensions containing live and sterile
inocula and uninoculated controls were performed as described for the
previous pure culture experiments (Table 17). Under the conditions of
the assay, the microorganisms slowly released Hg®. The rate of loss
measured for the sterilized controls indicated some chemical reduction,
i.e., non-biological reduction also occurred. The major difference be-
tween the Colgate Creek and Eastern Bay samples (B-2 and EB~1), respec-
tively) was, in each case, the amount of mercury bound to non-filterable
material, Lowering the initial Hg2* concentration by an order of magni-
tude decreased the loss ot Hg°, but increased binding.

Analysis of Seasonal Date

Figures 9 and 10 illustrate changes in proportions of HgCl2-resistant
bacteria in water and sediment during the period from January 1972
through May 1973. A distinct periodicity was evident for both water and
sediment, with a major peak in mercury-resistant bacteria observed in the
Spring months of both 1972 and 1973 and a possible secondary peak in the
Fall ot 1972. Figures 9 and 10 also show the relative differences in
resistance among three of the stations. Several physical parameters
were found to demonstrate a seasonal periodicity (Fig. 11) characteris-
tic of the Chesapeake Bay estuary (32).

The data were subjected to mltiple stepwise regression analysis to de-
tect possible relationships between the selected physical parameters and
number of resistant organisms and to establish a basis for comparison of
the three sites. The percentages of bacteria resistant to HgCl2 in
water and sediment were treated as dependent variables, respectively.
Independent variables, whose partial regression coefficients were sig-
nificantly greater than zero (t test of significance), are presented in
Table 18. Standard partial regression coefficients were also calculated
to show the relative contribution of each variable to the regression
equations (29). When each site was considered separately, it was evi-
dent that there were differences in factors affecting mercury resistance
among the sites (sample 1, 2 and 3). When data for all six stations
were added to the regression equation, the individual measured parame-
ters did not adequately account for changes in resistance (sample 4).
Three stations were selected on the basis of similarities in sediment
type and differences in sediment concentrations (sample 5). Percentage
of resistance in water and sediment for each site were observed to be
related, but differences between water and sediment were not significant
(0.05 significance level). However, average differences in percentage
of resistance in water and sediment, respectively, among the three loca-
tions were significant (0.05 significance level). Resistance in sedi-
ments was found to be correlated positively with water transparency
(Secchi disc), dissolved oxygen, and total mercury concentration in the
sediment. Resistance was not observed to be related to total viable
count.

34


1.

12,

13.

14.

15.

16.

17,

18.

19.

20.

Furukawa, K. and K. Tonomura, Induction of metallic mercury-releas-
ing enzyme in mercury-resistant Pseudomonas. Agr. Biol. Chem,
(Japan) 36:2441-2448. (1972)

Summers, 4.0. and S. Silver. Mercury-resistance in a plasmid bear-
ing strain of Escherichia goli. J. Bacteriol. 112:1228-1236. (1972)

Magos, L.A., A. Tuffrey, and T.W. Clarkson. Volatilization of mer-
cury by bacteria, Brit. J. Ind. Med. 21:294-298. (1964)

Komura, I., T. Fumaba, and K. Izaki. Mechanism of mercuric chloride
resistance in microorganisms. II. NADPH-dependent reduction of mer-
curic chloride and vaporization of mercury from mercuric chloride by
a multiple drug resistant strain of Escherichia coli. J. Biochem.
(Japan) 70:895-901. (1971)

Nelson, J.D. and R.R. Colwell. Effects of tropical storm Agnes upon
the bacterial flora of Chesapeake Bay. Chesapeake Research Consor-
in #27, U. S. Army Corps of Engineers, Baltimore, Maryland.
1974,

Tonomura, K., K. Maeda, and F. Futai. Studies on the action of
mercury-resistant microorganisms on mercurials. II, The vaporiza-
tion of mercurials stimulated by mercury-resistant bacterium. J.

Ferment. Technol, £6:685-692. (1968)

Olson, B.H. and R.C. Cooper. Methylation of mercury by estuarine
sediments. In: Abstracts. Ann. Meeting Amer. Soc. for Microbiol.,
Miami, Florida. p. 48. (1973)

Spangler, W.J., J.L. Spigarelli, J.M. Rose, and H.H. Miller. Methyl-
mercury: Bacterial degradation in lake sediments. Science 180:
192-193. (1973)

Bothner, M.H. and R. Carpenter. The rate of mercury loss from con-
taminated estuarine sediments in Bellingham Bay, Washington. In:
Proc. First Ann. National Sci. Foundation Trace Contaminants Confer-
ence, Oak Ridge National Laboratory, Oak Ridge, Tennessee. (1973)

Gilmour, J.T. and M.S. Miller. Fate of a mercuric-mercurous chloride
fungicide added to turf grass. J. Environ. Qual. 2:145-148. (1973)

Kimura, Y. and V.L. Miller. The degradation of o: jomercury fungi-
cides in soil. J. Agric. Food Chem. 12:253-257. (1964)

Vostal, J. Transport and transformation of mercury in nature and
possible routes of exposure. In: Mercury in the Environment (L.T.
ore) J. Vostal, eds.). CRC Press, Cleveland, Ohio. pp. 15-

15


Table 11, EFFECT OF MAGNESIUM ION CONCENTRATION ON PMA
RESISTANCE IN PSEUDOMONAS STRAIN 244

Magnesium Growth (days)®
concentration PMA concentration (ppm)
(mM) 0 10 15 20 25 30
0.1 + +3 -- - - -—
1.2 + + +3 +3 +3 _
5.6 +1 + +H +3 +3 +3
11.2 + + + +3 +3 +3
22.3 +3 + nal] +3 +3 +3

80,01 ml of culture was transferred to tubes of basal
broth containing varying concentrations of MgClg and
PMA, The tubes were incubated at 25 C, with shaking,
and observed up to 7 days for growth.

Ce Site of Merc’ ne id_ Metal

The effects of several inhibitors of oxidative phosphorylation were
tested in Pseudomonas strain 244 PMA-metabolizing system to see if
active transport of PMA into the cells was essential for metabolism
(Table 10). Potassium cyanide (10 mM) caused considerable inhibition of
the initial rate of reaction, but none of the inhibitors caused more
than a 30% net decrease in the PMA metabolized after 30 min. The partial
inhibitions may have resulted from the formation of complexes of phenyl-
mercuric ions with the inhibitors, Cells treated with sodium arsenate
or sodium azide bound considerably more label than did the control, but
cyanide-treated cells bound less label. These results suggested that
PMA metabolism takes place on, or near, the cell surface. A similar con-
clusion was reached by Tonomura et al. (10), who used a PMA-resistant
Pseudomonas. The cellular site for binding of PMA was examined in the
following . Cells of P; strain 244 were grown in the pres-
ence of ig-labeled PMA for 2/3 generation time, after which the cells
were fractured by mechanical shear. PMA degradation was stopped immedi-~
ately by the breakage, and the cells were separated into crude subcellu-
lar fractions by differential centrifugation (Table 12). Supernatant
solutions were separated from pellets, after sequential centrifugation
at 3,000 x G for 15 min, 35,000 x G for 15 min and 126,000 x G for 1 hr.
Approximately 75% of the cell-bound radioactivity was present in partic-
ulate fractions sedimented by 3,000 and 35,000 x G forces. On the basis
of these findings, it was concluded that the majority of the PMA binds
to components of the cell envelope.

al


Representative colonies from platings of water and sediment on HgClo-
and PMA-containing media were selected for further study. Cultures were
purified and identified to genus for each of the individual samples
examined during 1972 (Table 19). The majority of HgCl2-resistant bac-
teria were cytochrome oxidase positive, non-pigmented, Gram-negative,
asporogenous rods which produced H2S, but did not utilize glucose or
require added sodium ion for growth. PMA-resistant bacteria differed
primarily in the utilization of glucose and production of diffusible
pigments. Upon comparison of paired HgClg-resistant and randomly
selected isolates from medium without Hg, it was found that differences
in salt requirements and H2S production were not significant (0,05 level
of significance).

Preliminary results, however, indicated that there were detectable dif-
ferences in the population distributions of the mercury-resistant and
the total viable, aerobic, heterotrophic bacterial populations (34).
The total population is now being more fully characterized to verify
this observation. A total of 539 pure cultures isolated on 6 ppm
HgClg-containing medium, collected in the period from October 1971
through December 1972, were characterized. On the basis of our modifi-
cation of the scheme proposed by Shewan et al. (27), the cultures were
placed into one of seven generic groups or an eighth category (less than
4% in the majority of cases) into which were lumped yeasts, molds, and
unidentified bacteria, Chi-square analysis of the data (0.01 level of
significance) indicated that there were cant differences in dis-
tribution spatially (Fig. 12), seasonally (Fig. 13), and between water
and sediment (Fig. 14). The apparent diversity of genera was greatest
at the Eastern Bay stations. The most conspicuous seasonal changes in
population structure were in the Pseudomonas and Bacillus spp. (Fig.
12). Minima in percentage of the former occurred in the spring and
fall, while a maximum of the latter occurred during the fall destratifi-
cation. Water and sediment differed primarily in the proportions of
Pesndomonaa and Gram positive rods (Bacillus spp. plus coryneform bac-
teria). The genus Pgeudomonas accounted for 66% of all HgCl2-resistant
bacteria in water and sediment. A much higher proportion, 93%, of the
70 PMA-resistant isolates were Pseudomonas spp., the majority of which
were type II (27).

DISCUSSION

Experimental Sampling Stations

The range of mercury concentrations found in sediments of the regularly
sampled stations appears to be related to the nature of the sediments
and their locations (Table 13). The highest concentrations were found
in the fine. silty-clay sediments located near industrial activity or
industrial plant outfalls. There was no significant difference in the.
mercury level of the sandy sediments of Station A-1 and the silty-clay
sediments of Eagtern Bay. The difference in mercury concentrations

39


metabolism, The magnesium effect is particularly interesting in view
of the estuarine locale from which the organism was isolated. The opti-

mum magnesium concentration approximated the average in situ concentra-
tions of magnesium. We have shown that metabolism of PMA by Pseudo-
Ronas strain 244 is dependent upon cell concentration and exhibits
saturation kinetics. At 25 C, the pH optimum was between pH 6 and 7,
the half saturation constant (Km) was 16.7 ppm PMA, and the maximm
velocity of degradation was 10.6 ug/mg dry weight of cells/min.

Table 10. EFFECTS OF SALTS AND INHIBITORS ON PMA METABOLISM BY PSEUDO-
MONAS STRAIN 244

PMA metabolized (percent of control*)

Experimental condition Initial rate Net/30 min
82.0% sea water, Tris buffer 99.3 _
8.2% sea water, Tris buffer 15.0 --
8.2% sea water, no buffer 56.1 -
Tris buffer, alone 5.6 --
NaCl, phosphate buffer? 26.1 -_-
KCl, phosphate buffer? 0.0 ~
MgClp, phosphate buffer? 99.3 -
0.01 M KCN® 56.6 The
0.01 M NaN3® 100.0 82.4
0.01 M Nag As, -7H20° 72.0 73.5

20.01 M buffered-"three salts" (pH 7.0) containing 6 ppm PMA and ap-
proximately 0.1 gm dry weight of cells/ml, aerated at 25 C.

concentration equivalent to total ionic strength of "three salts" so-
lution.

“Inhibitors incubated with cells 5 min before reaction started in phos-
phate-buffered three salts.

20


Figure 22.

Thin section of culture #85 cells immediately after addition
to broth containing 2 ppm of HgC12. The cells show dispersed
chromatin, normal ribosomes and cell envelope constituents,
as well as typical gram negative cell division by constric-
tion.

Thin section of culture #85 cells after 2 h incubation in
broth containing 2 ppm of HgCl . Similar in appearance to
zero time, except for condensed chromatin, and the appear-
ance of electron dense groups or ribosomes (arrow).

58


16

17

18

19

20

23

25

B

Growth of Bacillus, species 394, in the presence and
absence of 5 ppm of HgCl2

Thin section of cells of culture #394 grown in the pres-
ence of 5 ppm of HgCl,

Thin section of cells of culture #394 grown in the pres-
ence of 15 ppm of HgCl2

Growth and uptake and metabolism of mercuric ion by
Enterobacter, strain 85

Colonial variation in Egterobacter, strain 85, grown in
the presence of 2 ppm of HgCl,

Growth and uptake and metabolism of mercuric ion by
Enterobacter, strain 85

Thin section of culture #85 cells immediately after
addition to broth containing 2 ppm of HgClo

Thin section of culture #85 cells after 2 h incubation
in broth containing 2 ppm of HgClo

Thin section of culture #85 cells after 6 h incubation
in broth containing 2 ppm of HgClo

Thin section of culture #85 cells after 24 h incubation
in broth containing 2 ppm of HgCl,

Thin section of culture #85 cells after 26 h incubation
in broth containing 2 ppm of HgCl,

Thin section of culture #85 cells after 28 h incubation
in broth containing 2 ppm of HgCl,

Sterilizable oyster aquarium
Oyster aquarium in operation
Oyster dissection

vii

52

53

53

54

55

56

58

58

59

61

67


The following experiments describe tke resistance characteristics of
pure cultures of bacteria grown in the presence of selected mercury com-
pounds. Inoculum size, physiological age, and aeration of broth cul-
tures were all found to be factors influencing mercury resistance.
Hence, these parameters were carefully controlled in each experiment.
Two hundred and forty-nine pure, freshly isolated, HgCl2-resistant cul-
tures were tested for resistance to a series of types and concentrations
of mercury compounds by replicating from a master agar plate to a set of
meroury~containing plates using sterile Velvetine cloth (Table 6).
Groups of cultures which were strongly resistant to a selected compound
showed a corresponding high resistance to other compounds of mercury.
This suggests that the acquisition of resistance to a single compound of
mercury confers a generalized resistance to mercury compounds. The con-
parative resistance of bacteria from water and sediments were estab-
lished in an experiment with 131 cultures isolated from samples collect-
ed at a single station in Baltimore Harbor, Maryland (Fig. 2). In this
particular sample, sediment populations were more resistant to different
types and concentrations of mercury compounds. Similarly, the percent
of total mercury-resistant bacteria was greater in sediment samples than
in water samples. Differences in resistance can be explained by the
fact that sediment and water bacterial population distributions are sig-
nificantly different, but not always by intrinsic differences in mercury
resistance, because the relationship between water and sediment in terms
of percent resistance is not consistent (Section V).

Table 6, CROSS RESISTANCE OF BACTERIAL CULTURES TO MERCURY COMPOUNDS

Percent of cultures resistart

Group?
2k, 15 3 3 100 50
Test compound® PMA PMA PMA Me HgCl HgClp = gClp
24 ppm PMA - -- - 27.9 36.4 9.4
15 ppm PMA _- -- _ 90.8 63.6 20.8
3 ppm Me HgCl 85.8 89.6 6.1 -- 100.0 29.2
100 ppm HgCly 28.6 hel 0.0 25.6 -- --

®Fach compound was incorporated into a solid growth medium.

P Rech culture was classified according to the maximum amount of mer-
cury tolerated (ppm).

11


23.

2h.

25.

26.

29.

30.

31.

32.

33.

35.

Jernelov, A. Factors in the transformation of mercury to methylmer-
cury. In: Environmental Mercury Contamination (R. Hartung and B.D.
Dinan, eds.). Ann Arbor Science Publishers, Inc., Ann Arbor,
Michigan. pp. 167-172. (1972)

Lockwood, R.A. and K.Y. Chen. Chemical transformation of mercury in
the aquatic environment. Presented at the 35th Annual Meeting of the

American Society of Limnology and Oceanography, March 19-22,
Tallahassee, Florida. (1972

Jernelov, A. Studies in Sweden on feasibility of some methods for
restoration of mercury-contaminated bodies of water. Environ. Sci.
and Technol. 7:712-718. (1973)

Hatch, W.R. and W.L, Ott. Determination of sub-microgram quantities

of mercury by atomic absorption spectrophotometry. Anal. Chem. 40:
2085-2087. (1968)

Shewan, J.M., G. Hobbs, and W. Hodgkiss. A determinative scheme for
the identification of certain genera of gram-negative bacteria, with

special reference to the Pseudomonadaceae. J. Appl. Bacteriol. 23:
379-390. (1960)

Colwell, R.R. and W.J. Wiebe. "Core" characteristics for use in
classifying aerobic, heterotrophic bacteria by numerical taxonomy.
Bull. Georgia Acad. Sei. 28:165-185. (1970)

Steel, R.G.D. and J.H. Torrie. Principles and Procedures of Statis-
tics. McGraw-Hill Book Company, Inc., New York, N.Y. (1960)

Balkwill, D.L. and L.E. Casida, Jr. Microflora of soil as viewed by
freeze-etching. J. Bacteriol. 114:1319-1327. (1973)

Westoo, G. Determination of methyl mercury compounds in food stuffs.
I, Methyl mercury compounds in fish, identification and determination.
Acta Chem, Scand. 20:2131-2137. (1966)

Schubel, J.R. The physical and chemical conditions of the Chesapeake
Bay, J. Wash. Acad. Sci. 62:56-87. (1972)

Gaby, W. and C. Hadley. Analytical laboratory test for the identi-
fication of Pseudomonas seruginoga. J. Bacteriol. 74:356-358. (1957)

Nelson, J.D., H.L. McClam, and R.R. Colwell. The ecology of mercury-
resistant bacteria in Chesapeake Bay. In: Preprints. Proceedings
of the Sth Annual Conference of the Marine Technology Society,

Sept. 11-13, Washington, D.C. (1972)

Walker, J.D. and R.R. Colwell. Mercury-resistant bacteria and petro-
leum degradation. Appl. Microbiol. 27:285-287. (1973)

16


of mercury employed in the study were selected so that correlation of
results obtained with in sity conditions in Chesapeake Bay could be
achieved.

MATERIALS AND METHODS

Organisms and Cultural Methods

Organisms were cultured in an artificial estuarine salts nutrient broth
and agar medium described previously. (See Section V.) Cultures used
in the study were isolated by spreading suitable dilutions of water and
sediment on media supplemented with varying concentrations of HgCl, or
PMA.

Electron Microscopy

Cells in late log phase cultures were transferred to fresh broth (0.1
ml/m1) in shaker flasks with and without sublethal concentrations of
HgClg added and were incubated at 25 C for 3 to 4 generation times, or
until equal turbidities were obtained during early logarithmic phase of
growth. The cultures were fixed according to the procedure described by
Kellenberger and Ryter (58). Ten ml of culture was washed once in bar-
bituric acid (K-R) buffer (pH 6.2), following which the cells were fixed
in 1.0% osmium tetroxide in K-R buffer and prestained in 0,5% uranyl
acetate, in K-R buffer, prior to stepwise dehydration through a 50%,
15%, 85%, 95%, and 100% ethanol and propylene oxide series, The speci-
mens were embedded in Epon resin (Miller-Stephenson Chemical Co., Inc.,
Danbury, Conn.) (59), and ultra-thin sections were prepared using an IKB
800 Ultramicrotome III (LKB-Producter AB, Bromma 1, Sweden), fitted with
a diamond knife (E. I. DuPont de Nemours and Co., Wilmington, Del.).

The sections were placed on collodion-coated 200 and 300 micron mesh
size copper grids and stained with a saturated solution of uranyl ace-
tate (60) and lead citrate (61). An RCA EMU-3E or Hitachi HU-11A micro-
scope (accelerating voltage of 50 kv) was used to view the sections.

2034 5_ C1.

Culture 85, grown without mercury, was added to shaker flasks containing
broth (0.1 ml inoculum/5 ml of broth). The first, i.e,,,control flask
contained no mercury, the second flask, 2 or 4 ppm of -labeled HgCl2
(approx. 200,000 dpm/ml), The flasks were incubated at 34 C (first ex-
periment) or 25 G (second experiment) and optical densities relative to
an uninoculated 203Hg-labeled blank control were measured using a Klett-
Sommerson Photoelectric Colorimeter (Klett Manufacturing Co., New York,
N.Y.). Aliquots of cells were removed aseptically from the unlabeled
medium and fixed for electron microscopy or diluted and plated on
nutrient agar for viable counts. Duplicate 0.1 ml volumes of culture
were removed at appropraite intervals for determination of total and
non-filterable radioactivity by liquid scintillation.

hg


eh

Table 25, TISSUE DISTRIBUTION OF MERCURY AMONG TREATED OYSTERS®
A, Relative uptake

Percent?
Mantle e Visceral Adductor Whole
Experiment fluid Mantle Gills mass muscle oyster
_ oT 1.05 + 1.43 14.51 + 3.81 51.79 + 11.63 29.57 + 12.71 3.06 $1.11 4019 + 2.49
II 1.17 40.52 12.14 + 5.86 67.50 + 9.36 16.87 + 7.89 2.34 + 0.51 9.84 + 4.77
IIr 1.25 40.54 20.67 + 5.63 57.84 + 5.44 17.70 + 6.45 2.46 + 1.20 9.20 + 5.91

B. Mercury concentration®

Mean mereury concentration (ppb HgClp - wet weight)

Mantle Visceral Adductor Whole

Experiment. fluid Mantle Gills® mass muscle oyster
I 4.13 + 5.69 162,10 + 128,90 619,50 + 474.40 231.90 + 182.90 52.80 + 44.10 173.30 + 120,00
II 10.35 + 8.59 397.05 + 290.00 2974.10 + 1703.70 338.30 + 262.00 153.25 + 105.76 424.60 + 267.00
TII 8.49 + 6.23 714.78 + 414.67 2778.65 + 1060.78 408.77 + 238.93 200.02 + 197.69 389.77 + 277.59

'after four days in the presence of HgCl, (initial concentration of 10,2 ppb).

b
Tissues expressed relative to total uptake of HgCl2 per oyster. For whole oysters, expressed relative to total
initial amount of HgCl2 added to the aquarium, Ninety-five percent confidence interval for mean of 4 determinations.

°Ineluding labial palps.

Ninety-five percent confidence interval for mean of 4 determinations. Underlined values are significantly different

from control values (experiment I).


62.

63.

70.

1.

72.

73.

The

Troger, R. Uber den Metallnachweis in quecksilber-oder kupfer-
behandelten Bakterien. (Detection of metals in bacteria tested with
mercury or copper.) Arch. Mibrobiol. 33:186-190. (1959)

Murray, A. and D, Kidby. Mercury distribution and transformations
in growing yeast. In: Abstracts. Ann. Meeting Amer. Soc. Microbiol.,
Philadelphia, Pennsylvania. p. 17. (1972)

Gillespie, D.C. Mobilization of mercury from sediments into guppies
( . Jd. Fish, Res. Bd. Canada 29:1035-1041.
1972,

Anderson, A.A., J.M. Anderson, and L.E. Mayer. System simulation to
identify environmental research needs: Mercury contamination. OIKOS
242231-238. (1973)

Barber, R.T., A. Vijayakumar, and F.A. Cross. Mercury concentrations
in recent and ninety-year-old benthopelagic fish. Science 178:
636-639. (1972)

Weiss, H.V., M. Koide, and E.D. Goldberg. Mercury in a Greenland ice
sheet: Evidence of recent input by man. Science 174:692-69%4. (1971)

Parsons, T.R., C.A. Bawden, and W.A. Heath. Preliminary survey of
mercury and other metals contained in animals from the Fraser River
md flats. J. Fish. Res. Bd. Canada 30:1014-1016. (1973)

leatherland, T.M., J.D. Burton, F. Culkin, M.J. McCartney, and R.J.
Morris. Concentrations of some trace metals in pelagic organisms
and of mercury in Northeast Atlantic Ocean water. Deep Sea Res. 20:
679-685. (1973)

Westoo, G. Methylmercury as percentage of total mercury in flesh and
tn) of salmon and sea trout of various ages. Science 181:567-568.
1973

Windom, H., R. Stickney, R. Smith, D. White, and F, Taylor. Arsenic, —
cadmium, copper, mercury, zinc in some species of North Atlantic fin
fish, J. Fish, Res, Bd. Canada 30:275-279. (1973)

Holden, A.V. Mercury in fish and shellfish. A review. J. Food
Technol, §:1-25. (1973)

Klemmer, H. and S.N. Iuoma. Mercury levels in marine biota. Project
Bull. No. 6, Water Resources Research Center, University of Hawaii,
Honolulu. (1973)

Peakall, D.B, and R.J. Lovett. Mercury: Its occurrence and effects
in the ecosystem. BioScience 22:20-25. (1972)

19


The experiment was repeated using an elevated concentration of mercury
(4 pn) to test the hypothesis that the length of the lag phase demon-
strated by this organism was dependent upon reduction of mercury con-
centration to a specific threshold concentration. The patterns of
growth and loss of mercury were as observed in the first experiment,
except that the lag phase was found to be almost three days (Fig. 20).

ood

JOACTIVITY
8

19 RADI

PERCENT H,
rs
3
\ °
°
a
°
LOG TOTAL VIABLE COUNT

20

a
10) 20Fe a 4. —e
ON
a es fb
05}
.
Pree ns

10 20 30 40 60 60 70
INCUBATION TIME (hr.)

LOG TURBIDITY (KLETT UNITS)

Figure 20. Growth and uptake and metabolism of mercuric ion by Entero-
r, strain 85. Shaker flasks containing broth with
soa and un-labeled HgCl2 (4 ppm) were inoculated with an
18 h culture and incubated at 25 C. Turbidity (A) and total

(A) and non-filterable (TM) radioactivity in the labeled
culture were measured. An uninoculated, labeled control
flask (1) was also assayed for total radioactivity. Total
viable counts (Q) and numbers of small colonial variants
(85-S, Fig. 19) (@) were obtained by spreading suitable di-
lutions of unlabeled mercury containing medium upon basal
nutrient agar.

However, during the lag phase, the mercury concentration dropped 70%, to
a level of 1.2 ppm, a concentration closely approximating the concentra-
tion of 1.1 ppm HgClg concentration at which growth commenced in the

56


sources of mercury. The hypothesized relationship between mercury re-
sistance and metabolism was suggested by the published observations
cited previously, and was substantiated by a series of experiments.

| CHESAPEAKE BAY

NAUTICAL MILES.
2 5 10 15 2

| 6

10" 20" 30" 40
KILOMETERS, es

Figure 7. Experimental sampling sites in upper Chesapeake Bay. Station
designations are: 2 = B-2 (Baltimore Harbor); 3 = B-1 (Balti-
more Harbor); 4 = A-1 (Annapolis); 5 = A-2 (Annapolis); 7 =
EB-1 (Eastern Bay); 8 = EB-2 (Eastern Bay); and 20 = Elizabeth
River.

28


RESULTS AND DISCUSSION

Three experiments, each using four oyster replicates per experiment, were
completed (Table 22). The oysters were harvested at the same time of
year from the same location, There was some variation in size, but it
was determined that there was no correlation between size and the amount
of mercury accumulated. Total mercury concentrations in the oysters
prior to each experiment were nearly equal to the initial concentration
of mercury in the aquarium water (Table 23). The significantly greater
concentrations of mercury in treated oysters indicated that tracer uptake
was truly a measure of accumulation and not exchange.

The purpose of the first experiment was to establish a base line for mea-
suring the effect of Hg-resistant bacteria on Hg uptake by the oysters.
In the second experiment, culture #14 (see Section V), which accumlates
Hg (12 u gm of HgClg accumulated/mg of cells/15 min from a solution of

6 ppm HgClg) was added. In experiment three, culture #5 (see Section V),
which actively reduces Hg’* to Hg° (25.4 ug of HgClg reduced to Hg°/mg of
cells/15 min from a solution of 6 ppm HgCla) was added. In experiments
II and III, a quantity of cells approximately equal to that of the mixed
flora in the aquarium was added.

Table 22. EXPERIMENTAL OUTLINE

Oyster tissue wet weight (gm)*

Mantle Visceral Adductor
Experiment fluid Mantle Gills mass muscle Total
p> 12.3 5.7 5.0 7.6 3.6 34.1
r° 12.3 3.2 2.3 5.0 1.6 Bed
rz1¢ 15.3 2.7 1.9 4.4 1.2 25.2

average of four oysters.

bo bacteria were added. Total viable bacterial count increased from
3.4 x 10/1 to 2.5 x 109/m) after 4 days.

c
Culture #14 added (1.2 oh 109 cells). Total viable bacterial count
increased from 7.2 x 104 to 9.6 x 104 after 4 days.

Acuiture #5 added (1.2 x.109 cells). Total viable bacterial count
increased from 1.0 x 109/m1 to 1.4.x 10°/ml after 4 days.

69


Table 13. TOTAL MERCURY CONCENTRATIONS AND PERCENTAGE OF THE TOTAL VIABLE
BACTERIAL COUNT RESISTANT TO 6 ppm OF HgCl,

Mercury concentration? Percent resistant’

Jecation Sediment (ppm) Water (ug/l) Sediment Water
0.220 + 9.004 ,

peBalbioore. Kastor (2-2) 0.800 + 0.070% 0,090 + 0.010 22.8 28.5

3-Baltimore Harbor (B-1) 0.590 + 0.620° 0.370 + 0.010 8.2 4.2

4-Annapolig (A-1) 0.060 + 0.040° 0,080 + 0.010
S5-Ammapolig (A-2) 0.200 + 0,060" 0.490 + 0.010 2.9 2.8

6 0.170 + 0.010

TeEastern Bay (EB-1) 0.080 + 0.0307 0,100 + 0.020 1.3 0.5
8-Eastern Bay (EB-2) 0.040 + 0.0308

9 0.094 + 0.002

10 0.015 + 0,007

11 0.104 + 0,013

12 0.120 + 0.010 2.6 0.2
13 0.073 + 0.002

14 0.100 + 0.010 0.8 9.4
15 0.052 + 0.011

16 0,060 + 0.020 1.8 0.7
17 0.023 + 0.009

18 0.070 + 0,010 3.5 0.5
19 0,007 + 0.002

20 (May 1972) 0.860 + 0.030 6.3 5.7
20 (February 1973) 0.280 + 0.013 0.070 + 0.010 2.2 2.3

®Numbers refer to sampling stations. Regularly sampled stations are under-
lined. Other stations were sampled in May 1972 and February 1973.

Mean mercury concentration (dry weight) + average deviation of one sample,
except where designated otherwise. Water values based upon unfiltered
samples taken October 1972.

Sone standard deviation is approximately 10% of "Percent resistant." Sta-
tions 3, 12, 14, 16, 18, and 20 assayed on May 1972 cruise and stations 2,
5, 7, and 20 assayed on February 1973 cruise.

Wyinety-five percent confidence interval of the mean mercury concentration
of 8 samples.

°Ninety-five percent confidence interval of the mean mercury concentration
of 6 samples.

f Ninety-five percent confidence interval of the mean mercury concentration
of 9 samples.

Eninety-five percent confidence interval of the mean mercury concentration
of 4 samples.

29


in bacterial mercury metabolism in the spring is inconsistant with the
elevated suspended sediment load normally present during that season.
However, the lack of coincidence of the two peaks may explain the ap-
parent paradox. In contrast, the elevated levels of dissolved oxygen
during the spring season, in conjunction with organic complexing agents,
may increase the solubility of Hg (23, 24).

Although analysis of variance tor the miltiple linear regressions for 3
stations and 6 stations gave significant F values (0.01 and 0.05 levels
of significance, respectively), the mltiple correlation coefficients
(0.74 and 0.88, respectively) indicated that the parameters which were
measured could not account for all of the variation in numbers of resis-
tant bacteria. There are other seasonal influences in Chesapeake Bay
which deserve mention but could not be considered in this study for lack
of time, personnel and funds. For example, in Chesapeake Bay there is
no spring phytoplankton bloom (47), but total nitrogen and zooplankton
populations do peak during the spring months (32, 48).

The observed relationship between mercury-resistant bacterial popula-
tions and mercury concentration in sediment is compatible with the con-
cept of bacterial "adaptation," However, because of differences in
either the chemical form or the availability of mercury and the seasonal
fluctuations in mercury-resistant bacterial population levels, a clear
and unequivocal relationship cannot be established. That there is, in-
deed, a spring peak in the mercury-resistant bacterial population was
confirmed by the monitoring data obtained during the first half of the
second year. Untortunately, because of the plan of work which had been
laid out, the field work had to be terminated before the secondary fall
peak could be monitored. The secondary peak in the mercury-resistant
bacterial population which was observed in the Fall of 1972 may have

been a consequence of the effects of tropical storm, Agnes, which occurred
in June 1972. Correspondingly, data for the months preceding and follow-
ing the storm suggest that there were significant, temporary changes in
the bacterial flora following the storm (15).

D: ib

Using a taxonomic scheme similar to that employed in this study,
Murchelano and Brown (40) obtained a seasonal distribution of hetero-
trophic bacteria in Long Island Sound which was much like that reported
here for mercury-resistant bacteria in Chesapeake Bay. Total viable
counts reached minima in the summer in both cases, and Pseudomonas spp.
were predominant in long Island Sound samples (40.6%), as in the case
for mercury-resistant bacteria in Chesapeake Bay (66.0%). Our results
and those of an earlier study of bacteria found in Chesapeake Bay water
and sediment (49) suggest that the mercury-resistant bacterial popula-
tion is richer in Pseudomonas spp. relative to the total population.

Yet, the total number of Pseudomonas spp. did not correlate with seasonal
mercury resistance (see Figs. 9, 10, and 13). However, when the Pseudo-
monas spp. were separated into physiological sub-groups, according to the
method of Shewan et al. (27), it was evident that the proportion of

45


Table 9. METABOLISM OF PHENYIMERCURIC ACETATE BY MERCURY-RESISTANT

BACTERIA
Ieolate Generic Percent radioactivity remaining*
number identification 1 bour® 4, days®
244e Pseudomonas sp. 52.0 42.54
187° Peeudomonas sp. 4.8 87.1
one Pseudomonas sp. 88.3 73.7
127° Pseudomonas sp. 82.6 59.9
72 Arthrobacter sp. 9468 63.2
132 Citrobacter sp. 94.6 65.7
85 Enterobacter sp. 92.3 85.1
2 Vibrio sp. 98.2 87.5
119 Flavobscterium sp. 42.7 43.3

90.4 ml PMA_in 95% ethanol, final concentration = 0.4 ug PMA

(5.75 x 10° pg), was added to 0.9 ml of pH 7.0 PES buffer con-
taining approximately equal quantities of cells. The suspensions
were incubated at 25 C, and 100 ul samples were withdrawn.

Pusth aeration.
°
Stationary.
isolate 244 was incubated 2 days.

°Tgolates 244, 187, 94, and 127 were Pseudomonas sp. types I, II, III,
and IV, respectively.

(Reproduced with the permission of the American Society for Micro-
biology. )

1T


SECTION VI
EFFECTS OF MERCURIC CHLORIDE UPON GROWTH AND MORPHOLOGY
OF SELECTED STRAINS OF MERCURY-RESISTANT BACTERIA

INTRODUCTION

Preceding sections of this report have included experimental evidence
linking bacterial mercury resistance with in situ generation of Hg®.

The positive correlation observed between numbers of aerobic, hetero-
trophic bacteria resistant to given levels of mercury under laboratory
conditions and ambient mercury concentration found in Chesapeake Bay
sediments suggests that environmental mercury contamination can exert
an effect upon the population structure of the estuarine microflora.

To elucidate the mechanisms by which mercury manifests selective pres-
sures under natural environmental conditions, we examined the effects of

inorganic mercury upon growth and morpholoty of representative strains
of bacteria isolated from Chesapeake Bay.

Investigations of mercury resistance in microorganisms have revealed
several possible mechanisms for this resistance. Phytoplankton (53) and
various bacterial strains (9, 12, 54) are capable of adapting to growth
in the presence of mercury. In some bacteria, resistance is attained
through the acquisition of plasmid, an extra chromosomal element which
can be transferred intra- and inter-generically (5, 6, 7, 8, 9). Micro-
organisms detoxify mercury metabolically by formation of volatile Hg®
(9, 12, 53, 54, 55), or of mercury mercaptides (50, 51).

Comparatively little information is available concerning the morphologi-
eal effects of mercury upon microorganisms. Tingle et al. reported
mitochondrial damage and swelling, pellicular membrane disruption, and
loss of motility in jotrahmmens pyriformis incubated in a solution of
0.5 ppm of HgCl2 (56). Exposure of cells of Pseudomonas aeruginosa to
HgCl2 caused swelling which was reversible by addition of su. 1
compounds (57). Also, extremely low levels of PMA (0.9 - 3 ppb) causing
gross changes in the cellular form of growing cultures of Phaeodactylum
tricornutum and Chorelja have been described by Nuzzi (52).

The research work described in this report was carried out using cul-
tures of bacteria isolated from Chesapeake Bay. In addition, the levels

48


first experiment. An increased isotope effect was observed in the second
experiment, where the difference between the lag phase for labeled and
unlabeled cultures was nearly 10 hours. Again, a drop in viable count
was accompanied by a drop in cell-bound Hg. As in the first experiment,
the rate of loss of Hg from the inoculated flask accelerated at the end
of lag phase. Numbers of small colonial variants comprised approximately
1% of the total viable count, and these small colony variants increased
in parallel with the total population (Fig. 20).

Fixed and stained thin sections prepared from cells harvested, in the
first experiment, from unlabeled broth with, and without, mercury were
examined, Control cells examined at the early, mid, and late logarithmic
phase of growth were found to be normal in all respects, viz., array and
morphology of the DNA fibrils, ribosomes and cell envelope, as well as
mode of division, At Oh, the mercury-containing culture was found to
be identical to the 0 h control (Fig. 21). After 2h (Fig. 22), the
cells appeared normal in overall morphology. The DNA fibrils in some
cells appeared condensed, rather than dispersed throughout the cytoplasm,
and electron dense areas among the ribosomes were seen. Intact 6 h
cells (Fig. 23), resembling 2 h cells, were accompanied by swollen and
plasmolyzed cells. Approximately one-third of all the cells at this
stage showed signs of lysis. At 24 h (Fig. 24), the culture was char-
acterized by debris arising from lysed cells, as well as viable but
pleomorphic cells demonstrating abnormal cross-wall formation. Electron
dense clusters of ribosomes were more frequently seen in these cells.

As growth progressed through the 26th and 28th hours, the cells became
relatively more rod-shaped, although irregular cell walls and electron
dense ribosome clusters were still present (Figs. 25, 26). The latter
clusters are consistent with the preferential intracellular binding of
Hg, but the technique of electron probe microanalysis, with sufficient
resolution, would be required to clarify this point.

Properties of Mercury-selected Cultures

After repeated serial transfers in mercury-free medium, the small colo-
nial variants lost their pleomorphism and regained motility, but retain-
ed their colonial morphology. If the mtants resulted as an "adaptation"
to mercury, they should differ from the parent stock, with respect to
mercury resistance and/or the ability to reduce Hg@t, Comparative data
on mercury resistance and ability to produce Hg from HgClg of strains
85, 85-L and 85-S are presented in Table 21. It was evident that the
isolates previously grown in mercury, specifically strain 85-S, were
more resistant to mercury and demonstrated increased capability to me-
tabolize Hg2+, Thus, it appears that, during the lag phase of growth,
mercury-resistant mutants are selected which are capable of releasing
Hg° from the growth medium. It is probable that these cells are iden-
tical to the pleomorphic, but viable, cells observed in thin sections
of those cells examined after 24 h. When the mercury concentration was
reduced to ca. 1 ppm via chemical and biological reduction of He2*, the
entire population: initiated growth at a normal rate.

5T


DISCLAIMER

This report has been reviewed by the Environmental Research Laboratory,
U.S. Environmental Protection Agency, and approved for publication.
Approval does not signify that the contents necessarily reflect the
views and policies of the U.S. Environmental Protection Agency, nor
does mention of trade names or commercial products constitute
endorsement or recommendation for use.

ii


a

\

DISSOLVED OXYGEN (ppm)
o
Pt

\
(
3

TEMPERATURE (SURFACE)

/
\
f

——

J F M A M

Figure 11. Seasonal variation in salinity, temperature, and dissolved
oxygen concentration in surface waters from three locations
in Chesapeake Bay. Station B-1 (A); Station B-2 (A);
Station A-2 (m); and Station EB-1 (@).

Analysis of plankton indicated wide fluctuations in mercury levels, but
no significant differences (0.05 significance level) in average concen-
trations among the three areas. The relationship between mercury con-
centration in plankton and numbers of mercury-resistant bacteria in
water or sediment was not found to be significant. Limited data, how
ever, indicated possible seasonal parallels between the sizes of the
Hg2t resistant bacterial microflora of the plankton and the water and

sediment.

Because phenylmercurials are now the only organomercurials registered
for industrial use (T. Adamezyk, Registration Division, Environmental
Protection Agency, personal communication), phenylmercuric acetate (PMA)
was also used as a test compound in these studies. Three ppm of PMA
(1.79 Hg) proved to be much more toxic than 6 ppm of HgClg (4.43
ppm ig) vhen incorporated in the growth medium. In fact, poor precision
in viable counts were obtained because of few colonies and poor survival
on the PMA medium. Nevertheless, the data suggest that bacterial re-
sistance to PMA in water and sediment exhibit a periodicity paralleling

that of ‘+ resistance.

37


Table 17, Hg® EVOLUTION BY NATURAL POPULATIONS OF BACTERIA ISOLATED FROM WATER

AND SEDIMENT
Percent change in (203xg)&
Decrease

0 HgCl. J TVC per Time Uninocu- Live sus- Sterile Cellular

Sample (ppm ml (nh) lated pension suspension uptake
B-2 sediment 6.0  1.2x10 14 6.6 1.70 38.3
B-2 sediment 6.0 1.2x109 2 10.3 0.0 38.7
Be2 sediment 6.0 1.2x10 3 11.0 3.0 40.2
B2 sediment 6.0 1.2x10 2% 11.6 6.3 46.4
Be2 sediment 6.0 1.2x10° 48 13.7 9.9 50.5
EB-1 sediment 6.0 1.0x10° 1 va 1.72 9.4
EB-1 sediment 6.0 1.0x109 2 10.5 3.9 10.9
EB-1 sediment 6.0 1.0x109 3 8.8 2.2 11.7
EB-1 sediment 6,0 1.0x109 % 9.1 9.1 15.6
EB-1 sediment 6.0 1.0x105 48 11.8 10.7 16.8
B-2 water 6.0 7.0x102 1 17 1.3 6.8
B-2 water 6.0 7.0x102 2 9.3 1.0 7.2
B-2 water 6.0 7.0x102 3 Wa 0.1 9.0
B-2 water 6.0 7.0x102 2% 15.6 0.7 9.7
B-2 water 6.0 7.0x10° 48 20.6 24 10.7
B-2 vater 6.0 1.8x106 2 5.3 5.8 4.6° 5.8
B-2 water 6.0 1.8x 106 18 8.0 10.5 7.8 6.0
B-2 water 6.0 1.8x 10° 42 4.6 10.2 1.5 7
B-2 water 6.0 1.8x10° 66 4.5 12.7 7.2 9.6
B-2 sediment 0.6 4.6x102 1 0.3 1.4) 95.0
B2 sediment 0.6 4.6x102 2 1.8 4.1 101.0
B-2 sediment 0.6 4.6x102 3 1.8 5.5 96.5
Be2 sediment 0.6 4.6x 109 2% 4.1 71 9.9
EB sediment 0.6 2.5% 10° 1 4.0 47 40.0

EB sediment 0.6 2.5x10° 2 4.2 5.7 4h.3 |
EB sediment 0.6 2.5x100 3 5.5 17 4.7
EB sediment 0.6 2.5.x 10° 24 7.7 13.6 57.2

89.1 ml of 203Hg01 in PES was added to a 4.9 ml PES suspension of seston or sedi-
ment extract (prepared as described in Materials and Methods). The tubes of sus-
pension were aerated, via a capillary tube, with moistened air for 15 min prior
to each sampling. Radioactivity in 0.1 ml samples of suspension and non-filter-
able material was measured and corrections for quenching and decay were made.

Sterilized by heating at 121 C for 15 min, No attempt was made to maintain
sterility after the start of the incubation.

Sterilized with chloroform. No attempt was made to maintain sterility after the
start of the experiment.

35


Figure 24. Thin section of culture #85 cells after 24 h incubation in
broth containing 2 ppm of HgClg, Viable, pleomorphic cells
and fragments of lysed cells are seen. Cellular division
occurs by cross wall formation (arrows), rather than by nor-
mal constriction. Numerous electron dense groups of ribo-
somes are also seen.

Thin section of culture #85 cells after 26 h incubation in
broth containing 2 ppm of HgCl2. Predominately viable, pleo-
morphic cells are seen. Electron dense clusters of ribosomes
are present in all ceils.

60

Figure 25.


SECTION IV
PHYSIOLOGICAL AND CULTURAL CHARACTERISTICS
OF MERCURY-RESISTANT BACTERIA

INTRODUCTION

The major objective of this research project was to determine the mecha-
niem and extent of influence of bacteria in the transformations of mer-
eury which take place in the estuarine environment. Isolated reports by
other investigators (cited in later sections) of bacterial synthesis and
metabolism of mercury compounds stimulated the initiation of the work
summarized in this report. The first major premise was that bacterial
resistance to mercury is a function of ability to detoxify mercury by
metabolic transformation. A corollary was that measurement of numbers
of mercury-resistant bacteria in gitu should provide an index of real,
or potential, microbiological mercury transformation in Chesapeake Bay.
The following section is a description of the experimental plan followed
in the characterization and isolation of mercury-resistant bacteria from
Chesapeake Bay.

MATERIALS AND METHODS
Isolation, Culture and Identification of Bacteria

Media us methods are described elsewhere (Materials and Methods, Seo-
tion V).

Meroury Analysis

Samples of sediment were allowed to settle, supernatant liquids, if any,
were removed, and the sediments were air dried. Unfiltered samples of
water were acidified to 0.5 N HNO3, and plankton samples were freeze
dried. Al] samples were wet ashed and analyzed for total mercury by
flameless atomic absorption according to "EPA Provisional Method for
Mercury in Sediment and Water," January 1972. The vapor phase analyses
of PMA-metabolizing bacteria are described elsewhere (1). Six plates of
basal medium containing 0.3 ppm of PMA were inoculated with cultures
grown on slants of the same medium. The cultures were placed in a her-
metically sealed glass container provided with an on-line dual beam
atomic absorption spectrophotometer. Six uninoculated plates served as


reduction of mercuric ion to the metallic state was established by sur-
veying a number of HgClo-resistant cultures. The reaction was also ob-
served in microorganisms isolated by differential centrifugation ‘of
water and sediment samples. Mercuric ion exhibited an average half life
of 12,5 days in the presence of approximately 10° organisms/ml.

Cultures resistant to 6 ppm of mercuric chloride and 3 ppm of phenylmer-
curic acetate (PMA) were classified into eight generic categories.
Pseudomonas spp. were the most numerous of those bacteria capable of
metabolizing both compounds; however, PMA was more toxic and was more
selective for Pseudomonas. The mercury resistant generic distribution
was distinct from that of the total bacterial generic distribution and
differed significantly between water and sediment, positionally, and
seasonally. The proportion of non-glucose~utilizing mercury resistant
Pseudomonas spp. was found to be positively correlated with total bac-
terial mercury resistance.

It is concluded from this study that numbers of mercury-resistant bac-

teria ag established by plate count can serve as a valid index of in
gitu metabolism.

47


Figure 16. Thin section of cells of culture #394 grown in the presence
of 5 ppm of HgCl2. In comparison to cells wn without
mercury, irregular septa were tormed (arrow). Scale in
this and subsequent micrographs is one micrometer.

Figure 17. Thin section ot cells of culture #394 grown in the presence
of 15 ppm of HgCl2. Cells show irregular cell division and
pleomorphism.

53


effects upon bacterial populations. It is logical to assume that con-
centrations of mercury actually encountered in the environment should
induce detectable effects in the corresponding bacterial flora.

Table 1. COMPARISON OF RESISTANCE OF SEDIMENT AND WATER POPULATIONS TO
DIFFERENT LEVELS OF HgCl2 AND PMA

Percent of
Station Test medium tvc® comprising mercury resistant bacteria
Water Sediment
Patuxent 6 ppm HgClp 0.2 2.6
R, mouth
0.12 ppm HgClo - 15.3
5/15/72
1.2 ppb HgClo 72.0 -
3 ppm PMA . < 0.09 0.05
0.12 ppm PMA - 3.7
1.2 ppb PMA 48.3 -
Potomac 6 ppm HgClo 9.4 0.76
R, mouth
0.12 ppm HgCl, - 93.4
5/6/72
3 ppm PMA <0.1 < 0.005
0.12 ppm PMA - 2.5
1.2 ppb PMA 19.2 -

8TVC = total viable, aerobic, heterotrophic bacterial population. (Re-
produced with the permission of the Marine Technology Society.)

The effect of incubation temperature on numbers of mercury-resistant bac-
teria is shown in Table 2. As incubation temperature was increased to
37 C, apparent mercury resistance increased greater than ten-fold. This
result may have been a consequence of increased volatilization of the
mercury from the medium or a selection for mercury-resistant populations.
The known non-biological reduction of mercuric ion which occurs in this
medium (see Section VI) tends to favor the former explanation. At the
temperature (15 C) most closely approximating the in situ temperature,
the largest TVC was obtained.

Spangler et al. (2) have reported that mercury-resistant, methyl mercury-
degrad: bacteria metabolize methyl mercury to methane and elemental
mercury (Hg°) relatively more extensively under anaerobic rather than
aerobic conditions. Consequently, plates of HgClp medium were inoculated


EPA-600/3-75-007
October 1975

METABOLISM OF MERCURY COMPOUNDS

IN MICROORGANISMS

by

Rita R, Colwell and John D. Nelson, Jr.
University of Maryland
College Park, Maryland 20742

Grant No. 802529

Project Officer

Cc. S. Hegre
Environmental Research Laboratory
Narragansett, Rhode Island 02882

U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
ENVIRONMENTAL RESEARCH LABORATORY
NARRAGANSETT, RHODE ISLAND 02882


RESULTS

Experimental Sampling Stations

Chesapeake Bay was traversed longitudinally during two cruises, one in
May 1972 and the other in February 1973, to survey total mercury concen-
trations and mercury resistant populations of bacteria in sediments (see
Fig. 7). The results (Table 13) showed that a range of mercury concen-
trations exists in Chesapeake Bay sediments, with highest levels present
in the two most industrialized areas, Baltimore Harbor (stations 2 and 3,
Fig. 7) and the Elizabeth River near Norfolk, Virginia (station 20, Fig.
7). Bacterial resistance to HgCl2, expressed as the proportion of the
total viable, heterotrophic, aerobic population capable of growth in a
medium containing 6 ppm of HgCl2, was also greatest at these two sites.
The data strongly suggest a possible causal relationship between ambient
mercury levels and numbers of mercury resistant bacteria.

A total of 6 stations at three locations in the upper Bay, shown in Fig.
7 (B-1, 23 A-1, 25 and EB~1, 2), were monitored at approximately 1.5
month intervals over a 17 month period, Fall 1971 through Spring 1973.
Samples of surface water (10 cm depth) and the upper sediment layer were
routinely analyzed for numbers of bacteria resistant to 6 ppm of HgCl2-
and 3 ppm of PMA and for total mercury concentration. The three stations
encompassed a spectrum of environmental quality and ambient mercury con-
centrations from heavily industrialized Baltimore Harbor to relatively
clean Eastern Bay, the site of important fin and shellfish fisheries.
Differences in mercury concentrations in sediments among the three loca-
tions were significant at the 0.05 level and did not change appreciably
during the 17 month monitoring period (15). Techniques of sufficient
accuracy and precision for detection of mercury in water samples were
developed somewhat later in the study. Levels of from 0.00 to 0.68 ppb
were detected in the water from the three sites. However, there were
insufficient data to draw conclusions as to the relationship of water to
sediment mercury concentration or to location. Colgate Creek (station
B-2), a subestuary in Baltimore Harbor, was found to contain the highest
concentrations of mercury found in Chesapeake Bay during the course of
this study. This site was subsequently found to be heavily contaminated
with high levels of other heavy metals and oil pollutants as well (0.
Ville and J. Marks, personal commmication). The sources of mercury at
this station have not been identified directly, but the indications are
that point sources, such as a paint factory or a hospital sewage outfall,
in the area may be implicated. The two sites of study which are located
near Annapolis Harbor, A-1 and A-2 shown in Fig. 7, are subject to heavy
commercial marine traffic.

erial

From the onset of this investigation, the working hypothesis was that the
percent of bacteria in the autochthonous microbial population which
demonstrated resistance to mercury could be used as an index of potential
microbial activity in the generation of Hg° from inorganic or organic

eT


Table 23, AVERAGE TOTAL MERCURY CONCENTRATION IN UNTREATED OYSTERS®

Total mere concentration Methyl mercury
Experiment Dry weight (opp) Wet weight (ppb)¢ (

spike recovery (%)

I 324 + 23 48.6 100

I 7 +14 14.5 109
II 201+ 6 30.2 76
II 103 + 25 15.5 83
III 239 +99 35.8 100
III 111417 16.7 90

ato oysters were selected from each experimental group of six before
treatment with mercury.

Mean + average deviation of three determinations.

°Extrapolated from dry weight concentration. Dry weight = 15% of wet
weight (average).

The results of the three experiments are summarized in Table 24. Approxi-
mately twice as much mercury was accumulated by the oysters when mercury
resistant bacteria were added to the aquarium (experiments II and III).
The less-than-quantitative recovery obtained may have been a result of
adsorption of mercury onto the aquarium and shell surfaces. Trapping of
volatilized mercury by the first trap was essentially quantitative, with
less than 0.10% of the total added radioactivity accumlating in the second
trap. Approximately 10% of the added Hg was volatilized, even in the ab-
sence of added mercury metabolizing bacteria. The volatilization of Hg
and the apparent excretion of Hg in fecal material suggest that the
oysters, themselves, may actively metabolize Hg. During the course of
each experiment, it was observed that the total concentration of mercury
in the water approached the concentration of mercury in the microparticu-
late fraction of the water. Particulate mercury increased during the

first 24 h and then decreased. During this period, the rate of decrease
in dissolved Hg was most rapid.

The relative and absolute accumulations of mercury by individual oyster
tissues are presented in Table 25. Generally, the largest proportion

of the accumulated Hg was found in the gills (Table 25A). In experiments
II and III, the amount found in the gills was statistically significantly
greater than all other tissues examined. In the latter experiments, the
mantle and visceral mass accumulated significantly greater proportions
of Hg than either the adductor mscle or mantle fluid. Table 25B shows
the tissue concentrations of mercury. Comparison of experiment I with

TO


resistance to 24 ppm of PMA rose to only a transient 19-fold increase.
This difference may be the result of loss of Hg inducer, for it has
been observed that Hg is chemically reduced and volatilized in the
growth medium employed (Section VI).

22 an ; 80

20 nen
a

ist ° A 60

ZO
. ta 50

GROWTH (LOG KLETT UNITS)
a
LOG NO. PMA RESISTANT (24ppm)

a“ \
r
14 . LO 40
a Se
°
12 3.0
.
Coa
10 1 2 3 4 5 6 7 Ve
TIME (HOURS)
Figure 3. Induction of resistance to PMA by P. ©) 244. Broth

with (triangles) and without (circles) 6 ppm of PMA was
inoculated with cells. Growth was measured turbidimetrically
(open symbols), and numbers of cells resistant to 24 ppm of
PMA/ml were determined (closed symbols).

To ascertain the fate of PMA, above experiment was repeated, using
Pseudomonas 244 growing in a labeled PMA medium (Fig. 4). Dupli-
cate flasks, with and without inoculation, were assayed for cell~bound
and total radioactivity at hourly intervals. During the period, in
which it was shown in the previous experiment (Fig. 3), that resistance
to PMA increased rapidly, a net loss of 39% and a cell accumulation of
4% of the label in the inoculated flask occurred. The uninoculated
flask showed no significant decrease during the same period of time.

A group of representative bacteria wes surveyed for the ability to
volatilize Hg from labeled PMA-containing suspensions (Table 3. Analy-
sis of the vapor phase of cultures on PMA agar by atomic absorption
spectrophotometry and gas liquid chromatography (1) indicated that the
cultures degraded PMA to Hg° and benzene vapors (Fig. 5). The apparent
periodicity of Hg evolution was an artifact of the sampling procedure.
No organomercurials were detectable among the gaseous products. The

15


RESEARCH REPORTING SERIES

Research reports of the Office of Research and Development,

U.S. Environmental Protection Agency, have been grouped into
five series. These five broad categories were established to
facilitate further development and application of environmental
technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in
related fields. The five series are:

Environmental Health Effects Research
Environmental Protection Technology
Ecological Research

Environmental Monitoring
Socioeconomic Environmental Studies

MV FwWNP

This report has been assigned to the Ecological Research series.
This series describes research on the effects of pollution on
humans, plant and animal species, and materials. Problems are
assessed for their long- and short-term influences. Investi-
gations include formation, transport, and pathway studies to
determine the fate of pollutants and their effects. This work
provides the technical basis for setting standards to minimize
undesirable changes in living organisms in the aquatic,
terrestrial and atmospheric environments.

This document is available to the public through the National
Technical Information Service, Springfield, Virginia 22161.


on

Table 19, PHYSIOLOGICAL CHARACTERISTICS OF HgClg~ AND PMA-RESISTANT BACTERTA
soluble Bacto OF medium plus 1% glucose
Cytochrome® Gram- Pigment Hs? Nat® 9b:

oxidase _negative Produced produced required Acid/——  Acid/Acid Alkaline/---__-—-/-—-
A, HgCly (6 ppm) resistant®
No, of cultures 288 353 55 233 32 35 217 103
Percent 68.0 83.5 13.0 55,0 7.6 hed 8.3 51.3 hed
B. PMA (3 ppm) resistant!
No. of cultures 64, 69 15 40 6 38 2 28 te)
Percent 92.8 100.0 21.8 58.0 8.7 55.0 2.9 40.6 0

Method of Gaby and Hadley (33).
prom sodium thiosulfate or L-cysteine.

Requirement for 1% NaCl for growth.

a
Anaerobic tubes were layered with sterile mineral oil.

"Total of 423 isolates tested.

£
Total of 69 isolates tested.

Dash indicates no change in pH.


Aerobic, heterotrophic bacteria resistant to mercuric ion were isolated
from water, sediment, and plankton. They are predominately of the
genus, Pseudomonas. Many of these organisms are also resistant to
organomercurials, PMA (phenylmercuric acetate) and Me HgC1 (methylmer-
curic chloride), deriving resistance from their ability to degrade mer-
cury compounds with the formation of Hg®. The capability of the organ-
isms to produce Hg° from Hg2t and PMA is correlatea with their ability
to grow in the presence of compounds of mercury. Mercury resistance is
also related to conditions of incubation, such as aerobiosis and tempera-
ture of incubation, as well as the salt content of the growth medium and
the physiological age of the cultures.

23


Table 16, CHARACTERIZATION OF THE VOLATILE Hg METABOLITE PRODUCED BY

PSEUDOMONAS STRAIN 5
Percentage of
Experinent Sample added C°3xg )®
re Suspension after 60 min 0.67
Cells after 60 min 0.98
HgBro-KBr trap after 60 min 85.00
Benzene extract of trap 0.00
II® a. Suspension after 90 min 0.80
b. Cells after 90 min 0.60
c. HgBro-KBr trap after 90 min 96.80
a. First dithizone extract of trap 22.80
e. Second dithizone extract of trap 76.20
T1I? a, Dithizone extract of II c. 44.00
b. Thin layer chromatography of III a.
spot plus origin 44.00

3, flaak containing 9.0 ml of PES containing 2°3Hg-labeled HgCl2 was
connected via plastic tubing to a second flask containing 10.0 ml of
HgBr2-KBr trapping solution. The experiment set-up was such that
the atmosphere was continually recirculated by means of a peristaltic
pump as the flasks were shaken. After 15 min incubation, 1.0 ml of
washed cells in PES was added to the first flask and incubation was
continued. The trapping solution was examined for radioactivity
before and after addition of cells to the system. Transfer of radio-
active Hg to the trapping flask was insignificant in the absence of
the bacterial suspension, At the end of the incubation, cells were
filtered through a 0.45 micron Millipore filter and washed thrice
with 1.0 ml volumes of PES.

by 14 ml dry weight of cells with 1.8 ppm °2Agclg (131,000 dpm/ml).
Five volumes of trapping solution were acidified with concentrated
HCl to a concentration of 1 N, and were extracted with 1 volume of
benzene.

°1 19 ng dry weight of cells with 1.5 ppm 2°3ugc1, (202,000 dpm/m2).
One volume of trapping solution was extracted with 2 volumes of a 1%
solution of dithizone in benzene. The extraction was repeated.

one volume of trapping solution (Expt. II) was extracted with 2
volumes of 0.8% dithizone in benzene. The dithizonate derivative was
gpotted on a silica gel G plate (250 p thickness) and developed with
petroleum ether:diethyl ether (70:30) solvent. Spots were scraped
off and collected in scintillation vials and suspended in an Aquasol
(New England Nuclear) gel for counting. Dithizone extraction and
chromatography were according to the methods of Westoo (31).

33


Term
GFU

Hg?
MeHg 62
PES

PMA
Three salts

Tvc

SECTION X
GLOSSARY

Definition
Colony-forming units
Elemental mercury
Methylmercuric chloride

A 0.01 M phosphate-buffered "three salts solution"
(pH 7.0)

Phenylmercuric acetate

An artificial estuarine salt water mix of 1% NaCl,
0.23% MgClo-6H20, and 0.03% KC1

Total viable, aerobic, heterotrophic count of bac-
teria by spread plate enumeration

83


[rete] uotovan w aowves anavaae

----en7

<—% o001—>

|

gg ng
ge LaNK
7 20 40 60 80 100,

Figure 5. The change in mercury contained in the bioreactor atmosphere
as a function of time is plotted for formation of Hg® from
nine bacterial isolates from the Chesapeake Bay, vach growing
on six agar plates of basal media containing 0.3 ppm PMA. The
small open circles represent + std. dev. from the mercury cal-
ibration curves used. Hg values plotted here are not addi-
tively corrected for portions of reactor atmosphere removed at
each sampling period. Note that in order to avoid overlapping,
the individual curves are not referred to a common zero on the
ordinate, but the 1,000 ng intervals shown on that scale pro-
vide an indication of the relative change in the amount of Hg
present above each isolate referred to a control blank (un-
inoculated plates).

(Reproduced with the permission of the American Society for
Microbiology. )

18


TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)

1. REPORT NO. 2. fs, RECIPIENT'S ACCESSION NO,
EPA-600/3-75-007

4, TITLE AND SUBTITLE

Ib. REPORT DATE
October 1975 (Issuing Date)

METABOLISM OF MERCURY COMPOUNDS IN MICROORGANISMS 6. PERFORMING ORGANIZATION CODE

17. AUTHOR(S) ls. PERFORMING ORGANIZATION REPORT NO.

Rita R. Colwell and John D. Nelson, Jr.
9. PERFORMING ORGANIZATION NAME AND ADDRESS: 10. PROGRAM ELEMENT NO.

Department of Microbiology Se NTORANT NO.
University of Maryland

College Park, Maryland 20742
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Research Laboratory

R 802529-01
13. TYPE OF REPORT AND PERIOD COVERED

Office of Research and Development 14. SPONSORING AGENCY CODE
U.S. Environmental Protection Agency EPA-ORD
Narragansett, Rhode Island 02882

15. SUPPLEMENTARY NOTES.

16, ABSTRACT

This report describes the physiology and ecology of mercury-resistant and mercury-
metabolizing bacteria from Chesapeake Bay. Evidence is presented which establishes
a role for bacteria in the cycling of mercury in the estuarine environment. From
the results of a survey of Hg° production among a group of randomly selected,
HgClo-resistant bacteria and mixed natural microbial populations, it was established

that the enumeration of mercury-resistant bacteria by plate counting is a valid index
of potential Hg*+ metabolism in situ.

The distribution of mercury-resistant bacteria was significantly different in water
and sediment, from station to station, and seasonally; the proportion of Hg@+-
resistant bacteria among the total, viable, heterotrophic bacterial population
reached a reproducible maximum in Spring and was positively correlated with water
turbidity, dissolved oxygen concentration, and mercury concentration in the sediment.

These findings and the observation of the evolution of Hg° from freshly collected
water and sediment suggest that bacteria may contribute substantially to the mobili-
zation and transformation of mercury from existing deposits in Chesapeake Bay,
specifically, and in the aquatic environment, in general.

H7. KEY WORDS AND DOCUMENT ANALYSIS
fa. DESCRIPTORS lb. IDENTIFIERS/OPEN ENDED TERMS |c. COSATI Field/Group
Marine microorganisms Mercury degrading bacterial 6M
Mercury mobilization 6P
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SECTION II
RECOMMENDATIONS

We have identified and characterized a group of bacteria which are po-
tentially significant in the mobilization and transformation of mercury
in Chesapeake Bay. Our results and the reports of others indicate that
Hg is continually mobilized from existing deposits of mercury in the in-
shore marine and estuarine environments. As mercury levels did not de-
crease significantly during the 17 months in which we monitored the
several stations in Chesapeake Bay, which were included in our study, we
assume that processes of addition and mobilization maintain a steady
state level of mercury. A mercury budget should be calculated to deter-
mine whether production of Hg° correlates with theoretical seasonal
fluctuations predicted from our results. Clearly a "microbial mobiliza-
tion index" (MMI) will then prove to be a useful parameter for assessing
the environmental impact of presently existing or new reservoirs of mer-
cury.

The fate of Hg®, following release into the aqueous medium, should also
be investigated to determine: 1) the proportion of Hg lost from the en-
vironment through volatilization; and 2) the chemical species and ulti-
mate destination of mercury in solution under in situ conditions. This
information mst eventually be obtained in order to establish the
sources and routes of mercury in marine and estuarine food webs.

Finally, our findings underline the importance of an understanding of
the microbial population structure in the natural environment. An un-
equivocal conclusion from our studies is that the bacteria play a sig-
nificant role in the cycling and introduction of mercury and other

pollutants to the marine and estuarine food webs. More attention must

be placed on understanding and managing the bacteria, fungi, and other
microorganisms in the environment.


walls of the flask since only 8.9% of the radioactivity was recovered in
sequential washes of the vessel using water, 0.5 N HNO3, and 10% Radiac
wash (Atomic Products Corp., Center Moriches, N. Y.). An initial, small
loss of cell-bound mercury coincided with the drop in viable count, in-
dicating lysis of the cells may have occurred. When the mercury concen-
tration dropped approximately 45.0%, to 1.1 ppm, growth was initiated.
At this point, the rate of loss of Hg from the broth accelerated in the
inoculated flask, and the rate of cellular uptake, per unit volume of
broth, increased. The possibility that the former effect resulted from
biological reduction of Hg2t was subsequently investigated.

After several hours of incubation, small colonial variants (85-S) of the
HgCl2-containing culture began to appear on the spread plates prepared
for determination of total viable counts (Fig. 19). These colonies con-
tained non-motile, pleomorphic cells, in contrast to the large colonies
(85-L) comprising the remainder of the population.

Figure 19. Colonial variation in Enterobacter, strain 85, grown in the
presence of 1 ppm of HgClp. Samples of culture grow in 2
ppm unlabeled HgCl2 containing broth were diluted and plated
on basal nutrient medium (Fig. 17) and incubated at 25 ©
for 7 days. Small (85-S) and large (85-L) colonial forms
were observed.

55


experiments II and ITI indicates that significant increases in mercury
accumalation in gill tissue occurred when mercury-resistant bacteria
were added to the system. The distribution of mercury in the tissues
found in this study is consistent with the data published by others.
Pentreath (79) showed that the stomach and gills were the primary sites
of both concentration and exchange of heavy metal nuclides in the mssel,
Mytilus edulis. Kopfler (80) reported that in oysters continuously ex-
posed to 50 ppb of Hg as HgClo, the accumilation of Hg in gills was mre
than ten-fold higher than any other tissue. Mercury accumlated in the
gills is probably entrapped in the microparticulate or dissolved forp via
the mous secretions of the gills. Similarly, Penreath found that ““Fe
was associated with the mucous covering of the gills (79).

The results of experiment III were umexpected, in that relatively less
accumlation would have been predicted due to increased volatilization
of Hg through bacterial metabolism, Although less Hg was accumlated
than in experiment II, significantly more was accumulated than in the
control experiment, The relatively high half saturation constant (20
ppm of HgCl ) (see Section IV) for the evolution of Hg by the bacterial
culture is a likely explanation. At the low concentration (10.2 ppb) of
HgCl in the water, the bacterial cells will evolve Hg at a relatively
low rate. However, culture #5 does accumlate Hg, albeit at 1/3 the
rate of culture #14, and may also enhance Hg uptake by the oysters.

CONCLUSION

In short term experiments, mercury resistant, mercury accumulating bac-
teria caused relative increases in Hg accumlation in all of the oyster
tissues examined, Increases in mercury concentration of the gills were
statistically significant, with concentration factors in excess of three
thousand. It can be concluded that bacteria may have a demonstrable

effect upon mercury accumulation in those food chains which include fil-

ter feeding components.

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Figure 12.

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Distribution of HgCl2 resistant, aerobic, heterotrophic bac-
teria isolated from water and sediment of Chesapeake Bay.
Four hundred and eighteen pure cultures found to be resis-
tant to 6 ppm of HgCl2 were isolated between January 1972
and December 1972. These cultures were classified into 8
genera or generic groups: 1 = Pseudomonas, 2 = Vibrio and
Aeromonas, 3 = Bacillus, 4 = coryneform, 5 = Oytophaga and
Aghromobacter, i

Elavobacterium, 6 = » and Acineto~
T= and 8 - unidentified, The

bacter,
cultures were isolated from B~-1 and B-2 ("Baltimore"), A~1
and A-2 ("Annapolis") and EB-1 and EB-2 ("Eastern Bay"),

AL


observed for the two Baltimore Harbor stations may be related to differ-
ences in hydrocarbon content (35), for it has been shown that mercury is
associated with the hydrocarbon fraction of sediments in Baltimore Har-
bor. Although no attempt was made to characterize the chemical form of
the mercury found in the sediments in Chesapeake Bay, apparent contrasts
in bioavailability of mercury, as in the case of Baltimore Harbor, might

well prove to be attributable to the concentration of oil in the sedi-
ment of a given site.

We have established previously that the effect of acquisition of resis-
tance to a single mercury compound is pleiotropic, in terms of resis-
tance to other mercury compounds (15). It was also shown that metabolic
conversion of PMA to Hg® and benzene (1), an inducible mechanism, is
associated with resistance (15). Similar inducible systems have been
hypothesized for other organisms (11, 14). These findings concur with
those relating to inorganic mercury and reported in this paper. Along
this line of reasoning, the synthesis of methyl mercury, which also is
a conversion of mercury to a more mobile state, is thought to be a re-
sistance mechanism in Neurospora (35). The apparent adaptability of
microorganisms to growth in the presence of mercury or mercury compounds
suggests a possible causal relationship between mercury in the environ-
ment and the degree of biotransformation and mobilization of mercury.
Our findings support the concept of a plate count index of potential in
cr mercury metabolism. We have used concentrations of HgClg and PMA
6 ppm and 3 ppm, respectively) routinely in preparing the media used
in our investigation. These concentrations are considerably higher than
the mercury concentrations encountered in Chesapeake Bay. However, our
observations show that concentrations as low as 1.2 ppb of HgCl2 or PMA
will inhibit a measurable portion of the total population (34).

The phenomenon of bacterial reduction of Hg is probably related to the
evolution of Hg° from water and sediment which can be directly measured
using fresh, raw sediment from Baltimore Harbor (F. Brinckman and W.
Iverson, National Bureau of Standards, personal communication) and the
Patapsco River (34) in a closed system fitted with a flameless atomic
absorption spectrophotometer. Bothner and Carpenter (19) reported that
total mercury concentrations in sediments of Bellingham Bay of Puget
Sound, Washington, decreased according to first order kinetics, follow
ing the reduction of mercury discharges trom a chlor-alkali plant. They
found that the apparent half life of mercury in these sediments was 1.3
years. From data published by others, they also calculated half lives
for the conversion of Hg2t to methyl mercury to be 2.9 to 18 years. Our
experiments, using similar concentrations of Hg and natural microbial
populations isolated without selective enrichment for mercury resistance,
also indicated a first order process (Table 17). An average half life
of 12.5 days for Hg in aerated suspensions was caleulated. It should
also be noted that, in most cases, heat- or chloroform-sterilized in-
ocula also induced a significant loss of Hg. The rate of loss observed
in our experiments should be treated as an estimate of the upper limit

43


controls. Samples of the vapor phase were analyzed for benzene using a
flow splitter for simultaneous thermocouple and flame ionization detec-
tion. Samples were injected under the following conditions: isothermal
at 50 C, No carrier flow at 30 ml/min, 10% Apiezon L on 80/100 Supelco-
port in 0.225 in. by 6 ft. stainless steel columns. Samples were cali-
brated against 2.0 ml benzene-air mixtures from which croas-integration
of thermocouple and flame ionization detector peak areas yielded a limit
of detection of 0.01 ppm for benzene in air.

Radio Isotope Experiments

203qg-1abeled HgClg or PMA were added to suspensions of cells in basal
broth or 0.01 M phosphate or TRIS-buffered (pH 7) "three salts" solutions
and the suspensions were incubated with shaking or agitation with a mag-
netic spin bar or aerated through capillary tubes. Samples were removed
and added directly to liquid scintillation cocktail or filtered through
0.45 micron Millipore filters. The filters were washed with three 1 ml
volumes of salts and placed in scintillation cocktail to assess cell-
bound activity. Corrections for chemical quenching and decay of the
isotope were applied.

RESULTS AND DISCUSSION

Resistance of natural populations of aerobic, heterotrophic bacteria was
measured by ability to form colonies on a simple solid growth medium
supplemented with selected inorganic and organic mercury compounds. The
preparation and incubation of the medium was varied.

Total mercury analysis of natural materials provides only limited infor-
mation with respect to chemical form and biologically available concen-
trations of mercury in a microenvironment. Consequently, mercury
concentrations used in our selective media were arbitrarily chosen
initially. Samples of water, sediment, and homogenized plankton were
routinely d on agar containing 6 ppm HgCl2 2.41 microgram atoms
of Hg/liter) or 3 ppm phenylmerouric acetate (PMA) (3.9 microgram atoms
of Hg/liter). PMA was found to be significantly more toxic on a per-
mole-of-Hg basis than HgCl2 and was subsequently reduced in concentra-
tion to 0.3 ppm. The media were prepared by addition of either aqueous
solutions of HgCl2 in sterile “three salts" solution or alcoholic solu-
tions of PMA to the sterile, molten agar medium. It was found that
sterilization of the solutions by membrane filtration was unnecessary
and caused a reduction in final mercury concentration of the solutions.

The concentration of resistant bacteria in each sample was expressed as

a@ percent of the "total viable, aerobic, heterotrophic bacterial count"

(TVC) obtained by counting colonies appearing after 7 days incubation at
25 C. The comparative effects of selected concentrations of mercury and
concentrations approximating environmental levels are shown in Table 1.

Levels as low as 1.2 ppb of PMA or 1.2 ppb of HgClo showed measurable