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This article was downloaded by: [Monash University Library]On: 10 May 2013, At: 10:48Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number:1072954 Registered office: Mortimer House, 37-41 Mortimer Street,London W1T 3JH, UK
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Effects of salinity on
sedimentation and ofparticipates on survival
of bacteria in estuarine
habitatsMargaret M. Roper
a& K. C. Marshall
a
aSchool of Microbiology, University of New
South Wales, Kensington, 2033, New SouthWales, Australia
Published online: 28 Jan 2009.
To cite this article:Margaret M. Roper & K. C. Marshall (1979): Effects of
salinity on sedimentation and of participates on survival of bacteria in estuarine
habitats , Geomicrobiology Journal, 1:2, 103-116
To link to this article: http://dx.doi.org/10.1080/01490457909377727
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Effects of SalinityonSedimentation
andof Participates
on SurvivalofBacteria
in Estuarine Habitats
Margaret M. Roper
and K. C.
Marshall
School of Microbiology, University of New South Wales
Kensington,
2033,
New South Wales, Australia
Coliform bacteria have been considered
as a
model
for
studies
on
the deposition and survival of microorganisms in estuaries. These
bacteria were deposited
in
bottom muds
of an
estuarine system
once the salinity exceeded a specific conductivity of 2.5 mS cm
-1
.
Survivalof the bacteria appearedto beenhanced in thesediments.
Studiesof bacterial survival in specially constructed chambersim-
mersed in an estuary indicated that sediment particulates have a
protective effect, prolonging the survival of the bacteria compared
with that inseawater.A similar protectionof thebacteria was ob-
served in the presence of a montmorillonitic clay.The interaction
of microorganisms with both colloidal
and
larger particulates
is
considered
in
relation
to
such protective effects.
The
role
of
salinity
in microbial sorption-desorption phenomena,
as
well
as the
role
of
particulates
in
inhibiting biological control
of
alien bacteria, must
be
of
general significance
in the
geomicrobiology
of
sediments
in
estuaries.
Introduction
There is a general lack of information on the survival, metabolism and
sorption-desorption characteristics of microorganisms in estuarine sedi-
ments.
Roper and Marshall 1974) have demonstrated that
scherichia
Geomicrobiology Journal,
Volume1,Number2
0149-0451/79/0301-0103 $02.00/0
Copyright
1979
Crane, Russak & Company,
Inc.
103
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104 M argaret M . Roper and K.C . Marshall
coli,
its specific bacteriophage, and even a portion of the natural sedi-
ment microbial population were sorbed to sediments at high electrolyte
concentrations but were rapidly desorbed following dilution of the
electrolyte below a critical salinity. These authors presented evidence
that
E. coli
was protected from bacteriophage attack by the presence of
sediment, montmorillonitic clay, or organic matter at both high and low
electrolyte concentrations. Fecal bacteria disappear rapidly in waters
due to prdation and parasitism (Ketchum et al., 1952; Mitchell et al.,
1967;
Mitchell, 1968, 1971; Enzinger and Cooper, 1976; Roper and
Marshall, 1978a) and to a lesser extent due to the physical environment
(Orlob, 1956; Carlucci and Pramer, 1960; Klock, 1971). However,
several authors have detected larger numbers of fecal bacteria in sedi-
ments, compared with overlying water, and this led them to suggest that
sorption to sediments may prolong bacterial survival (Rubentschik et
al., 1936; Rittenberg et al., 1958; Hendricks, 1971; van Donsel and
Geldreich, 1 97 1; Gerba and McLeod, 1 97 6). Such increased survival
could result from an inhibition of prdation and parasitism by sediment
particulates as indicated by the preliminary results of Roper and
Marshall (1974).
The purpose of this investigation was to examine the sedimentation
of fecal bacteria in relation to the salinity gradient existing in an estua-
rine system and to establish whether sediment particulates upset natural
biological control mechanisms.
Materials and Methods
Bacterial counts
E. coli
strain M13 (Ro per and Marshall, 1974) was used in all
appropriate experiments. Viable counts of coliform bacteria or E. coli
M l 3 were made by diluting water or sediment samples in 1% peptone
and plating on MacConkey agar (Difco). The plates were counted after
18 h incubation at 37C .
Tamar R iver sampling
The Tamar River in Tasmania (Fig. 1) is heavily contaminated
with domestic sewage near its source at the confluence of the North and
South Esk Rivers. There is a considerable input of silt and mud, the
deposition of which has created extensive mud flats along the river. The
Tamar River is subject to a strong tidal influence, sometimes with
a
difference of 3.5 m between high and low tide .
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Survival of bacteria in estuarine sediments
105
Water samples were collected in sterile 100-ml glass bottles, and
sediment samples were obtained by means of a Petersen dredge (Welch,
1948). The salinity was measured in terms of specific conductivity
BASS STRAIT
2-5
2-7
North Esk River
LAUNCESTON
South Esk River
Fig. 1.
Map of Tasmania, Australia, showing the location of
the
Tamar River,
which flows 60 km from Launceston to Bass Strait. Sampling sites along the
length of the river are indicated as well as distances from the sewage outfall at
Launceston.
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106 M argaret M . Roper and K .C. M arshall
(L
s
=msiemens cm
1
or mS cm
1
) using a Townson Portable Water Qual-
ity Monitor. Turbidity, as a measure of suspended solids, was read on
the same instrument. Water samples were plated to enumerate coliform
bacteria. In examining sediment samples for the presence of coliform
bacteria, a weighed amount of wet sediment (1-2 g) was suspended in
approximately 100 ml of distilled water to desorb bacteria from saline
samples (Roper and Marshall, 1974). The suspension was diluted,
plated on MacConkey agar, and the total volume of liquid was re-
corded. The number of coliform bacteria per gram of dry sediment was
calculated after determining the percent dry sediment to wet sediment
following drying a weighed amount of wet sediment at 110C for 48 h.
In situ survival of E. co li in the Georges River
Chambers based on a modification of those used by McFeters and
Stuart (1972) were used for in situ studies of the survival of E. coliin
the Georges River, New South Wales. The central piece of plexiglass in
the chambers (Figure 2) was thicker to create a larger chamber volume
and to provide space for two small holes (10 mm diam) for sampling
purposes. These holes were plugged with rubber stoppers. The mem-
brane filters, which were M illipore microweb sheets (WHW P304F1
Millipore Corp., Massachusetts), were cut into circular pieces and auto-
claved before use. The chambers were sterilized by exposure to ultra-
violet light for 30 min and the apparatus was assembled aseptically.
The chambers, which were supported in a stainless-steel frame covered
with a nylon mesh to protect the filters, were held approximately one
meter below the water surface by attaching the frames to a raft situated
about 200 m offshore in the Georges River. Water at this point of the
Georges River has the same salinity as that of seawater, since the area
is subject to tidal movements and there is a regular exchange of out-
side water.
Samples of seawater and sediment were collected near the raft in the
Georges River. Each of the samples was divided into two portions, and
one portion of each was autoclaved at 121C for 15 min. When the
sterilized samples were cool, all four samples were inoculated with
E.
colias follows: (a ) E. coli in sterile seawater, (b) E. coli in seawater,
(c) E. coli in sterile sediment, (d) E. coliin raw sediment. There were
three replicates of each treatment. Initially, an aliquot from each treat-
ment was plated to determineE. colinumbers before the chambers were
filled. The chambers were immersed in the Georges River and, at daily
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Survival of bacteria in estuarine sediments
107
intervals, 1 ml of water or sediment sample was withdrawn from each
chamber and added to 9 ml of 1% peptone in a 25-ml McCartney
bottle. The sample bottles were placed in ice and immediately trans-
ported back to the laboratory, where they were plated on MacConkey
agar. Microscope observations of samples taken from chambers contain-
ing seawater were made at various intervals.
In a second experiment, none of the seawater or sediment samples
were autoclaved. Seawater taken from near a sewage outlet was used as
an enriched source of predators and parasites in some treatments. The
four treatments were (a) E. coli in seawater, (b) E. coliin seawater +
sewage-enriched seawater, (c)
E. coli
in sediment, (d)
E. coli
in sedi-
ment -f- sewage-enriched seawater. In all other respects the methods
were the same as used previously.
Fig. 2.
The chamber used for in situ studies of
E. coli
survival in the Georges
River. The chambers consisted of three circular pieces of plexiglass. The outer
pieces were 6.5 mm thick and the central piece was 25 mm thick. The central
lumen was 6 cm in diameter an d an enclosed chamb er (volume = 70.7 ml) was
made by sandwiching two Millipore microweb filters (poresize 0.45 /m between
the inner side of each outer piece and the central piece of plexiglass. Two 10-mm-
diam sampling ports cut in the central piece of plexiglass were plugged with
rubber stoppers.
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108
Margaret M. Roper and K.C. Marshall
100
so-
so
70
. 50
30
U 20-
10-
0
1S00
S
E
10
5 10 15 20
Distance from Se w ag e Outfall km )
Fig. 3. Numbers of coliform bacteria in water and sediments, and measure-
ments of specific conductivity and suspended solids for water samples, as a func-
tion of distance from the sewage outfall in the Tamar River. Sampled at high
tide.
O numbers of coliform bacteria in water; V coliform bacteria in sediment;
# specific conductivity; suspended solids.
Effect of m ontmorillonite on survival of E. coli
Montmorillonite was used in laboratory model experiments on sur-
vival of
E. coli,
the treatments were (a)
E. coli
-f- sterile seawater,
(b ) E. coli
j
seawater, (c ) as in (a ) -f- montmorillonite, (d ) as in (b )
+ montmorillonite. Seawater samples were taken from near a marine
sewage outfall to provide an inoculum enriched with predators and
parasites. The effects of two different forms of montmorillonite were
examined. A colloidal montmorillonite sample was prepared by sus-
pending Wyoming bentonite in distilled water, separating the coarse
fraction by centrifugation at 12,000
g
for 20 min, and finally concen-
trating the fine fraction by centrifugation at 23,000
g
for 45 min (Lahav,
1962 ). A final concentration of 150 ^g ml
1
was used in all treatments
containing colloidal montmorillonite. A crude sample of montmoril-
lonite was prepared by mixing powdered Wyoming bentonite with sea-
water to form a thick slurry with a final concentration of 500 mg ml
1
.
A volume of slurry equal to that of seawater used in the minus-clay
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Survival of bacteria in estuarine sediments
109
5
750
S 10 15 20
Di s t a n c e f rom S ewa g e Out fa l l ( k m )
Fig . 4. Sam e as for F ig. 3, but samp led at low tide .
10 15
Time (Days)
20
E
1
25
i
S
.a
a
30
Fig. 5. Survival of E. coli enclosed in chambers immersed in the Georges River.
E. coli
in sterile seawater;
E. coli
in seawater; D
E. coll
in sterile sediment;
O
E. coli
in sediment.
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110
Margaret M. Roper and K.C. Marshall
treatments was used for the clay treatments. All treatments were incu-
bated at 26C on a rotary shaker. Viable counts of
E. coli
were made
at daily intervals until the numbers became stable.
Results
Tamar River sampling
Water samples taken by B. Pike and M. Morris (Tasmanian Depart-
ment of Agriculture, Launceston) along the entire length of the Tamar
River indicated that high numbers of
E. coli
persisted in the water until
the specific conductivity (L
s
) attained a value of approximately 2.5 mS
cm
1
. At this point the numbers of bacteria began to decrease, and, as
10 15
Time Days)
20 25
Fig. 6. Survival of E. coli enclosed in chambers immersed in the Georges
River.
E. coli
in seawater; 0
E. coli
in sewage-enriched seawater; D
E. coli
in sediment; O
E. coli
in sewage-enriched sediment.
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Survival of bacteria in estuarine sedim ents 111
the salt concentration increased still further, the numbers dropped to an
insignificant level. This decline in numbers corresponded to a decrease
in suspended solids, suggesting that bacteria were being sedimented
along with suspended particulates.
To confirm that the bacteria were being sedimented in to muds and not
being removed by some other means, we carried out a more extensive
sampling of both mud and water over a smaller area of the river, where
the salinity corresponded to an
L
s
of approximately 2.5 mS cm
1
.
The results of sampling at high tide (Fig. 3) indicated a rapid
decline of coliform bacteria in water with increasing salinity and in-
creasing distance from the sewage outfall. This corresponded to a rise
in the numbers of coliform bacteria in the sediments located between 5
and 18 km from the sewage outfall. Beyond the 18-km point, however,
the numbers of coliforms in the sediments decreased, since the bulk of
the bacteria were sedimented further upstream.
Sampling at low tide produced results similar to those found at
high tide (Fig. 4). As might be expected, the increase in salinity and
corresponding decline of coliform bacteria in water occurred further
downstream. Large numbers of bacteria were found in the same area of
sediment (5-18 km from sewage outfall) as reported above for the
high-tide sampling. This suggests that bacteria sedimented further up-
stream at high tide remained viable in the sediments, at least for a short
period of time.
In situ survival of E. coli in the Georges River
In situ survival studies of
E. coli
in chambers immersed in the
Georges River estuary indicated that sediment particulates prolong the
survival of
E. coli
when compared with the survival in seawater alone
(Fig. 5). In sterile sediment, there was was no decline of E. colinum-
bers. Nonsterile sediment provided reasonable protection, although
there was a gradual decrease in bacterial numbers with time.
E. coli
numbers in seawater declined rapidly after an initial lag period, and
bacterial survival in sterile seawater was little better than in the natural
seawater. Microscope observations on samples from chambers contain-
ing only seawater, made on day 7, indicated that both sterile and
nonsterile samples contained fast-moving bacteria (possibly pseudo-
monads and bdellovibrios). This suggested that the sterile seawater
samples became contaminated with natural seawater containing antago-
nists when the chambers were being sampled. The nonsterile samples
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112
Margaret M. Roper and K.C. Marshall
also contained a wide range of active protozoans, such as dinoflagellates
and euglenas.
In a second experiment, where seawater enriched with predators and
parasites was added to some of the chambers, sediment materials again
were found to prolong the survival of E. coli (Fig. 6). The addition
of more antagonists to the sediment material did not alter the protec-
tive effect. However, the decline ofE . colinumbers in seawater enriched
with antagonists was more rapid than in natural seawater.
Effect of monttnorillonite on survival of E. coli
Montmorillonite, both in the colloidal and crude form, had a consid-
erable effect on the survival ofE. coliin seawater under laboratory con-
ditions (Fig. 7). There was more than a 10-fold improvement in the
recovery of E. coli in the presence of colloidal montmorillonite com-
pared with the treatment containing seawater alone (Fig. 7a). An
even greater recovery was achieved in the presence of crude mont-
morillonite (Fig. 7b). Numbers of E. coli in the sterile controls, either
with or without montmorillonate, remained almost constant throughout
the test period.
*
a) Colloida l Clay
\ ba
\
\