effects of salinity on.pdf

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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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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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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


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


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

\

\

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