FEMS MicrobiologyEcology85 (1991) 161-168

1991 Federation of European Microbiological Societies 0168-6496/91/$03.50

Published by Elsevier
ADONIS 016864969100069K

161

FEMSEC 00324

Survival of Escherichia coli K12 in seawater

Soren J o h a n n e s S o r e n s e n
Department of General Microbiology and Institute of Biological Chemistry B, University of Copenhagen, Copenhagen, Denmark

Received 13 May 190

Revision received 16 November 1990
Accepted 19 November1990

Key words: Auxotrophic mutant; Filter-sterilized seawater; Microzooplankton grazing

1. S U M M A R Y
The fate of an auxotrophic Escherichia coli K12
strain (NF1830) in coastal water was investigated.
The E. coli K12 were enumerated after incubation
for varying times in seawater. Incubated in raw
seawater at 15 and 20C, the NF1830 decreased
f r o m 10 6 c f u / m l to below detection within six
days of incubation, but when incubated at 7C it
persisted longer. The NF1830 was capable of cell
division in sterile seawater. Growth was also shown
to occur in raw seawater in the presence of autoclaved sediment. The E. coli K12 decreased in
number at a much lower rate when incubated in
seawater treated with eukaryotic inhibitors. These
findings suggest that the die-off of the auxotrophic E. coli K12 strain seen in the raw seawater
was caused by grazing of bacterial predators in the
seawater.

Correspondence to." S.J. Sorensen, Department of General Microbiology and Institute of Biological Chemistry B, University
of Copenhagen, Solvgade 83H, DK 1307 Copenhagen, Denmark.

2. I N T R O D U C T I O N
It is a common assumption that the Escherichia
coli K12 strains used in scientific work and in the
biotechnological industry are so weakened by mutations, that they cannot survive outside the
laboratory settings. It is therefore remarkable that
only scant information on the survival, establishment and growth of E. coli K12 in the natural
environment is available [1].
The survival of E. coli K12 has recently been
investigated in the intestinal canal of mice and
human subjects [2,3], in a laboratory and farm
environment [4], in soil [5], and in seawater,
sewage, lake water and soil [6]. In contrast to these
few recent investigations concerning the survival
of E. coli K12, the fate of enteric bacteria in
general has been the subject of investigation for
about 100 years. In studies of the factors influencing the survival of pathogenic bacteria, De Giaxa
(1889) found that Vibrio c o m m a lived for several
days in heat-sterilized seawater, but in untreated
seawater it soon disappeared [7]. Since then,
numerous investigations have been conducted to

162

elucidate the reason(s) for the low viability of

non-marine bacteria in this medium.
Two theories have been put forward to explain
the background of the bactericidal activity of
seawater. One is physico-chemical, and the other a
biological explanation. The first ascribes the
bactericidal activity to various physico-chemical
parameters such as: p H [8], temperature [9], salinity [10], solar radiation [11] and toxic chemicals
[12]. The second theory suggests that the bactericidal activity results from activity of macromolecules [13] associated with algae blooms [14], lack
of nutrients [15] or is caused by competition [16]
and predation [17]. In general, any of the above
mentioned factors may cause a change in enteric
indicator populations, if it becomes the limiting or
excessive variable in the bacterial environment.
The determining factor in the marine environment
may change due to seasonal, climatic, and local
conditions. This could easily be a major reason for
the seemingly contradictory results found in the
literature.
A third, and quite new theory, concerning the
fate of enteric bacteria in marine environments
claims that the die-out of enteric bacteria in
seawater is not necessarily caused by the death of
the allochthonous bacteria, but may instead reflect
the fact that these bacteria enter a non-culturable
state [18].
The main objective of the present study is to
elucidate the potential fate of an auxotrophic E.
coli K12 in seawater sampled near shore on the
east coast of Zealand, Denmark.

3. M A T E R I A L A N D M E T H O D S
3.1. Bacterial strain and growth media
The E. coli K12 strain used throughout the
experiment was NF1830 (provided by N. Fiil),
a
recA
derivative of MC1000
with a
F'laclQZ::Tn5. The relevant phenotype is: L e u - ,
recA , K m r, and Str r.
The method used for enumeration of E. coli
K12 after its introduction into samples of raw and
sterile seawater, was a modification of the antibiotic resistance selection method [19]. Strains were
cultured in Luria Bertani (LB) broth [20] at 37C

with constant agitation. (A + B) medium [21] was

used as diluent, except when otherwise noted. LB
broth with 1.5% agar was used as solid medium
for spread plating in all experiments. LB-plates
with 30 /~g/ml kanamycin (Km) and 200 /~g/ml
streptomycin (Str) were used as selective media for
samples from the raw seawater incubations.
Total bacterial counts were obtained by counting DAPI-stained bacteria in epifluorescence microscope [22].
3.2. Water samples
Seawater samples were collected near shore (depth: 0.3 m) in Koge Bugt on the east coast of
Zealand, Denmark. Seawater was sampled in 10-1
plastic containers, and sediment samples were collected in plastic buckets (approximately 5 kg sediment + 5 1 seawater in each bucket). Samples were
stored in the dark at 5C until use. There was
always less than 4 h between collection and initiation of the incubation.
Raw seawater samples were sterilized by
vacuum filtration through three sterile layers of
filter pads, and finally through a sterile 0.2-/xm
membrane filter into sterile 1-1 bottles. The sterile
seawater was stored in the dark at 5C until use.
To test the effectiveness of this procedure a 1-ml
subsample of the sterile seawater was inoculated
into a test tube with 5 ml LB and incubated at
room temperature for 14 days. N o growth was
seen, neither in broth nor when plated on LBplates.
To assess the effect of predation on survival of
the E. coli K12 in seawater, eukaryotic inhibitors,
cycloheximide (0.25 m g / m l ) and nystatin (30
/~g/ml), were added to raw seawater 24 h before
starting the experiments [23]. Sediment was
screened through a stainless steel 0.5 m m 2 sieve
and sterilized by autoclaving (1 h, 120C) in 2-1
preserving jars and stored in the dark at 5C until
USE.

Salinity of the seawater was determined from

the conductivity of the seawater compared with a
standard curve for NaCI concentration versus conductivity measured at 0C. The conductivity of
the seawater was measured by a 'flow and pipette'
(CDC314) conductivity cell at 0C. Salinity varied
between 7%0 and 15%o in the sampling period.

163
M e a s u r e m e n t of p H (7.0-7.5) was b y use of a p H
i m m e r s i o n electrode at r o o m t e m p e r a t u r e . T h e
t e m p e r a t u r e was m e a s u r e d in situ. It v a r i e d f r o m
4 C on 2.20.1989 to 15C at the 5.20.1988. T o t a l
bacterial c o u n t v a r i e d between 10 6 a n d 10 v cfu p e r
ml of seawater d u r i n g the s a m p l i n g period.

107 -

3.3. Incubation

105 -

Before all i n c u b a t i o n s a fresh E. coli K12 overnight culture was w a s h e d twice b y c e n t r i f u g a t i o n

t h r o u g h sterile seawater, a n d finally s u s p e n d e d in
5 ml sterile seawater. T h e r e were always m o r e
t h a n 10 9 c e l l s / m l in the final suspension. This
s u s p e n s i o n in sterile seawater will b e referred to as
SSS.
Test tubes c o n t a i n i n g 5 ml of a 103-105 dilution of SSS in either raw seawater, sterile seawater,
s e a w a t e r + 5 g sterile s e d i m e n t or s e a w a t e r +
e u k a r y o t i c inhibitors, were i n c u b a t e d in the d a r k
with c o n s t a n t a g i t a t i o n at 15C. In one experim e n t test tubes with N F 1 8 3 0 in raw seawater were
i n c u b a t e d at 7C, 15C, a n d 20C. Test tubes
with 5 ml seawater w i t h o u t a d d i t i o n of SSS served
as controls.
The c o n t e n t s of three i n c u b a t i o n tubes a n d one
c o n t r o l were h a r v e s t e d at each s a m p l i n g time a n d
100 ~tl of a p p r o p r i a t e dilutions in (A + B) m e d i u m
were s p r e a d on selective LB-plates. T h e test tubes
with s e d i m e n t were shaken vigorously for 30 s
b e f o r e the s a m p l e s were taken.

106"

104

. . . .
0

~ . . . .
5

~ . . . .

10

. . . .

15

. . . .

20

25

Day

Fig. 1. Survival of E. coli K12 (NF1830) in sterile seawater at

15C with two different inocula. The data shown are the mean
values of three replicate microcosms with error bars indicating
the standard deviation.

e x p e r i m e n t , with a slight increase in n u m b e r from

d a y 1 to d a y 3.
4.2. R a w seawater

W h e n the 13". coil K12 was i n c u b a t e d in raw

seawater at 7 C n o m a j o r changes in p o p u l a t i o n
d e n s i t y were r e c o r d e d d u r i n g the five d a y i n c u b a tion. T h e viable cell p o p u l a t i o n of N F 1 8 3 0 was
3.5 106 c f u / m l at the start of this e x p e r i m e n t
a n d after 5 d a y s of i n c u b a t i o n it was still 1.5 10 6
c f u / m l (Fig. 2).

lo 7

4. R E S U L T S
10 5 .

4.1. Sterile seawater

W h e n i n c u b a t e d at 15C in sterile seawater, the

N F 1 8 3 0 p o p u l a t i o n grew e x p o n e n t i a l l y f r o m an
initial p o p u l a t i o n of 10 4 c f u / m l to a 15-fold higher
level (equivalent to four generations) d u r i n g the
first 5 d a y s of i n c u b a t i o n . The p o p u l a t i o n stabilized at this level for the r e m a i n i n g 20 d a y s of the
e x p e r i m e n t (Fig. 1). A g e n e r a t i o n - t i m e (T2)
c a l c u l a t e d for the g r o w t h p e r i o d f r o m d a y 2 to d a y
4 was 16 h. If the E. coil K12 was i n o c u l a t e d at a
d e n s i t y a b o v e 10 6 c f u / m l no clear g r o w t h c o u l d
be detected. T h e p o p u l a t i o n r e m a i n e d a p p r o x i m a t e l y stable at the initial level t h r o u g h o u t the

104,
~-

2
o

w sediment

~o3"
1o 2
lO 1 .
lO o
o

Day

Fig. 2. Survival of E. coli KI2 (NF1830) in raw seawater

incubated at different temperatures (7, 15 and 20C) and with
raw sediment added to the seawater incubated at 15C. At
15C two different initial densities were used. The data shown
are mean values of three replicate microcosms with error bars
indicating the S.D. The arrow indicates the limit of detection.

164

107
106"

104.
E 103'
~ 102.

~ : ] _ _ _ ~ Ew~er~;~tl
e rr~
hlbltors

101
10 0

day
Fig. 3. Survival of E. coli K12 (NF1830) in raw seawater with and without eukaryotic inhibitors incubated at 15C and total direct
count of DAPl-stained bacteria in raw seawater with and without eukaryotic inhibitors. The data shown are the mean values of three
replicate microcosms with error bars indicating the S.D. The arrow indicates the limit of detection.

At 15C and 20C the density of NF1830 remained essentially constant for the first day but
from day 1 to day 5 the number of cfu declined
rapidly, from an initial density of 1_06 c f u / m l to
below detectable levels. No appreciable difference
was found for incubations at 20C and 15C. The
half-life of NF1830 in raw seawater at 15C and
20C (Defined as: T_ 2 = In 2 / ( l n X 0 - In Xt) x t
days x 24 h) calculated for the period from day 1
to day 5 was approximately 5 h. The same pattern
was seen when the initial density was changed to
104 cfu/ml.

4. 3. Eukaryotic inhibitors
Incubations of NF1830 at 15C were performed in raw seawater with and without
eukaryotic inhibitors (Fig. 3). Without inhibitors
the population declined as previously shown to
below detectable levels within five days of incubation. The calculated half life ( T 2 ) of the E. coli
population during this period was approximately
6.5 h.
When eukaryotic inhibitors were added to the
seawater, the number of NF1830 decreased only
very slowly from the initial level of 2 x 105 c f u / m l
to 7 x 103 c f u / m l after 5 days of incubation. The
calculated half-life for the NF1830 in seawater
with eukaryotic inhibitors was 24 h. When sterile
seawater was used (experiments not shown) no
effect of eukaryotic inhibitors was observed.

4.4. Effect of sediment

N o effect of raw sediment addition on the
survival of NF1830 in raw seawater at 15C was
noted (Fig. 2). The cell density remained at its
initial level during the first day of incubation, but
from day 1 the density declined from 106 c f u / m l
to below the detectable level at the fifth day of
incubation in the presence or absence of sediment.
The half-life ( T 2) of NF1830 under these conditions was approximately 5 h.
Incubations in which autoclaved sediment was
included showed that the NF1830 population de107 I

101/ -

104

102 I . . . .
0

~ ....
6
Day

~ ....
10

Fig. 4. Survival of E. coli K12 (NF1830) in (O) raw and (4.)

sterile seawater incubated at 15C in microcosms with sterile
sediment added. The data shown are mean values of three
replicate microcosms with error bars indicating the S.D.

165
creased 10-fold during the first day in both raw
and sterile seawater, and then increased more than
100-fold during the following 3 to 5 days (Fig. 4).
In incubations containing autoclaved sediment and
sterile seawater, the NF1830 population stabilized
after five days of incubation, at a final level more
than 500-fold higher than the initial density. The
generation-time ( Tz) found in the growth period
from the third to the fifth day of incubation was
approximately 10 h.
Cell densities in incubations with sterile sediment + raw seawater reached a m a x i m u m (100fold higher than the initial density) on the fifth
day of incubation. The generation time ( Tz)
calculated for the growth period from day 1 to day
5 was 15 h. Thereafter the population declined, at
first slowly, but from day 9 to day 15 (Fig. 4) the
NF1830 population decreased to a level approximately 25-fold lower than the initiation density. The half-life (T_2) of NF1830 during this
period was approximately 20 h.

5. D I S C U S S I O N
In raw seawater the auxotrophic E. coli K12
declined to below detectable levels within 6 days
of incubation at 15C. The persistence of E. eoli
K12 in raw seawater was shown to be inversely
related to the incubation temperature. This is in
good agreement with the fate of fecal bacteria in
seawater. Low temperatures often appear to increase survival time of enteric bacteria [24]. In
sterile seawater NF1830 grew exponentially to a
level 15-fold higher than the initial density. This
growth could only be detected if the start density
of NF1830 was low; at higher density no clear
growth was detected. This might be because the
available nutrition in sterile seawater only can
support growth equivalent to approx. 106 c f u / m l .
If so, one would expect only a small increase in
number when the start density was high, e.g. 10 6
c f u / m l . The data in Fig. 1 actually indicate such a
small increase from day 1 to day 3. A pattern
similar to the results presented here has been
reported for Klebsiella pneumonia, Salmonella
typhimurium and Saccharomyees cerevisiae in

sewage, as well in lake water for the latter two

organisms [25].
In this study the added E. coli were able to
grow in sterile seawater even though the used
strain was an auxotrophic mutant. This may be
because eutrophic seawater contains a broad selection of organic compounds, leachates from algae,
decomposing organic material etc.. Thus the use of
auxotrophic mutants, as a containment system
preventing s u r v i v a l / g r o w t h of genetically engineered microorganisms in the environment, may
not be an effective strategy.
Many different factors have been suggested to
explain the antibacterial effect of seawater. In this
study, it was shown that filter-sterilization delayed
this antibacterial effect for more than 25 days. It
was also shown that low temperature diminished
this effect significantly. Furthermore the antibacterial effect of raw seawater was reduced by
the addition of eukaryotic inhibitors.
An important question is whether the decline,
seen in this study, is a die-off of the E. coli K12,
or the decline is artificial and only reflects that the
E. coli K12 added to the seawater has entered a
'non-culturable' stage [26]. In the present study no
decline was seen in sterile seawater, while the
decline observed by Xu and co-workers [26] was
from incubation experiments in sterile seawater.
There are no obvious reasons why the E. coli in
raw seawater should enter a dormant stage, while
the same E. coli in sterile seawater and seawater
with eukaryotic inhibitors does not become
dormant.
The decrease in numbers of NF1830 followed
the same pattern both with and without raw sediment included. If the effect of sterile sediment
addition is due to the presence of additional nutrients liberated by autoclaving, then nutrient addition can decrease or postpone the die-off of E.
coli K12 in seawater. Competition with indigenous
bacteria has been emphasized as one of the main
factors responsible for the decline in numbers of
allochthonous bacteria in natural environments
[6,15,16]. The rapid die-off seen in this study
cannot be explained by competition with the indigenous seawater bacteria alone, since eukaryotic
inhibitors showed no negative effects on the density of indigenous bacteria. In fact, indigenous

166

bacteria actually grew to higher levels when

eukaryotic inhibitors were added to raw seawater
(results not shown).
Predation is therefore the simplest explanation
for the decline of E. coli K12 in raw seawater
from Koge Bugt. This is in good agreement with
conclusions from other work. It has been reported
that E. coli was eliminated by protozoa in
estuarine waters [27,28] and similar results have
been reported from experiments with freshwater
[23]. It has been pointed out that predators do not
eliminate their prey, and that the threshold
bacterial density for predation by protozoa is assumed to be between 1 0 4 - 1 0 6 cells per ml [29]. In
the present study the E. coli population was also
suppressed when its cell densities were below
threshold. Such an observation can be explained
by assuming that indigenous bacteria in raw
seawater provided sufficient numbers to exceed
this threshold and supplied alternative prey [30].
When the indigenous seawater bacteria were at
levels above the threshold for grazing, E. coli
added to the seawater would be eliminated if its
multiplication rate was less than the grazing rate.
In a study of microzooplankton grazing on
planktonic marine bacteria from estuary-coastal
water samples it was concluded that there is a
daily turnover of the standing crop of bacteria in
seawater [31]. Zooflagellates are known to be the
major predators on bacteria in seawater. In most
offshore and coastal waters (during summer), zooflagellates on the average clear 20-50% of the
water for bacteria per 24 h [32]. If these grazing
rates also are valid for the seawater from Koge
Bugt, added E. coli K12 must multiply with more
than one generation per day otherwise its population will be eliminated according to the above
mentioned 'predation theory'. E. coli K12 was
shown to have a generation time of 16 h during
the first days of incubation in sterile seawater.
The lag-period seen during the first day or two
after E. coli incubations in raw seawater (Figs. 2
and 3), may therefore reflect a situation where E.
coli K12 actually multiplies with a generation time
equal to the clearing rate of the zooplankton.
After a day or two the nutrients are probably
depleted, and growth of the E. coli K12 population stops, as seen in the incubation in sterile

seawater (Fig. 1). When growth of the E. coli

population has ceased, its elimination would take
1-5 days with a clearing rate of 20-100% per day.
The results presented in this study are consistent
with this consequence of the 'predation theory'.

ACKNOWLEDGEMENTS
This research was supported, in part, by grants
from the National Food Agency, Denmark. The
views expressed in this report are not necessarily
those of the Agency. I acknowledge Annelise
Kjoller and Sten Struwe for advice and helpful
suggestions, and Jesper Damgaard for technical
assistance.

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