Annals of RSCB
Vol. XVII, Issue 1/2012
ENVIRONMENTAL FACTORS INFLUENCES ON
BACTERIAL BIOFILMS FORMATION
Aurelia Manuela Moldoveanu
FACULTY OF NATURAL AND AGRICULTURAL SCIENCES
„OVIDIUS” UNIVERSITY, CONSTANTZA
Summary
Biofilms are a very important aquatic microcommunity due to their main role in
micro and macrofouling formation. The first phases of biofilms formation are influenced
by a series of environmental factors like: temperature, salinity, water dynamism,
nutrients concentration that can change biofilms growth, density and thickness. The aim
of this study was to determine some of the main factors that influenced biofilms
formation in laboratory conditions. The experimental systems were 100 ml sterile
containers filled with sea water as culture media in static conditions. The total bacterial
growth was between 49 ·104 cells/mm2 - 79 ·104cells/mm2 for the salinity variation and
between 40.7 ·104 cells/mm2 + 68 · 104 cells/mm2 for the various temperatures that
influenced the bacterial growth and density. The low temperatures determined a lower
bacterial cell density, sea water salinity variations influenced the biofilm formation and
low concentrations of salt also determined a low bacterial growth than normal and
medium salinity values. Bacteria forms in the first phase microcolonies in lower
numbers, but in the late phases they form large colonies on the sample surfaces.
Keywords: biofilm, static methods, temperature, salinity
[email protected]
Introduction
then d) maturation of the biofilm matrix
(Zobell, 1943; Kjelleberg, 1982).
Marine bacteria grow optimally at a salt
concentration between 33-35%, do not
develop or some time develop poorly when
there is no NaCl in the water. Bacteria can
be oligotroph, psychotrophe or psychophile
with the exception of those that growth in
the surface layers in tropical waters (Parsek
and Fuqua, 2004). Depending on the
habitat, they are barophobe (at the sea
surface), barotolerant and barophile. They
display a marked pleomorphism, probably
determined by oligotrophs and the effects of
high hydrostatic pressure (Hinrichsen,
2011). Most of them (80%-95%) are Gramnegative, mobile (75%-85%), aerobe,
optionally aerobe and obligatorily anaerobe,
those living in the depth of the sediments
(Zarma, 1959).
In laboratory conditions, the variation of
environmental factors is essential in the
formation of biofilms.
Free-living or a planktonic mode of
growth of microorganisms is usually
observed in laboratory cultures. But this
growth mode is infrequent in the natural
environment were bacterial cells may seek
out advantageous niches (Costerton, 1978;
Zarnea, 1994). Once planktonic cells meet
such surfaces, they attach and eventually
develop biofilms through growth and
division (Donlan, 2002). Although the
attachment is initially reversible, it becomes
stronger and irreversible with time.
Although the chemical and biological
processes are poorly understood, it is
thought that the early process of biofilm
formation occurs through a sequence of
processes; a) it begins with the adsorption
of organic and inorganic particles on the
surface, b) attachment of pioneer
microorganisms,
c)
growth
and
reproduction of primary colonizers, and
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Annals of RSCB
Vol. XVII, Issue 1/2012
This is important to know which
factor has the most influence on the
adherent bacterial cells. Thus, the growth
and multiplication of microorganisms is the
result of a number of coordinated metabolic
reactions whose normal development is
ensured by an optimal environmental
temperature or salinity (Characklis 1990;
Stoica, 2004).
The aim of this study was to observe
marine bacteria from the initial stage of
biofilm formation on the artificial surfaces
of glass slides exposed to natural seawater
to the late irreversible phases of biofilms
formation when the bacteria are inbeded at
the surface interface under the influence of
two very important environmental factors
for the marine environment
like
temperature and salinity.
Temperature and salinity are very
important environmental factors involved
in the bacterial growth and density but also
for the bacteria attachment and biofilm
formation and for this reason some
variations of these two factors similar with
those of seawater conditions were
necessary. At first the salinity variations
were chosen similar to those of the
seawater in various area, a low
concentrations of 5 g/l frequently obtain in
the Danube tributary area, 10 g/l and 15 g/l
which are medium values for the Black Sea
waters (Celik et al. 2009; Nita et al. 2011)
and 25 g/l for similar to the area of contact
with de Mediterranean Sea waters
(Gavrilovic et al. 2011). The variations
were obtained by additions of distillate
water in order to decrease salinity and by
marine salt additions per liter in order to
increase or decrease salinity.
The temperature variations were chosen
between 6ºC, a low temperature commune
in winter obtain in a refrigerator, 18ºC as a
medium but constant temperature obtain in
a thermostat room of Biodiversity
Laboratory and a high temperatures of
23ºC, a room temperature commune in
summer in the Laboratory of Microbiology
(“Ovidius” University Constanta).
The information about the first phases
in biofilms formation under environmental
factors influences were investigated from 2
hours to 72 hours, with the harvesting of
the slides at different intervals of 4 hours
in the first phases up to 24 hours and 8
hours in the later phases up to the 72 hours
of the experiments.
The slides were analyzed under bright
field microscopy by the aid of the Hund
Wetzlar Microscope with 100X objective
and 10X ocular (Hulea, 1969, Morató et al.
2004). The bacterial density and the total
cell number were determined by means of a
10mm X 10mm micro-ocular grid
(macroscopically). An area of 10
microscopic fields per harvested slide with
thee repetitions per chosen time interval
were chosen (Fry, 1990; Surman et al.
1996).
Material and methods
The experiments analyzed biofilm
formation in static conditions using
containers and fresh sea water as culture
media. All the hydrophile surfaces
(microscope slides) were previously
degreased with 70% ethanol (Lazar et al.
2004) and sterilized by immersion in
sulfochromic mixture (K2Cr2O7/H2SO4)
to
avoid
contamination
with
microorganisms and organic matter prior to
the experiment (Mercier-Bonin, 2004).
In order to obtain biofilms on the
smooth surfaces of the microscope slides,
fresh seawater from the Black Sea (Eforie
Nord) was used as culture medium littoral
and kept in sterile containers as shortly as
possible the slides were stain immediately
without fixation whit one drop of 0.1 %
Methylene Blue between the slide and the
cover sleep in a capillarity staining.
In the experiments Henrici Slide
Technique was used as culture method in
order to obtain fresh biofilm on the
artificial
surfaces
as
shown
by
www.BiofilmsONLINE.com, 2008, and
the method was adapted for avoiding
debris according to Kuman and Prasad,
2006;
Moldoveanu, 2011 by oblique
position of slides in the containers.
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Annals of RSCB
Vol. XVII, Issue 1/2012
hours at the end of the interval the density
values are lower of 6.67 ·103 cells/mm2
(Fig.1).
The results prove that marine bacteria
react to a salinity reduction compare with
the values obtain at the normal and medium
salinity values. A low value of 0.47 for
equation of growth was observed but a high
linearity coefficient of 0.95 was observe in
this case in this case that determined a high
linearity of the cell density and growth.
After this fist experiment two more
salinity values were obtain similar to those
found in summer and winter in the costal
area compare with the medium values of
the salinity between 10 g/l - 15 g/l .
For the probe with 10 g/l salinity after
two hours after imersion the bacterial cell
density was 0.98 ·103 cells/mm2 after 6
hours this values is double with a value of
1.77 ·103 cells/mm2 at a first pick is reaches
14 hours the density reaches the value of
3.76 ·103 cells/mm2 a at 24 hours when the
density is 4.83 ·103 cells/mm2 at 48 hours
the pick is 6.45 ·103 cells/mm2 the highest
value in the control probe and after 72
hours at the end of the interval the density
values are lower of 5.08 ·103 cells/mm2
(Fig.2).
Results and discussions
The slides with biofilms formed on the
artificial surfaces were collected from the
containers with costal seawater and
analyzed under bright field microscopy
immediately after harvesting.
After the cell counting with the help of
the ocular grind showed that the bacterial
cell density values indicate that the biofilms
had a rapid growth under different
environmental factors.
The first factor was the salinity variation
of the marine seawater varied between four
concentations in order to observe de main
differences between the control probe and
the intervals of growth and development of
the biofilms.
In order to observe how marine bacteria
react to low salinity at first a value similar
to that of the seawater in case of large fresh
water addition like those found in the
Danube tributary area was used. This value
is important for the biofilms growth as a
manner of adaptation to the environment.
Bacterial biofilms under the influence of 5 g/l salinity
8
3
2
Density 10
cells/mm
7
y = 0.4756x + 0.823
R2 = 0.9588
6
5
Bacterial biofilms under the influence of 10 g/l salinity
4
3
8
2
7
3
2
Density 10
cells/mm
1
Salin. 5g/l
0
2
6
10
14
18
24
32
40
48
56
64
72
Time (hours)
Fig. 1 A low salinity value (5 g/l)
y = 0.446x + 1.6136
R2 = 0.766
6
5
4
3
2
1
Salin. 10 g/l
0
In the probe with 5 g/l after only two
hours after imersion the bacterial cell
density was 0.96 ·103 cells/mm2 after 6
hours this values is double with a value of
1.61 ·103 cells/mm2 at first pick is reaches
14 hours the density reaches the value of
3.12 ·103 cells/mm2, at 24 hours when the
density is 3.98 ·103 cells/mm2 at 48 hours
the pick is 5.24 ·103 cells/mm2 the highest
value in the control probe and after 72
2
6
10
14
18
24
32
40
48
56
64
72
Time (hours)
Fig. 2 A first medium salinity value
(10 g/l)
The values obtain shown that the fist
medium salinity value determined an
optimal cell growth and attachment but the
equation of growth was low of 0.44 and
also the linearity coefficient had a lower
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Annals of RSCB
Vol. XVII, Issue 1/2012
48 hours the pick is 7.47 ·103 cells/mm2 the
highest value in the control probe and after
72 hours at the end of the interval the
density values are lower of 8.62 ·103
cells/mm2 (Fig.4).
The results obtain shown that the
medium salinity values determined an
optimal cell growth and attachment and the
equation of growth was high of 0.62 and
also the linearity coefficient had a higher
value of 0.92 with high linearity of the
bacterial cell growth and attachment.
value of 0.76 this is due to a low linearity of
the density values.
For the case of the probe with 15 mg/l
salinity after two hours after imersion the
bacterial cell density was 1.05 ·103
cells/mm2 after 6 hours this values is double
with a value of 2.65 ·103 cells/mm2 at 14
hours the density reaches the value of 3.44
·103 cells/mm2 a first pick is reaches at 18
hours with a density of 4.94 ·103 cells/mm2
at 24 hours when the density is 4.84 ·103
cells/mm2 at 48 hours the pick is 7.32 ·103
cells/mm2 the highest value in the control
probe and after 72 hours at the end of the
interval the density values are lower of
7.12 ·103 cells/mm2 (Fig.3).
9
3
2
Density 10
cells/mm
3
Density 10
cells/mm2
8
y = 0.6283x + 1.7909
R2 = 0.9234
8
Bacterial biofilms under the infrlunce of 15 g/l salinity
9
Bacterial biofilms under the influence of 25 g/l
salinity
10
y = 0.5917x + 1.1858
R2 = 0.9353
7
7
6
5
4
3
2
6
1
5
Salin. 25 g/l
0
4
2
6
10
14
18
24
32
40
48
56
64
72
Time (hours)
3
2
1
Salin. 15 g/l
Fig. 4 A high salinity value (25 g/l)
0
2
6
10
14
18
24
32
40
48
56
64
72
Time (hours)
At last a final analyzes of the total
bacterial cell growth was observed for the 5
g/l cell density was 49 ·104 cells/mm2 on the
artificial surfaces. After the salinity was
higher 10 g/l the bacterial cell attachment
reached the value of 54.1 ·104 cells/mm2. A
high concentration of 15 g/l determined a
bacterial cell density of 60 · 104 cells/mm2.
The use of even higher concentrations of 25
g/l reached the value of 79 ·104 cells/mm2
(Fig.5).
The values obtain shown that the medium
salinity values determined an optimal cell
growth and attachment and the equation of
growth was high of 9.5 and also the
linearity coefficient had a higher value of
0.89.
This results show that the salinity
variation
is
an
important
factor
environmental factors for the marine
biofilms.
Fig. 3 A second medium salinity value (15 g/l)
The values obtain shown that the
medium salinity values determined an
optimal cell growth and attachment and the
equation of growth was high of 0.59 and
also the linearity coefficient had a higher
value of 0.93, due to this once again the
density values have a high salinity.
In the zone of contact the waters of the
Mediterranean Sea with a high salinity with
over 30 g/l is reached and the salinity is
much higher than the normal and medium
values for the Black Sea.
For this raison in the last probe a 25 g/l
salinity was chosen 0.99 ·103 cells/mm2
after 6 hours this values is double with a
value of 3.11 ·103 cells/mm2 at 14 hours
the density reaches the value of 4.67 ·103
cells/mm2 a first pick is reaches at 24 hours
when the density is 6.43 ·103 cells/mm2 at
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Annals of RSCB
Vol. XVII, Issue 1/2012
but not by salinity. Bacterial community
composition was analyzed by terminal
restriction-fragment length polymorphism
of polymerase chain reaction-amplified 16
S rRNA genes. In summer, community
compositions of both bacteria and diatoms
were strongly affected by salinity.
In addition, natural summer biofilms
that developed at three field sites, where
different salinities were found, harbored
appreciably different bacterial and diatom
community compositions. In contrast, in
winter, temperature exerted a major
influence on community compositions. This
expedients reveled are adds to the growing
evidence that environmental factors are
important determinants of both the quality
and quantity of marine biofilms (Chiu et al.,
2006). Results confirmed by the current
study as well.
The regulatory effects of salinity and
inorganic
nitrogen
compounds
on
nitrification and denitrification were studied
in intertidal sandy sediments and rocky
biofilms in the Douro River estuary,
Portugal, over a 12-month period.
Nitrification and denitrification rates were
measured in slurries of field samples and
enrichment
experiments
using
the
difluoromethane
and
the
acetylene
inhibition techniques, respectively.
Salinity did not regulate
denitrification in either environment,
suggesting that halotolerant bacteria
dominated the denitrifier communities.
However, nitrification rates were stimulated
when salinity increased from 0 to 15
practical salinity units (Magalhães et al.,
2005).
The temperatures were selected similar
to those find in the natural media in the
main season for the temperate water of the
Black Sea region.
The first temperature of 6ºC temperature
is found in situ usually as a medium
temperature of seawater in winter
conditions in the costal area.
Total bacterial growth under salinity variations
90
80
y = 9.584x + 36.58
R2 = 0.8919
3
2
Density 10
cells/mm
70
60
50
40
30
20
10
0
Salin. 5 g/l
Salin. 10 g/l
Salin. 15 g/l
Salin. 25 g/l
Fig. 5 Total bacterial density at different
salinity values
Some experiments were accomplished in
regards to the role of salinity (between
12g/l and 80g/l) in the surface corrosion
achieves some experiments using stainless
steel as substrate. They revealed the
existence of a drop of cellular density with
the increase of water salinity, noticing a
corrosion maximum at 35 g/l between 1.7
·109 CFU/cm2 and 2.1·10 CFU/cm2 for the
aerobe species analyzed (Chiu et al., 2006).
The experimental data have increased
values compared to those obtained this
experimental study.
Others studies examined quantitative
(dry weight, chlorophyll a content and C:N
ratio)
and
qualitative
(community
compositions of bacteria and diatoms)
changes in marine biofilms as a function of
season (summer 2003 and winter 2004),
temperature (16, 23 and 30 ◦C) and salinity
(20‰, 27‰ and 34‰) under laboratory
conditions. Biofilms were allowed to
develop for 20 days in the laboratory, using
natural sea water collected from Port
Shelter, Hong Kong.
At the end of the experiments the
following results were obtained: (1) biofilm
dry weight was greater in summer than in
winter, and greater at 34‰ than at 20‰; (2)
biofilm chlorophyll a content was affected
by all three factors (season, temperature and
salinity), with significant interactive effects
among the three factors; and (3) C:N ratio
was affected by season (winter>summer)
and temperature (30 ◦C>16 ◦C in summer),
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Bacterial biofilms under the influence of a
Bacterial biofilms under the influnce of a
6oC temperature
3
Density 10
cells/mm2
5
9
y = 0.3755x + 0.9202
R2 = 0.8035
y = 0.4992x + 2.4367
R2 = 0.8924
8
3
Density 10
cells/mm2
6
23oC temperature
4
3
2
7
6
5
4
3
2
1
1
Temp. 6 drg.
2
6
10
14
18
24
32
40
48
56
64
2
72
Time (hours)
18oC temperature
y = 0.4359x + 1.6274
R2 = 0.8413
6
5
4
3
2
1
Temp.18 drg.
0
2
6
10
14
18
24
32
14
18
24
32
40
48
56
64
72
In the probe with a 18ºC two hours after
imersion the bacterial cell density was
1.17·103 cells/mm2 after 6 hours this values
is double with a value of 2.52 ·103
cells/mm2 at 14 hours the density reaches
the value of 3.42 ·103 cells/mm2 a first pick
is reaches at 24 hours when the density is
4.56 ·103 cells/mm2 at 48 hours the pick is
6.55 ·103 cells/mm2 the highest value in the
control probe and after 72 hours at the end
of the interval the density values are lower
of 5.53 ·103 cells/mm2 (Fig.7).
This obtain results shown that the
medium salinity values determined a
optimal cell growth and attachment and the
equation of growth was high of 0.48 and
also the linearity coefficient had a higher
value of 0.84.
The temperatures of 25º C al media us
is found in the natural media usually in
summer and was obtain the laboratory by
exposing the sample at room temperature.
In the probe with a 25º C two hours after
imersion the bacterial cell density was 1.85
·103 cells/mm2 after 6 hours this values is
double with a value of 2.91 ·103 cells/mm2
at 14 hours the density reaches the value of
4.84 ·103 cells/mm2 a first pick is reaches at
24 hours when the density is 5.86 ·103
cells/mm2 at 48 hours the pick is 7.45 ·103
cells/mm2 the highest value in the control
probe and after 72 hours at the end of the
interval the density values are lower of
7.55 ·103 cells/mm2 (Fig.8).
Bacterial biofilms under the influence of a
7
10
Fig. 8 A medium temperature value
(25 ºC)
In the probe with a 6ºC after two hours
after imersion the bacterial cell density was
0.67 ·103 cells/mm2 after 6 hours this values
is double with a value of 1.12 ·103
cells/mm2 at 14 hours the density reaches
the value of 2.37 ·103 cells/mm2 a first pick
is reaches at 24 hours when the density is
3.87 ·103 cells/mm2 at 48 hours the pick is
5.13 ·103 cells/mm2 the highest value in the
control probe and after 72 hours at the end
of the interval the density values are lower
of 4.37 ·103 cells/mm2 (Fig.6).
This values obtain shown that the low
temperature chosen determined an optimal
cell growth equation of growth with high
value of 0.49 and also a linearity coefficient
with a high value of 0.89.
8
6
Time (hours)
Fig.6 A low temperature value (6ºC)
3
2
Density 10
cells/mm
Temp. 23 drg.
0
0
40
48
56
64
72
Time (hours)
Fig. 7 temperature 18 ºC
The 18ºC temperature is found usually
as a medium temperature value in late
spring and autumn start in the marine
temperate environment.
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Annals of RSCB
Vol. XVII, Issue 1/2012
The values obtain shown that the high
temperature determined a optimal cell
growth and attachment and the equation of
growth was high of 0.49 and also the
linearity coefficient had a higher value of
0.89.
In the total bacterial growth determined
by the temperature variation of 6 ºC
temperature cell density of 40.7 ·104
cells/mm2 on the artificial surfaces. After
the supply of sea water with 18 ºC of the
bacterial cell attachment reached the value
of 53.53 ·104 cells/mm2. A higher
temperature 23 ºC determined a bacterial
cell density of 68 · 104 cells/mm2 (Fig.9).
The values obtain shown that the
medium temperature values determined a
optimal cell growth and attachment and the
equation of growth was high of 13.74 and
also the linearity coefficient had a higher
value of 0.98.
by temperatures between 5-35 ºC and salt
contents up to 20 g/L NaCl. At these high
DOC loadings an appreciable ammonium
conversion could also be observed. The
ammonium conversion proved to be
sensitive to temperature and salinity. At 5ºC
the ammonium removal rate decreased by a
factor of five compared to 25-35 ºC. Under
many operation conditions investigated
more than 50% of the converted ammonium
was transformed into gaseous nitrogen. The
addition of 20 g/L NaCl caused a strong
inhibition of the ammonium removal rate
over the whole temperature range
investigated (Chapanova et al., 2007).
Some experiments regarding the
colonization of surfaces by the bacterium
Bdellovibrio
bacteriovorus
were
accomplished by Kelley et al. under the
influence
of
different
temperatures
(between 4 and 29 ºC), using clam valves,
glass and polystyrene as substrate, and
observing the existence of positive
correlations in the case of the factor
temperature and the formation of biofilms,
with maximum association of cells in the
biofilms at 18 ºC and a minimum one at 14
ºC, as well as a significant decrease of
density at temperatures below 5 ºC after 24
hours, followed by a progressive increase of
density 120 hours after the beginning of the
experiment.
The
values
obtained
demonstrated a logarithmic increase of the
number of adherent cells from 1.1 ·105
CFU/cm 2 to 1.4·105 CFU/cm 2 for the clam
valves, 1.7·103 CFU/cm 2
and 1.8·104
CFU/cm 2 for glass and 5.4·103 CFU/cm 2
and 1.0 ·104 CFU/cm 2 for polystyrene
Kelly, 1997. A number of experiments
regarding the capacity of accomplished
certain isolates of Stenotrophomonas
maltophilia to form biofilms in variable
temperature conditions (18 ºC, 32 ºC, 37
ºC) by were realized by Di Bonaventura et
al. in 2007 using different strains. There is
an increase of the quantity of biofilms for
the strains exposed to 32 ºC after one day to
0.680 BPI compared to those exposed to 18
ºC (0.557 BPI) and 37 ºC (0.491 BPI). In
what regards the used strains, the
Total cell bacterhial biofilm growth under temperature variations
80
70
y = 13.74x + 26.657
R2 = 0.9985
3
2
Density 10cells/mm
60
50
40
30
20
10
0
Temp. 6ºC
Temp. 18ºC
Temp. 23ºC
Fig. 9 Total density at different temperature
values
The influence of temperature (5-35 C)
and salinity (up to 20 g/l NaCl) on the
wastewater
purification
process
in
completely mixed and aerated submerged
fixed bed biofilm reactors (SFBBRs) were
studied. C- and N-conversion in SFBBRs
designed according to the DWA (German
Association for Water, Wastewater and
Waste) rules for carbon removal was
investigated for several months on synthetic
wastewater. The DOC degradation rate was
even at, according to the DWA, high
DOC/BOD loading rates not much affected
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Annals of RSCB
Vol. XVII, Issue 1/2012
temperature did not modify significantly
their distribution: 82% of those used formed
biofilms and only 2% did not form them.
One strain formed biofilms only at 18 ºC
and two strains only at 32 ºC. The capacity
to forms biofilms is important even at room
temperature (18 ºC), but the adherence
value is lower. Data in specialized literature
confirm the existence of a growth in
bacterial density depending on the exposure
time of the surfaces to aquatic environment
and the increase of temperature.
According to Chiu et al. in 2006, at 18
ºC, up to 25-30 ºC, there is an optimal
bacterial growth. But temperatures over 35
ºC, 40 ºC and 50 ºC affect the formation of
biofilms because the optimum limits for the
survival of certain bacterial species are
exceeded nMDS ordination comparing
community compositions of bacteria in
biofilms developed in a 3×3 array of
temperature (16, 23 and 30 ◦C) and salinity
(20‰, 27‰and 34‰) treatments in (a)
summer and (b) winter. n=3 for all
treatments. Open squares (30 ◦C and 34‰);
open diamonds (30 ◦C and 27‰); filled
squares (30 ◦C and 20‰); open triangles
(23 ◦C and 34‰); open inverted triangles
(23 ◦C and 27‰); filled triangles (23 ◦C
and 20‰); open circles (16 ◦C and 34‰);
open stars (16 ◦C and 27‰); filled circles
(16 ◦C and 20). A stress value<0.2 indicates
that conclusions can be drawn from the
nMDS ordination with confidence. In
summer (a), group I communities include
those developed at the low salinity (20‰)
at all temperatures, while group II
communities include those developed at
high salinities (34‰ and 27‰) at 23 and 30
◦C. In winter (b), group I communities
include those developed at the low
temperature (16 ◦C) at all salinities, while
group II communities include those
developed at the higher temperatures (23
and 30 ◦C) at all salinities (Chiu, 2006).
The influence of water temperature,
salinity, and pH on the multiplication of
toxigenic Vibrio cholerae serovar 01 cells
and their attachment to live planktonic
crustaceans, copepods, was investigated by
using laboratory microcosms. By increasing
water temperatures up to 30°C, a
pronounced effect on the multiplication of
V. cholerae was demonstrated, as was
attachment of the cells to live copepods.
These were measured by cultivable counts
on agar plates and direct observation by
scanning electron microscopy, respectively.
Of the three salinities examined (5, 10, and
15Mc), maximum growth of V. cholerae
and attachment to copepods occurred at
15%o. An alkaline pH (8.5) was optimal
both for attachment and multiplication of V.
cholerae, as compared with pH 6.5 and 7.5.
It is concluded that conditions affecting
attachment of V. cholerae serovar 01 to live
copepods observed under laboratory
conditions may also occur in the natural
estuarine environment and, thereby, are
significant in the epidemiology of cholera
(Huq, 1984).
This results confirm that bacteria attaché
on the surfaces are influenced by the
salinity and temperatures variations in situ
but also in laboratory conditions
Conclusions
The environmental factors have a very
important role in the biofilm growth, at low
values the cell density were low with values
of 103 cells/mm2.
The variations of temperature and
salinity influence
bacterial
growth,
metabolism and cell attachment on artificial
surfaces due to marine bacteria various
types.
The high values and concentations of
the growth determined higher values
bacterial growth and attachment.
At low temperatures and salinities the
marine bacteria tend to adapt to the
environment and the bacterial cell density
were lower.
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