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International Journal of Microbiology and Application 2015; 2(2): 50-55
Published online March 20, 2015 (http://www.openscienceonline.com/journal/ijma)
Effects of Growth Conditions on Hydrophobicity in Marine Obligate Hydrocarbonoclastic Bacteria
Santina Santisi1, 2
, Maria Genovese1, Francesca Crisafi
1, Gabriella Gentile
1, Anna Volta
1, 3,
Martina Bonsignore1, Simone Cappello
1, *
1Institute for Coastal Marine Environment (IAMC) – CNR of Messina, Messina, Italy 2Dep. of Biological and Environmental Sciences, Faculty of MM. FF. NN. University of Messina, Ph.D School in “Biology and Cellular
Biotechnology” of University of Messina, Messina, Italy 3Dep. of Industrial and Mechanical Engineering, Faculty of Engineering, University of Catania, Catania, Italy
Email address
[email protected] (S. Santisi), [email protected] (M. Genovese), [email protected] (F. Crisafi),
[email protected] (G. Gentile), [email protected] (A. Volta), [email protected] (M. Bonsignore),
[email protected] (S. Cappello)
To cite this article Santina Santisi, Maria Genovese, Francesca Crisafi, Gabriella Gentile, Anna Volta, Martina Bonsignore, Simone Cappello. Effects of Growth
Conditions on Hydrophobicity in Marine Obligate Hydrocarbonoclastic Bacteria. International Journal of Microbiology and Application.
Vol. 2, No. 2, 2015, pp. 50-55.
Abstract
The aims of this work is study the influence of different growth conditions (different concentration of carbon source) on the
variation of cellular hydrophobicity (capacity to adhere at polystyrene) of two obligate hydrocarbonoclastic bacteria,
Alcanivorax borkumensis SK2 and Thalassolituus oleivorans MIL-1. The strains were inoculated, separately, in ONR7a mineral
medium whit different concentration of sodium acetate. During all period of experimentation (10 days) measures of cellular
abundance and cellular hydrophobicity (capacity to adhere at polystyrene) were carried out. Data obtained revealed as addition of
carbon source and growth status are important factor in the regulation of adhesion to surface and as in these process Alcanivorax
present an advantage respect Thalassolituus in all experimental condition. The understanding of capacity to adhesion of these
bacteria for utilization of hydrophobic compounds is fundamental for their potential use in the mitigation of oil spills
phenomenon and the importance of these preliminary data resides mainly in biotechnological applications, especially in
reference to application of this bacteria in bioremediation strategies.
Keywords
Adhesion, Alcanivorax, Bioremediation, Polystyrene, Thalassolituus
1. Introduction
An important fraction of marine microbial community is
characterized from “Obligate HydrocarbonoClastic Bacteria”
(OHCB), ([1]). The group of OHCB is composed from
indigenous bacteria of marine ecosystems ([1], [2], [3]) able to
utilize hydrocarbons as exclusive source of carbon and energy
([4], [5]).
Presence of these bacteria in marine environment is directly
dependent on presence of hydrocarbons and/or crude oil that
determine, in specific condition, a change of microbial
community, whit the selection of these bacteria able to tolerate
and/or these chemicals.
Different study ([5], [6], [7]) showed that members of the
genera Alcanivorax and Thalassolituus are of the most
important microbes involved in removing of crude oil from
marine environment.
Alcanivorax sp. is a cosmopolitan marine bacterium ([1],
[8]) that exhibit a unique oligotrophic physiology with a
specialized metabolism adapted to the degradation of aliphatic
hydrocarbons ([9], [10]) even if not effectively involved in
degradation of aromatic compounds. However, members of
the group of Alcanivorax are indeed able to use some organic
substrates like n-paraffins, alkylic groups pertaining to
n-alkylbenzene and n-alkylcycloalkane as only carbon source
([7]).
Thalassolituus sp. is a chemoorganoheterotrophic
bacterium. Microbes related to this genus inhabiting both
marine and terrestrial environments subsurface caves and
ground waters ([1]) and were the dominant alkane degraders
51 Santina Santisi et al.: Effects of Growth Conditions on Hydrophobicity in Marine Obligate Hydrocarbonoclastic Bacteria
in n-alkane systems ([11]).
The importance of these bacteria resides mainly in
biotechnological applications, especially in reference to
bioremediation strategies. The problem in both
bioremediation and bioprocessing is that oil and water are
immiscible, but microbes grow only in an aqueous
environment. Consequently, the interface between the oil and
the water is of supreme importance for this technology.
Microbial attachment to hydrophobic interfaces has been an
area of interest for over 80 years, but there are relatively few
studies about the controlled use of microbial adhesion as a
means to promote reactions of oil.
Attachment to hydrophobic surfaces is a common and
natural strategy used by microorganisms to overcome
limitations of solubility of hydrocarbons.
Different studies ([12]) have demonstrated that
environmental factor (temperature, pH) ([13]) growth
conditions (nutrients concentration) and status of bacteria
(shape, dimension, metabolic status) were factors influencing
the process of adhesion and may determine variation in
hydrophobicity of the bacterial cell surface (CSH).
The aims of this work is study the influence of different
growth conditions (different concentration of carbon source)
on the variation of cellular hydrophobicity (capacity to adhere
at polystyrene) of two obligate hydrocarbonoclastic bacteria,
Alcanivorax borkumensis SK2 ([2]) and Thalassolituus
oleivorans MIL-1 ([14]).
2. Material and Methods
2.1. Strain in Study
A strain of Alcanivorax borkumensis SK2T ([2]) and a strain
of Thalassolituus oleivorans MIL-1T ([14]) were used in all
the experiments.
Strains used in this study belong to a collection of
hydrocarbon-degrading bacteria hold at IAMC-CNR of
Messina.
2.2. Growth and Experimental Set-up
Started cultures were carried out by inoculating one loop of
microbial cells into 10 mL of ONR7a mineral medium ([15])
containing 0.1% (w/v) of sterile of sodium acetate
(CH3COONa, Sigma-Aldrich, Italia). Culture were grown in a
rotary shaker (New Brunswick C24KC, Edison NJ, USA; 150
rpm) at 25±1°C for 5 days.
Mid-exponential-phase grown cells were harvested by
centrifugation at 11.250 ×g for 10 min, washed twice with
sterile medium and inoculated into different 250 ml sterile
Erlenmeyer flasks containing, each,100 ml of ONR7a medium
supplemented with 0.1% (w/v) of sodium acetate.
Mid-exponential-phase grown cells were harvested, a
second time as previously described, and inoculated at a final
concentration of 0.1 of optical density (OD600nm) in sterile
ONR7a mineral medium.
Four different series of experimentations have been
performed. In the first three experiment bacterial cultures
were carried out in ONR7a mineral medium added whit 0.6,
0.3 and 0.1% of sodium acetate, respectively. In negative
control, bacteria were cultivated in the same medium without
any source of carbon and energy.
The cultures were incubated (for 10 days) in a rotary shaker
(New Brunswick C24KC, Edison NJ, USA; 150 rpm) at
25±1°C.
All experiments were carried out in triplicate.
2.3. Biological Measurement
Every 24 hours, for a period of 10 days, sub-aliquots of
bacterial cultures were collected to measure total bacterial
abundance and variation in hydrophobicity (measured as
variation to adhesion at polystyrene). All the experiments
were triplicate.
2.4. Biomass Variations
The growth (biomass variations) of the cultures in study
was routinely assessed indirectly by measuring the turbidity
(OD600nm) using a UV–visible spectrophotometer
(Biophotomer Eppendorf).
2.5. Percentage of Hydrophobicity (%, HP)
For the assay of the adhesion to polystyrene a sub-aliquot of
bacterial culture has been directly withdrawn from the flask
daily.
The cells were washed twice in a phosphate-buffered saline
(PBS) buffer (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4,
1.4 mM KH2PO4, pH 7.4) a final concentration of 0.3
(OD600nm). The cells so suspended (10 ml) were put in a
polystyrene Petri plate (60 mm) and incubated for 30 min at
25±1°C without being shaken in order to study the initial
adhesion process.
The capacity of adhesion to polystyrene was analyzed, as
previously described ([13], [16]) well as the percentage of
hydrophobicity (% HP).
Cell surface hydrophobicity was determined by partitioning
the cell suspensions into the polystyrene plate and aqueous
phases after incubation time. The percentage of
hydrophobicity (%HP) of experimental bacterial suspension
was calculated using the following equation, adapted to our
purpose
% HP = (OD init - ODexp) × 100 / ODinit
where ODinit stood for the optical density of the suspension
before incubation in the polystyrene plate, and ODexp was the
optical density of the bacterial suspension after incubation.
3. Results
The quantitative estimation of the adhesion to polystyrene
measured as percentage of hydrophobicity (%HP) of A.
borkumensis SK2, on the various concentration of sodium
acetate as only source of carbon and energy, is shown in
Figure 1.
International Journal of Microbiology and Application 2015; 2(2): 50-55 52
Fig. 1. Dynamic of adhesion (percentage of hydrophobicity, %HP; open
squares) and bacterial abundance (filled squares) of Alcanivorax
borkumensis SK2 during growth in ONR7a medium whit 0.6% (a), 0.3% (b),
0.1% of sodium acetate (c) and without carbon and energy source (d).
During cultivation in ONR7a mineral medium whit 0.6% of
sodium acetate, Alcanivorax showed, in the first six days, an
exponential growth, passing from a value of optical density of
0.1 to 0.85 (OD600nm). After the first week of experimentation,
the cellular abundance remaining constant for all period in
study (10 days). During the cultivation, the value of %HP
showed an increment during the first 48 hrs, passing from
value of %HP = 50 (time zero) to %HP = 68 and remaining
constant just the 5th
day of experimentation. After the 6th
day
of cultivation a decrement of adhesion to polystyrene is
observed, whit values that passing from a value of %HP = 62
(T5) to %HP = 38 (T8) and remaining constant for all period of
study (Figure 1a).
Also during cultivation in ONR7a medium whit 0.3% of
sodium acetate A. borkumensis SK2 showed, for the first five
days, an exponential growth whit values that passing from a
0.1 to 0.7 (OD600nm). Successively, at stationary phase, the 1a).
decrement whit values starting from %HP = 62 (T2) until reach,
at the 6th
day of analysis, %HP value of 26. This value
remaining constant for all period of study (Figure 1b). On
ONR7a medium whit 0.1% of sodium acetate and without this
carbon source (Fig. 1c and 1d), Alcanivorax present, for all
experimental time, a stationary phase growth whit means
values of optical density of 0.25 (0.1% of sodium acetate) and
0.1 (no carbon source). In both experimentations a general
decrement, in the first two days, of %HP is observed, whit
means values of %HP = 14 that remaining constant for all
time.
During growth whit 0.6% of sodium acetate T. oleivorans
MIL-1 shown a long lag phase whit values of optical density
around 0.1 (OD600nm) just the 5th
day of experimentation. After
the 5th
day, a rapid exponential growth was observed whit an
increment of bacterial abundance that passing from a value of
0.1 (T5) to 0.6 (OD600nm) just at the end of experiment. During
the cultivation the value of %HP presented a decrement during
the first 3 day, passing from value of %HP = 50 (time zero)
to %HP = 12 (T3) and remaining constant just the 5th
day of
experimentation. After the sixth day of cultivation an
increment of adhesion to polystyrene is observed, whit values
that passing from a value of %HP = 12 (T5) to %HP = 60 (T7)
and remaining constant for all period of study (Fig. 2a).
Also during cultivation on ONR7a medium added whit the
0.3% of sodium acetate Thalassolituus oleivorans MIL-1
shown a long lag phase whit values around 0.1 (OD600nm) just
the 3th
day of experimentation (Fig. 2b); after the 3th
day of
experimentation an exponential growth is showed whit values
of cellular abundance that passing from 0.1 (T3) to 0.7 (T10).
Curve of percentage of hydrophobicity present a trend whit
value of %HP = 50 (time zero), 12 (T2 and T3), 60 (T5) and 30
(T10).
Similar to Alcanivorax, also Thalassolituus MIL-1, present
during cultivation in ONR7a medium whit 0.1% of sodium
acetate and without carbon source a induction of a stationary
phase whit means values of optical density (OD600nm),
respectively, of 0.17 and 0.1. In parallel, the values of %HP
presented a general decrement during the first 48h, passing
from a value of %HP = 50 in time zero to %HP = 14 and
remaining constant for all the period of study (Fig. 2c and 2d).
bacteria abundance remaining constant whit values around
0.75 (OD600nm) just the end of experimental period. After a
initial increment (%HP = 50 to 62 in the first 2 days) the of
percentage of hydrophobicity showed a progressive.
53 Santina Santisi et al.: Effects of Growth Conditions on Hydrophobicity in Marine Obligate Hydrocarbonoclastic Bacteria
Fig. 2. Effect of different concentration of carbon source on adhesion, as
percentage of hydrophobicity (% HP) of Thalassolituus oleivorans MIL-1.
Optical abundance (filled squares) and percentage of hydrophobicity (open
squared) during growth in ONR7a medium 0.6% (a), 0.3% (b), 0.1% of
sodium acetate (c) and without carbon and energy source (d).
4. Discussion
In this preliminary study, for the first time, we have
analyzed the effect of different concentrations of substrate
(sodium acetate) on hydrophobicity in two marine obligate
hydrocarbonoclastic bacteria, Alcanivorax borkumensis SK2
and Thalassolituus oleivorans MIL-1.
Among the hydrocarbon-utilizing bacteria, A. borkumensis
and T. oleivorans are two of the most frequently isolated from
hydrocarbon-contaminated environments.
Marine OHCB occupy a special trophic niche among
marine heterotrophic bacteria participating in the global
carbon cycle, as they mediate degradation of chemically stable
saturated and aromatic hydrocarbon species that are not
substrates for most bacteria ([1]). However, the
eco-physiology of these bacteria has not been studied
extensively for different motivations as discovery relatively
recent, difficult to isolation and maintaining of these
organisms in pure culture, etc.
The analysis of the variation of bacterial adhesion during
optimal growth conditions is a fundamental data in order to
understanding of capacity to adhesion of these bacteria and
utilization of hydrophobic compounds is fundamental for their
potential use for the mitigation of oil spills has been discussed
elsewhere.
Bacteria and other microorganisms have a natural tendency
to adhere to surfaces as a survival mechanism and bacterial
colonization of solid surfaces has been described as a basic
and natural bacterial stratagem in a wide variety of
environment ([12], [17], [18]).
As showed, during cultivation in ONR7a medium whit 0.6
and 0.3% of sodium acetate, Alcanivorax present, in the first
days, a exponential growth during which is possible observe a
increment of cellular hydrophobicity that decrease when
bacteria community enter in a stationary phase. Moreover it is
very important to remind that this growth-phase transition is
controlled by a genetic regulatory network integrating various
environmental signals ([19]) characterized by a reduction of
proteic synthesis ([20]) that favours the specific protein
synthesis ([21]) to disadvantage of the synthesis of proteins
and structures naturally implied in the processes of bacterial
adhesion.
Though genes putatively coding for transcriptional
regulators of various families in A. borkumensis SK2 were
identified and were characterized nine specifies sigma
involved in the ability of this organism to build adaptive
responses to environmental signals ([10]), little is known
about effective regulation of gene for cell surface
polysaccharide and for bifunctional protein for LPS
biosynthesis, although these have been identified ([10]). The reduction of cellular hydrophobicity is more visible
cultivation at 0.1% of sodium acetate and without source of
carbon and energy. The concentration of 0.1% of sodium
acetate is not sufficient to support bacterial growth and after
complete utilisation of this substrate the population enter in a
stationary phase presumably associate at a carbon starvation.
In this case, the drastic decrement of cellular hydrophobicity,
can be justified both from reduction of proteic sysntesis,
characteristic of carbon starvation, both through the
dimensional reduction that the bacterial cells endure in that
condition ([20]). The morphologic variation can determine a
International Journal of Microbiology and Application 2015; 2(2): 50-55 54
change in the contact angle that the bacteria have with the
surface of adhesion with consequent variation in the cellular
adhesion ([22]).
Also during cultivation of Thalassolituus is possible observe
a correlation between the status of growth and the cellular
hydrophobicity. During growth at 0.6 and 0.3% of sodium
acetate the growth of this bacterium is slow. The concentration
of sodium acetate showed a inhibitory effect in growth of
Thalassolittus indeed after a time lag of about 6 days (0.6% of
sodium acetate) and about 3 days (0.3% of sodium acetate); this
inhibition is also observed in adhesion to polystyrene that
values decrease rapidly. After this period of adaptation, the
bacterial growth incoming and in parallel also the values
of %HP increase. During cultivation in 0.1% of sodium acetate
and without carbon source the behaviour of Thalassolituus is
similar to that of Alcanivorax whit a drastic reduction on
hydrophobicity during the status of stationary growth.
The preliminary data obtained showed a correlation
between the physiological status (identified from curve of
bacterial abundance) and cellular hydrophobicity ([13], [15],
[23]). The maximum and minimum values of cellular
hydrophobicity were obtained during cultivation of
Alcanivorax borkumensis SK2 and Thalassolituus oleivorans
MIL-1 are similar although depending from bacterial status.
The importance of these preliminary data resides mainly in
biotechnological applications, especially in reference to
application of this bacteria in bioremediation strategies.
Acknowledgements
This work was supported by grants of National Counsel of
Research (CNR) of Italy and by: i) Italian Project
PRIN2010-2011 “System Biology”; ii) National Operative
Project PON R&C 2007-2013 "STI-TAM"; iii) National
Operative Project PON R&C 2007-2013 "SEA-PORT"; iv)
PNRA2013 "STRANgE"; v) European Project KILL-SPILL
(KILL-SPILL-FP7-KBBE-2013.3.4-01).
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