Effects of Growth Conditions on Hydrophobicity in Marine Obligate ...

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 Crisafi1, Gabriella Gentile1, Anna Volta1, 3, Martina Bonsignore1, Simone Cappello1, * 1

Institute for Coastal Marine Environment (IAMC) – CNR of Messina, Messina, Italy Dep. 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 3 Dep. of Industrial and Mechanical Engineering, Faculty of Engineering, University of Catania, Catania, Italy 2

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

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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 using a UV–visible spectrophotometer (OD600nm) (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

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

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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.

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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

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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