ࡱ;   !"#$%Root Entry F!,CompObjbWordDocumenty>ObjectPool!,!, 4@   FMicrosoft Word 6.0 Document MSWordDocWord.Document.6;  Oh+'0$ H l   D hC:\WINWORD\TEMPLATE\NORMAL.DOT ABSTRACT Alex Kolman Alex Kolman@9Cܥe= e$y>!8p8pp8p8p8p8p888888888=J 9 9 9 9 9 9 9 9,9>;>;>;[;s<==TG>2=p8 9 9 9 9 9= 9p8p8 9 9 9 9 9 9p8 9p8 9,98&8@p8p8p8p8 9,9 9" 9INITIAL MONOLAYER FORMATION IN MARINE BIOFILMS S. Kova*,V.uti, V.Svetlii Center for Marine and Environmental Research, Ruer Bokovi Institute, PO Box 1016, 10 001 Zagreb, Croatia ABSTRACT Studies of biofilm formation are essential for understanding and control of processes in natural aquatic systems and for industrial application. Experiments with expanding mercury electrode/seawater interface offer an approach to studies of initial monolayer in biofilm formation in seawater. Chronoamperometry of dissolved oxygen was used as a measuring technic. Dynamics of film formation is measured as a time which is necessary to reach a full coverage of the electrode by adsorption of dissolved biomolecules and/or adhesion of cells suspended in aqueous electrolyte solution. Phytoplankton organism D.tertiolecta, a free-living bacteria strain and hydrophobic Acinetobacter sp. were chosen as model organisms. Dextran and albumin were chosen as dissolved biomolecules, and triton T-X-100 as nonionic detergent. We have demonstrated direct competition of adsorption of dissolved biomolecules and adhesion of cells in the initial monolayer formation at a freshly exposed surface and a high rate of such processes. KEY WORDS: biofilm, D.tertiolecta, initial monolayers, marine bacteria, mercury electrode INTRODUCTION Zobell in 1943. alerted us to the importance of the attached microbial community in the sea, but until now it is not clear completely how cells sense surfaces nor the relative importance of biochemistry and physico-chemical interaction behind the mechanism of adhesion. A biofilm consists of cells immobilized at a substratum and frequently embedded in an organic polymer matrix of microbial origin (1,2). In marine environment marine biofilms cause loss of performance on ships, increase fuel consumption, and corrosion on a ship surface. It has been accepted generally that the first step in the formation of a biofilm on a clean surface is the adsorption of an organic layer onto the surface from the aqueous milieu (2). Adsorbed organic films affect adsorption of other dissolved compounds as well as microbial colonization and subsequent growth on surfaces (3). In mixed population (bacteria and diatoms) each organism had a chance to attached sequentially. In nature, both population, in theory, would have the opportunity to attach simultaneously (2). Mixtures of dissolved molecules and suspended microbial cells are typical for natural aquatic environment such as seawater. The interaction between the various components in biofilm systems occurs via transport and interfacial transfer processes. For dissolved components it occurs via molecular diffusion and volumetric displacement ( including cell motility ) for particulate components (3). From applied and fundamental points of view, the transport step is important since insufficient transport can be the limiting factor in biofilm formation (4). METHODS AND MATERIALS Adsorption of dissolved organic molecules and adhesion of phytoplankton cells at the mercury electrode/seawater interface, which we use as a model, results in coverage of the electrode with organic material that displaces counter ions and water molecules from the interface (Figure 1.). Chronoamperometry of dissolved oxygen at renewable mercury electrode (5,6) is used as a measuring technique. The experiments were performed under conditions of maximum attraction at positively charged and hydrophobic interface, and enhanced transport to the interface by convective streaming. Phytoplankton organism Dunaliella tertiolecta (8-10 (m) has been chosen as model organism because it forms stable suspensions, has fluid cell wall and adhesion of individual cells to the electrode results in well defined electrical attachment signals. Their duration is 0.06-0.2 s, and amplitudes 0.6-2.2 (A (Figure 2.). Biomolecules dextran and albumin, and nonionic detergent Triton-X-100 were chosen as soluble molecules in concentration range 0.05-500 mg/l. Measurements were performed in seawater and in 0.1 M NaCl. RESULTS AND DISCUSSION Dynamics of film formation is measured as a film formation time (() (Figure 2.) which is necessary to reach a full monolayer coverage of the expanding mercury electrode in the time scale 10-2000 ms. Figure 3. shows dependence of film formation in presence of dissolved molecules (dextran sulphate M=500.000), and in mixture with D.tertiolecta cells. Adsorption of dissolved dextran molecules causes a decrease of the current of streaming maximum of oxygen reduction, of attachment signals frequency of cells (Figure 2.), and a film formation time (() (Figure 3.). We have demonstrated direct competition of adsorption of dissolved biomolecules and adhesion of cells in the initial monolayer formation at a freshly exposed surface and a high rate of such processes. Figure 4. shows dynamics of film formation for monomolecular films of chosen molecules and in mixture with phytoplankton D.tertiolecta. The time needed to reach a full monolayer coverage decreases exponentially with increasing concentration of the molecules and for the same weight concentration depends strongly on the size of the molecules. Formation of the full monolayer can be attained within one second time by convective transport from the aqueous phase at following concentration of molecules: dextran sulphate M=500.000 140 mg/l, dextran M=70.000 80 mg/l, albumin 55 mg/l and triton T-X-100 1 mg/l, and cells: D.tertiolecta 108/l, Acinetobacter sp. (RAG-1) 1.3x1011/l, and a free-living bacterial strain 1.8x1011/l. We also studied dynamics of film formation by hydrophobic bacterium Acinetobacter sp. (RAG-1) (7,8) and a free-living marine bacteria strain in mixtures with D.tertiolecta. Acinetobacter sp. RAG-1 from ATCC collection was obtained by courtesy of Franco Baldi. In mixture of D.teriolecta and RAG-1, frequency of attachment of D.tertiolecta cells decreases with time, which indicated a slow aggregation process in the suspension. Hydrophobic interaction seem to be the main driving force in the aggregation between D.tertiolecta and RAG-1, as well as in their adhesion at the electrode. Free-living bacteria strain does not cause important decreasing of frequency of attachment signals of D.tertiolecta during the measurement, nor with time. REFERENCES: 1 - Characklis W.G., Marshal K.C., Biofilms: A basis for an interdisciplinary approach, In: Biofilms, Charaklis W.G., Marshal K.C., eds., John Wiley & Sons, Inc., New York, 3-15. 2 - Cooksley K.E., Wigglesworth-Cooksley B., 1995. Adhesion of bacteria and diatoms to surfaces in the sea: a review, Aquat. microb. Ecol., 9: 87-96. 3 - Kirchman D.L., Henry D.L., Dexter S.C., 1989. Adsorption of Proteins to Surfaces in Seawater, Mar.Chem., 27: 201-217. 4 - Van der Mei H.C., Meinders J.M., Busscher H.J., 1994. The influence of ionic strength and pH on diffusion of micro-organisms with different structural surface features, Microbiol., 140: 3413-3419. 5 - uti V., Kova S., Tomai J., Svetlii V., 1993. Heterocoalescence between dispersed organic microdroplets and a charged conductive interface, J.Electroanal.Chem., 349: 173-186. 6 - Barradas R.G., Kimmerle F.M., 1966. Effect of highly surface-active compounds on polarographic electrode processes, Part II. Influence on maxima suppression and diffusion currents, J.Electroanal.Chem., 11: 163-170. 7 - Van der Mei H.C., Cowan M.M., Busscher H.J., 1991. Physicochemical and Structural Studies on Acinetobacter calcoaceticus RAG-1 and MR-481 - Two Strains in Hydrophobicity Tests, Curr.Microbiol., 23: 337-341. 8 - Alon R.N., Mirny L., Sussman J.L., Gutnick D.L., 1995. Detection of (/(-hydrolase fold in the cell surface esterases of Acinetobacter species using an analysis of 3D profiles, FEBS Letters, 371: 231-235. FIGURE CAPTIONS Figure 1. Schematic presentation of interaction of a cell and a biomolecule with positively charged mercury electrode in aqueous solution and the current-time transient (attachment signal). Figure 2. Current-time curves of oxygen reduction in suspension of D.tertiolecta (5x107/l) alone (curve 1) and in mixture with 15 mg/l dextran sulphate M=500.000 (curve 2) in 0.1 M NaCl at -400 mV. The spikes are attachment signals of individual cells. Figure 3. Dependence of film formation time (() on concentration of dextran sulphate M=500.000 alone (curve 1) and in a mixture with D.tertiolecta 5x107/l (curve 2). Figure 4. Dynamics of film formation. Film formation time (() of monomolecular films of dissolved molecules (dextran sulphate M=500.000, dextran M=70.000, albumin and triton) and in mixture with phytoplankton cells D.tertiolecta (108/l. .ASummaryInformation(S@ȝ0ڼ@,@p=,DMicrosoft Word 6.062ࡱ; .089:$2aq56=Jkx\jmnr fsu wop9 T """"N#O#s#t#####'$($$$$$$$$$JJbJahJtJmVch^UZ$/u/0OX3,: H O x(l|E {!!I"F##$$p#-p#-p#-p#-p#-p#-p#-p#-p#-p#-p#-p#-p#-p#-p#-p#-p#-p#-p# p#-p#-p#s p#-p#-p#-p#-p#-p#-p#-p#-p#-p#-p#-p#p#-p#-p#-p#vp#p#-,$$K @ Normal ]a c"A@"Default Paragraph Font! $!!!!! L | !3$/$$" Alex KolmanC:\SOLVEG\CIESM.DOC U , $U5 &.U[ "A.U} c.U .U ".U .U .U  .U  /CAPF%AutoExecAutoOpenFileOpenF%OFileSaveF%S AutoClose FileCloseF%C FileSaveAsF%SA ToolsMacroF%35 FileTemplates CAPAUTOEXECAUTOOPENFILEOPENFILESAVE AUTOCLOSE FILECLOSE FILESAVEAS TOOLSMACRO FILETEMPLATES@HP LaserJet 4LLPT1:HPPCL5EHP LaserJet 4L D  ɓ3, HP LaserJet 4L D  ɓ3,  JTimes New Roman Symbol &ArialTimes New Roman CE"h&d(>$2ABSTRACT Alex Kolman Alex Kolmanࡱ;