INTERNATIONAL BIODETERIORATION& BIODEGRADATIONInternational Biodeterioration & Biodegradation 41 (1998) 93-100

Detachment studies on microfouling in natural biofilms on substrata with different surface tensionsKlaus BeckerBiolab Research Institute, Kieler Strasse 51, D-24594, Hohenwestedt, Germany

Received 1 April 1997; accepted 1 December 1997

Abstractstrength of bacteria, diatoms, choanoflagellates and four genera of ciliates (Corthuniu, Vorticella, Zoothamnion, biofilms was studied on artificial substrata by exposing them to laminar flow in a radial flow chamber. Seven artificial materials (PTFE: polytetrafluorethylene, FEP: fluorethylenepropylene, PFA: polytetrafluorethylene/pertluorcompoundscopolymer, ETFE: ethylenetetrafluorethylene, HC: acetalpolymer, PC: polycarbonate, and glass) with surface tensions between 19 Attachment

Ephelota) in natural

and 64SmNm- were used. Test panels were immersed between 3 hours and 8 days in the sea to grow biofilms in a natural environment. Attachment strength was studied by exposing the biofilms to 4 different laminar flow pressure intervals (between 3.9 and 16.9 Nmm2) in a radial flow chamber. The results showed that a minimum bioadhesive range between 20 and 25 mNm- exists for bacteria and diatoms during early colonization periods (up to 2 days). However, bacteria and diatoms possess compensation mechanisms to overcome weaker attachment strength on these materials. Protozoa were studied after 5 and 8 days only. Their attachment strength did not improve during that interval. Significant differences between the materials were observed for Corthunia, Vorticellu, and Ephelota. Highest detachment rates were usually recorded on materials between 20 and 25 mN m-. However, even after exposure to a flow pressure of 16.9Nm-* an average of more than 50% of the protozoans remained on each material. The present results indicate that although attachment strength of microfouling is affected by surface tension to some extent, within a few days most investigated microfouling groups resisted considerably strong flow pressure on every material tested. Therefore, surface tension cannot be considered a powerful long-term device to prevent microfouling. 0 1998 Elsevier Science Ltd. All rights reserved.Keywords: Microfouling; Bacteria; Diatoms; Protozoa; Attachment strength; Laminar flow pressure; Flow chamber; Biofilm.

1. Introduction Colonization of surfaces by bacteria, diatoms, protozoa and fungi (microfouling) leads to the establishment of biofilms. Biofilms are ubiquitous phenomena in aquatic environments. Biofilms are often a nuisance to technical applications, e.g., ship hulls, heat exchangers, pipes (Characklis and Cooksey, 1983; Lappin-Scott and Costerton, 1989). Therefore, many studies focused on the prevention of biofilm formation. Surface tension as a potential non-toxic antifouling measure has been paid some attention for several years because it influences attachment strength of organisms (Baier, 1973; Dexter, 1976, 1979; Fletcher and Loeb, 1979; Absolom et al., 1983; van Pelt et al., 1985; Meyer et al., 1988; Lindner, 1992; Hodson and Burke, 1994; Becker et al., 1997; Waterman et al., 1997). Several studies showed that densities of bacteria and diatoms are minimal on substrates with surface tensions between 20 and 25 mN m- (Baier, 1973; Dexter, 1976,1979; Characklis and Cooksey, 1983). Other studies found linear colonization patterns withSO964-8305/98/$19.00 0 1998 Elsevier Science Ltd. All rights reserved. PII: SO964C3305(98)00004-3

decreasing or increasing surface tension (Fletcher and Loeb, 1979; Absolom et al., 1983; van Pelt et al., 1985). However, varying colonization density on different substrates may level off with exposure time (Becker et al., 1997). The strength and mechanisms of attachment of bacteria (Fletcher and Loeb, 1979; Duddridge et al., 1982; Rittle et al., 1990), diatoms (Pyne et al., 1984; Woods and Fletcher, 1991), marine and freshwater fungi (Hyde et al., 1989; Read et al., 1991) to surfaces has been studied by several authors. These studies showed that bacteria, diatoms and fungi improve their attachment strength considerably within a few days. Very few studies considered attachment mechanisms of sessile protozoa (but see Brown et al., 1984) although they form a major part of biofilms (Gerchakov et al., 1976; Marszalek et al., 1979). So far, detachment studies on microfouling organisms have been conducted on laboratory cultures considering only one or two kinds of materials (e.g., glass, steel). The coexistence of different kinds of fouling organisms in natural, mixed biofilms (Cowan et al., 1991) and

94

K. Becker/International Biodeterioration & Biodegradation 41 (1998) 93-100

the ambient flow regime (Costerton et al., 1987) may influence biofilm development leading to strong adhesion of biofilm inhabitants. Therefore, this study was designed to show the influence of substratum surface tension on attachment strength of microfouling organisms (bacteria, diatoms, protozoa) in naturally grown biofilms. Do these organisms attach more strongly or more weakly to certain surfaces? To which extent are microfouling organisms able to compensate weak adhesion on certain surfaces? 2. Materials and methods 2.1. Description and operation of thejow chamber Attachment strength of bacteria, diatoms and protozoa in naturally grown biofilms was studied by using a radial flow chamber (RFC) which was originally designed by Fowler and McKay (1980). Its design, operational procedure and the calculation of the laminar flow pressure have been described by Fowler and McKay (1980). The RFC consists of two parallel disks with narrow spacing (Fig. 1). A spacing of 1 mm was used although some protozoa (Suctoria) were more than 1 mm long. However, it was necessary to keep the spacing as narrow as possible to obtain a high flow pressure. The spacing (h) between the upper and lower disk is an important variable in determining the flow pressure (P,): P,-=3Q@rh2; Q= flow rate, p = viscosity of the fluid, r = distance from the center of the disk). The maximum flow rate which could be obtained was 20 1min- . Water was pumped at a constant rate through the central inlet of the lower disk. The RFC was modified to insert test panels on which the

natural biofilms were grown. Four rows of circular hollows (1.5 cm diameter, 3 mm deep) were drilled in the upper disk (at r= 1.8, 3.8, 5.8, 7.8 cm). They were for the insertion of the test panels which were embedded in plasticine to avoid as far as possible laminar flow disruption. However, disruption of the laminar flow will eventually have occurred to some extent due to edgeeffects at the plasticine-panel border and the biofilm profile. Calculation of the flow pressure for turbulent flow (Reynolds number: > 2000) requires another formula. However, Fowler and McKay (1980) have never reported such values, even close to the central inlet. Therefore, the calculation for laminar flow was used. Flow pressure at the four radii using 20 1min- flow rate was accordingly: 3.9, 5.2, 8.0 and 16.9Nm-2. Samples were exposed for 10 minutes at each radius. 2.2. Preparation and surface tension determination of thesubstrates

Test panels of seven artificial substrata were used. All materials were inert, transparent or white and smooth. Except for polycarbonate (PC: 6 = 1.2 g cmp3), which was provided by the Richter Company (Kiel, FRG) and glass (6 = 2.5 g cmw3), one acetalpolymer (Hostaform C, HC: 6 = 1.41 g cme3) and four types of fluorpolymers from the Hoechst Company (Frankfurt, FRG) were employed: polytetrafluorethylene (PTFE: 6 = 2.16 gcmp3, (4) PTFE/ perlluor-copolymer (PFA: 6 = 2.17 g crn3), fluorethylenepropylene (FEP: S = 2.17 g cmp3), ethylenetetrafluorethylene (ETFE: 6 = 1.75 gcme3). Panels of PTFE, PC and glass were 2 to 3mm thick. FEP, PFA, ETFE and HC were less than 1 mm thick and were stuck on PC or

1Side view of the flow chamber Upper diskI I I

Hollows for test panelsI I I I \ I I

Outlet

Lower disk

Spacing (h)

Central inlet

Fig. 1. Schematic

view of the modified

version of the radial flow chamber

(RFC).

K. Becker/International Biodeterioration & Biodegradation 41 (1998) 93-100

95

glass with silicon glue. The panels were cleaned and surface tension was determined by contact angle measurements with two liquids (bidistilled water and analytical grade glycerin) as described by Becker and Wahl(l991). According to computer tables (Neumann et al., 1980), which are based on the equation of state approach, surface tension was calculated from contact angle data. Surface tension measurements in this study are based on the surface free energy concept. There are different concepts referring to surface tension. The present study uses the concept of surface free energy at interfaces (Y,,s= solid, v=vapor; see Neumann et al., 1974) like Absolom et al. (1983), Fletcher and Pringle (1985) and van Pelt et al. (1985). Another concept which will be of some relevance in that study is the concept of critical surface tension (y. see Zisman, 1964). Critical surface tension is an empirical determined parameter that is related to the surface free energy of a substratum. Dexter (1979), Baier (1973), Meyer et al. (1988) used the concept of critical surface tension. Nevertheless, both concepts may yield similar results because the values of the two terms approximate if the substrata are apolar to some extent (Rabel, 1971). 2.3. Sampling procedure and determination of microfoulingdensity

selected fields at 100 x magnification (field area: 7225pm). It was not possible to count the same panel before and after RFC operation due to the oil immersion technique. Three replicates, untreated panels and those exposed to laminar flow, were counted. Data from each treatment were compared to evaluate the original colonization and the remaining cells after exposure to flow. Diatom and protozoan samples were stained with a modified Gurrs stain, using Alcian blue and Ziehl Neelsen staining solution (Jackson and Jones, 1991). All materials were transparent enough to count the diatoms and protozoa directly using a light microscope (Olympus BH-2). The number of organisms was estimated on each sample by counts of diatoms, choanoflagellates (40 x magnification), peritricha (20 x magnification) and suctoria (10 x magnification). A total of 15 randomly selected fields in the center of each panel was counted before and after the flow chamber operation. Diatoms were counted on three, protozoa on two replicates. Statistical evaluation of the data which were obtained before and after the detachment experiments, was undertaken by using non-parametric statistics (Mann-Whitney test: Utest, Kruskal-Wallis test: H-test). 3. Results3.1. Surface tension of the substrata

Test panels (l*l cm) of each material were immersed in the sea (Gulf of Thailand) to obtain biofilms. All panels for one exposure interval were stuck together on a polyamide-plate. They were randomly distributed and the panels were at the same level with no gaps between. Samples were attached to a rope and fixed between two concrete poles close to the beach at Laem Than (Chonburi province). They were immersed at a depth of 1SO m at low tide (4.50m at high tide). Exposure experiments took place between January and September 1992. Samples for detachment studies were collected after 3 and 6 hours, 1, 2, 5 and 8 days. After more than 8 days many macroorganisms were present on the samples, whose presence may affect the detachment studies. Samples were preserved in a 4% formalin using artificial seawater. Before staining, every panel was rinsed under a pressure of 1Ocm distilled water to remove unattached particles and organisms. It was necessary to preserve biofilms because the organims had to be stained for counting. Formaline alters the structure of proteins. That may have some effect on attachment structures of organisms but a given strucure is fixed in its present state and all samples were treated equally. Thus, despite some biofilm modification due to preservation reliable results of detachment studies can be expected. Bacteria were stained with acridine orange (1: 10000 solution) and counted (oil immersion) by using an Olympus BH-2 epifluorescence microscope. The total number on the panels was determined by counts on 20 randomly

Surface tension data resulting from contact angle measurements showed that PTFE possessed the lowest (19 mN m-), and glass the highest surface tension (64.5 mNm_). FEP (20.5mNm-) and PFA (22 mN m-) belonged to the proposed minimum bioadhesive range (20 to 25 mN m-). Surface tension of ETFE was slightly above this range (25.5 mN m-). HC and PC were within the proposed bioadhesive range between 30 and 40 mN m- (Baier, 1973; Dexter, 1979). 3.2. Colonization of the substrates Bacteria and diatoms were the first organisms to settle. After 3 hours, bacterial densities ranged from 0.10 *lo3 (FEP) to 0.34 *lo3 (HC) cells per mm2 (Table 1). Between 18.6 *lo3 (FEP) and 28.4 *lo3 cells per mm2 were counted after 8 days. Materials within the 20 to 25 mN m- range were significantly (U-test: p < 0.05) less densely colonized than the other materials after each sampling interval. Diatom numbers ranged from 0.08 *lo3 cmp2 (FEP) to 0.66 *103cme2 (HC) after 3 hours (Table 2). Between 80.3 *103cme2 (ETFE) and 186.04 *103cmm2 (HC) cells were counted after 8 days. In- or decreasing densities with increasing surface tension were not observed. After 3 h, 5 d and 8 d, diatoms appeared at least on one material within the minimum range (FEP, PFA, ETFE) in significantly (U-test: p < 0.05) lower numbers than on other

96

K. Becker/International Biodeterioration & Biodegradation 41 (1998) 93-100 Table 1 Average densities (X = Number Material Exposure 3 hours X PTFE FEP PFA ETFE HC PC Glass 0.22 0.10 0.23 0.22 0.34 0.26 0.25 (S.E.) (0.02) (0.02) (0.03) (0.02) (0.02) (0.03) (0.03) time 6 hours X 0.56 0.41 0.33 0.33 0.37 0.49 0.33 (SE.) (0.04) (0.03) (0.02) (0.03) (0.02) (0.02) (0.02) 1 day X 2.56 2.09 2.09 2.25 3.25 3.79 2.56 (S.E.) (0.16) (0.16) (0.16) (0.40) (0.19) (0.24) (0.23) 2 days X 4.34 3.38 4.06 4.87 5.04 4.79 4.87 (S.E.) (0.34) (0.23) (0.22) (0.28) (0.27) (0.29) (0.35) 5 days X 24.16 13.58 13.95 13.99 20.66 20.12 15.35 (S.E.) (3.86) (2.19) (2.23) (2.22) (3.32) (3.21) (2.48) 8 days X 28.35 18.55 24.48 22.51 27.61 27.53 26.98 (S.E.) (1.31) (1.12) (1.39) (1.17) (1.16) (1.20) (1.81)

x lo3 per mm) of bacteria

on the substrata

(SE. = Standard

Error)

Table 2 Averge densities (X= Material Exposure 3 hours X PTFE FEP PFA ETFE HC PC Glass 0.30 0.08 0.17 0.03 0.66 0.22 0.17

Number time

x 10 per cm*) of diatoms

on the substrata

(SE.

= Standard

Error)

6 hours (SE.) (0.09) (0.04) (0.09) (0.03) (0.21) (0.08) (0.06) X 2.03 1.86 2.27 1.52 1.62 1.33 1.97 (S.E.) (0.21) (0.24) (0.20) (0.16) (0.19) (0.16) (0.28)

1 day X 7.76 0.90 1.52 0.84 2.93 6.23 1.93 (S.E.) (0.80) (0.12) (0.29) (0.15) (0.39) (0.67) (0.21)

2 days X 3.72 4.41 4.29 3.01 2.13 3.20 2.08 (S.E.) (0.29) (0.41) (0.34) (0.29) (0.23) (0.28) (0.22)

15 days X 17.96 15.19 17.97 26.54 21.93 20.20 21.22 (S.E.) (1.03) (1.12) (1.16) (2.02) (2.09) (1.60) (1.58)

8 days X (SE.)

123.7 (10.79) 95.73 (13.79) 97.32 (12.11) 80.27(11.07) 186.0 (22.20) 157.1 (18.94) 156.2 (28.65)

materials (PTFE, HC, PC, glass). Protozoan densities were too low for evaluation until the 5-days-interval. Thus, only samples which were collected after 5 and 8 days were considered (Table 3). Choanoflagellates were the most abundant protozoans with densities of up to

1288 per 10 mm2 (PTFE, 5 d). Significant differences (Utest: ~~0.05) between the materials which fit Dexters model (1979) were observed after 5 days. After 8 days, densities of choanoflagellates showed no significant differences between the materials.

Table 3 Average densities (X= Species of protozoa Time (days)

Specimens

or colonies

per 10mm) of the protozoa

on the substrata

(S.E. = Standard

Error)

Material PFTE X (S.E.) FEP X 916 478 119 13 371 230 114 89 5.5 0.4 (S.E.) (85) (42) (11) (9) (27) (19) (7) (8) (1.1) (0.1) PFA X 611 1029 47 10 339 256 139 83 7.6 1.4 (SE.) (49) (75) (7) (2) (24) (20) (14) (8) (2.1) (0.9) EFTE X 1242 425 93 9 247 325 139 94 4.4 0.7 (S.E.) (115) (44) (17) (2) (15) (26) (11) (7) (1.5) (0.2) HC X 1069 1170 33 7 158 133 113 77 4.7 0.5 (SE.) (83) (99) (4) (2) (16) (17) (7) (4) (0.9) (0.1) PC X 791 1004 27 9 147 201 53 127 6.5 8.4 (S.E.) (73) (86) (4) (2) (17) (21) (7) (8) (2.1) (1.9) Glass X 738 1016 41 7 233 327 112 79 13.3 0.8 (S.E.)

Choanoflagellata Corthunia Vorticella Zoothamnium Ephelota

5d 8d 5d 8d 5d 8d 5d 8d 5d 8d

1288 1036 32 14 167 295 138 90 8.0 0.4

(110)(129) (5) (3) (17) (28) (9) (7) (4.0) (0.2)

(50) (100)(5) (2) (20) (24) (6) (7) (4.1) (0.4)

K. Becker/International Biodeterioration & Biodegradation 41 (1998) 93-100

97

Among peritrichan ciliates, Vorticella occurred in greatest numbers while Corthunia appeared in lowest densities. Densities of Ephelota (Suctoria) remained below 10 per 10 mm2. The abundance of Corthunia, Zoothamnium and Ephelota showed no clear preference for a certain range of surface tension. Densities neither increased or decreased with surface tension nor was a clearly visible minimum range between 20 and 25 mNm_ existent. Only Corthunia showed after 8 days the pattern which one would expect from the Dexter model (1979) but the minimum occurred between 25.5 and 30 mN m-. 3.3. Detachment studies Detachment studies showed that a flow pressure of 16.9 Nmm2 was mostly required to remove numerous microfouling organisms from the test panels. Therefore, only results which were obtained under 16.9 Nme2 will be presented. Bacteria (Fig. 2) improved attachment strength considerably within 2 days. Over 60% of the cells were removed from every material after 3 hours. However, more than 50% of the cells remained on the substrata after 2 days. Significant different retention rates on the substrata employed were recorded between 3 hours and 5 days (H-test: p < 0.05). Lowest retention rates were found on one material within the 20 and 25 mN m-l range until 5 days. However, significant differences between the substrata disappeared after 8 days of exposure. Similar

results were obtained for diatoms (Fig. 3). Diatoms improved attachment strength on every material, on PTFE (19.0mNm-) and PC especially (33.5mNm-). After only 2 days of exposure less than 20% of the cells were detached from these two materials. Lowest retention rates were recorded on materials between 20 and 25 mN m- until the 5-days-interval. Significant differences between the substrata vanished after 8 days. Detachment studies on protozoa were conducted on 5 and 8 days old biofilms. Colonization of protozoa was too low for evaluation at intervals shorter than 5 days. The results obtained in both exposure intervals were summarized because detachment studies showed no increasing attachment strength between 5 and 8 days of exposure. More than 60% of the choanoflagellates remained on the different materials at 16.9 N rnp2 (Fig. 4). Significant differences between the substrata were not observed (H-test: p > 0.05). Detachment studies on protozoa yielded significant different detachment rates (H-test: p