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Enhancement of adhesion of the marine Chlorella vulgaris to glass1 T. R. TOSTESON Department of Marine Sciences, University of Plrerto Rico, Mayaguez, Plrerto Rico 00709 AND
W . A. CORPE Departrnent of Biological Sciences, Barnard College, Columbia University, New York, Nerv York 10027 Accepted February 12, 1975 TOSTESON, T. R., and W. A. CORPE.1975. Enhancement of adhesion of the marine Chlorella v~rlgaristo glass. Can. J. Microbiol. 21: 1025-1031. The adhesion of washed cells of a marine Chlorella vrrlgaris to solid surfaces was enhanced by non-diffusible material recovered from Chlorella exudate, marine bacterial cultures, natural seawater, and fouled marine surfaces. Materials isolated from certain bacterial cultures and from particulate materials filtered from seawater were three orders of magnitude more active than Chlorella exudate per unit weight. Active polymer materials from several sources were chromatographed on DEAE cellulose. The major fraction eluted with dilute base contained both protein and carbohydrate and enhanced adhesion more than the unchromatographed material. TOSTESON,T. R., et W. A. CORPE. 1975. Enhancement of adhesion of the marine Chlorella vulgaris to glass. Can. J. Microbiol. 21: 1025-103 1. L'adhesion B des surfaces solides de cellules delavees d'une Clllorella vulgaris marine est augmentee par I'emploi de substances non diffusibles obtenues d'exsudats de Chlorella, de cultures de batteries marines, de I'eau de mer naturelle et de surfaces agglutinees de matkriaux de mer. Les substances isolees de certaines cultures bacteriennes ou des particules obtenues par filtration de I'eau de mer s'averent trois fois plus efficaces par unite de poids que celles obtenues d'exsudats de Chlorella. Du materiel polymere actif des diverses sources fut chromatographit surcellulose DEAE. La fraction majeure obtenue, mise en presence d'une base diluee, contient a la fois des proteines et des hydrates de carbone et augmente I'adhesion plus que le materiel non soumis a la chromatographie. [Traduit par le journal]
Introduction The adhesion of marine Chlorella vulgaris cells to surfaces and the aggregation Of sing1e cells into large clumps is apparently mediated by a non-diffus;b]e materia( b n the cell surface ( T and ~ ~ ~l 19721.~~ ~ d ~ h en-~~ hancing has been found in non-diffusible fractions isolated from seawater and marine bacterial cultures. A comparison of adhesion enhancing activity of these products with that of Charella exudate is the subject of the present paper. Materials and Methods Chlorella Adhesion Assay System Chlorella ulrlgaris (Beijerinck) used in these experiments was '"lated from the sea in the area of the Marine Station a t La Parguera, Puerto Rico; and maintained as axenic, growing, batch cultures in the laboratory on a modified seawater medium, Cg-loy after Van (1967). Cultures were kept in constant illumination (3500 1x1 both a t laboratory temperatures (22-23C) and in a n incubator at 29C. 'Received September 23, 1974.
The assay system was that of Zaidi and Tosteson (1972). Washed cells of Cl~lorellawere introduced into glass tubes (3 x in. i.d.) and the open end sealed shut. The sealed tubes were placed on a horizontal rotating wheel and spun at about 72 rom for the duration of the e x ~ e r i m e n (5 t h). under constant illumination. of Chlorella cells in ~ ~The ifinal d ~ ~concentration ~ ~~ ~ ~ the individual tubes was 4 x lo7 cells in a final volume of 4 ml. At the end of the experiment the tubes were opened and the cell suspension discarded. The emptied tubes were rinsed by immersion sequentially into three 40-ml volumes of fresh media. After these washings the glass tubes were immersed into 40 ml of fresh medium and exposed to mild sonication for 5 min. The number of cells released from the glass surface by sonication was counted with the usual haemo~~tometer-type counting chamber. The cells removed from glass tubes by sonication were regarded as having been "adhered" to the surface. The of various materials on the adhesion of Cl~lorellacells to glass surfaces were determined by mixing known amounts of the materials with washed cells. The number of cells adhering to the inner walls of the tube after incubation was compared to the number of adherent cells in the unsupplemented corltrol tubes. The results were expressed in terms of the percentage increase- (or decrease) in the number of adherent cells in the Dresence of the test material as compared t o the control:
+
~
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CAN. J. MICROB IOL. VOL. 21. 1975
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% increase / adherent cells in \ / adherent cells \ - \experimental tube) - \in control tube) x 100. adherent cells in control tube The increase in the percentage of adherent cells reflects the adhesion enhancement activity (AE) of the materials under test. In the data t o be presented here, a total of 268 tubes were analyzed. In the control experiments ( N = 60) an average of 24 x lo4 cells adhered t o glass during the 5-h time period. The standard deviation was 2.97 x lo4 or 12.4% of the mean value; thus 3.5 x standard deviation was arbitrarily selected from statistical tables to represent a variation of 43.4z around this mean value. Since this degree of variation has a probability well below 0.01, experimental values falling outside of this variation are regarded as significantly different from the control. In the experimental tubes supplemented with various materials cell counts were done in duplicate when the adhesionenhancing activity was apparent by visual comparison with the control. Sources and Preparation of Material Eval~tated for Adhesion-enlrancement Activity Chlorella Exudates Chlorella cells grown in Cg-10 seawater medium were centrifuged at 5000 x g, washed in distilled water, resuspended in seawater medium, and incubated for 2 h in the light. The cells were again centrifuged and washed with distilled water two times. The washings were dialyzed against distilled water and freeze-dried. The white powder described hereafter as Chlorella exudate was used in the experiments to be reported. The cells showed no evidence of damage during the brief wash with distilled water. Bacterial Polymers Polyanionic carbohydrate materials were recovered from bacterial-culture filtrates and cell washings as previously described (Corpe 1972~).The dialyzed, desalted products contained non-diffusible, alcohol-insoluble material with both carbohydrate and protein components. The material was recovered from cultures of the following periphytic marine bacteria: Pseudomonos atlantica, strain T,C; Pseudomonas spp., Mag; Ca~tlobacter halobacteroides Poindexter (ATCC2 15269); and Saprospira grandis Gross (ATCC 23 116). Extracts of Marine Material Materials that had been scraped from wooden test panels exposed in the sea at various depths and locations for from 1-6 months were supplied by John DePalma of the United States Naval Oceanographic Office, Washington, D.C. The materials were described in an earlier report (Corpe 1972b). The samples identified as Hawaii (No. 1657) will be described in this paper. Insoluble material was recovered from large volumes of seawater pumped through Fulflow Honeycomb filter tubes (Commercial Filters Corp., Lebanon, Indiana) at the New Jersey shore, south of Sandy Hook State Park. Material collected in the filter included microorganisms grown at the expense of filtered material. Water-soluble 2ATCC, American Type Culture Collection.
substances were leached out with sterile seawater and steamed in an autoclave. The extract was desalted by dialysis against dilute HCI, exhaustively against distilled water t o remove chloride, and lyophilized to give a fibrous mass, light brown in color. The sample used in the present work is identified as AGDp. Recovery of Seawater-soluble Materials Water samples obtained at stations in the Caribbean were stored under refrigeration, transferred to the laboratory, and filtered through a 0.45-micron filter t o remove suspended materials. The water was reduced in volume by the addition of dry Sephadex gel (G-50). Active material was excluded and retained when dialyzed against distilled water. The desalted material was freeze-dried to a fine, light powder (Tosteson et a / . 1973) and is identified as "SW sol". Anion Exchange Clzromatography of'Exudates Lyophilized samples of a few of the recovered materials were dissolved in distilled water. Any insoluble residues, if present, were removed by centrifugation and the soluble material seated on the top of a column of diethylaminoethyl (DEAE) cellulose in the -OH form (Neukom and Kuendig 1965). Columns were eluted with ten 5-ml portions of distilled water, 0.05,0.1, and 0.5 N NaOH in that order; the fractions were examined for their absorption at 278 nm; and were analyzed for carbohydrate and protein as described below. Fractions containing carbohydrate and (or) protein were neutralized, dialyzed, lyophilized, and tested for adhesion-enhancing activity. Chemical Analyses The anthrone method for Carbohydrate determination as described by Neish (1952) was used. The standard curve was prepared using glucose. Uronic acid content of polysaccharides was estimated by the Dische carbazole -H2S04 method (Kabat and Mayer 1961). Glucuronic acid was used as the standard. Neutralized hydrolysates (in HZSO,, 6 h 100°C) containing amino sugar. were analyzed by the Rondel and Morgan method described by Kabat and Mayer (1961). Protein was determined using the procedure described by Lowry et al. (1951). Bovine serum albumin was the standard. Nucleic acids were analyzed using the hot perchloric acid extraction method described by Echlin and DeLamater (1962). Results were reported as 260 nm absorption per 3 millilitres containing 1 mg of sample.
Results The data presented in Table 1 show the percentage increase in the adhesion of cells of Chlorella uulgaris with an increasing concentration of Chlorella exudate. When theconcentration reached 0.2 ng/cell the percentage increase of cells adhering to glass as compared to the control, dropped. This effect was brought about by an increase in cell-cell adhesions at high exudate concentration. Large clumps of cells were found in the cell suspension when it was removed from the tube (Fig. 1). The large aggregates were easily removed from the glass surface by rinsing
-
1027
TOSTESON AND CORPE: ADHESION O F CHLORELLA TO GLASS
TABLE 1
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Effect of extracted preparations and exudates on the adhesion of Chlorella Exudate and extract concentrations, pg/cell 0.002 0.01 0.02 0.04 0.1 0.2 0.4 0.6 1 2 4
increase in Chlorella adhesion* produced by preparations from: Chlorella exudate
Hawaii No. 1657 Caulobacter Saprospira
T,C
Ma8
AgDp
SW,
- 19 8
- 17 46 25
- 19
124
29 63
148 171 310 608
150
119
26 58
-6
283 129
14
'Percentage increases ? 43% are significantly different from the control, p < 0.01
the tube resulting in a significant decrease in cell to glass adhesion when compared to the untreated cell control. Table 1 also shows the activity of various exudates and extracts from bacterial cultures and seawater compared with Chlorella exudate on a weight basis. The least active of these preparations was bacterial polymer Ma8. The Chlorella exudate and the preparations from Saprospira culture supernatant fluid were essentially an order of magnitude more active than Ma8. Activity was also observed in several other bacterial cultures and in extracts of scrapings from a heavily fouled wooden panel that had been submerged in the sea (Hawaii, No. 1657), but by far the most active of the crude preparations were bacterial polymer T6C and the extract of the seawater-insoluble material (AgDp). These preparations were essentially four orders of magnitude more active than bacterial polymer Ma8, in
terms of the concentration at which they doubled the number of adhering Chlorella cells. An elution curve of the polyanionic material (T6C) from DEAE cellulose by dilute base is shown in Fig. 2. The carbohydrate (Anthrone) and 280-nm absorbing material did not peak in precisely the same fraction, which was typical of all of the active materials. When the protein content of each fraction was determined with Folin phenol reagents, the resulting curve was essentially identical with the 280-nm absorption curve. Most of the material with adhesion-enhancing activity from each sample was found in a single peak. Fractions under the major carbohydrate peak were combined, neutralized, dialyzed to remove salt, and lyophilized. The quantitative and qualitative differences in the composition of the samples were marked (Table 2). The protein and nucleic acid contents were variable and not correlated with activity. Nucleic acid content
CAN. J. MICROBIOL. VOL. 21, 1975
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1028
FIG.1 . The enhancement of aggregation of washed Chlorellacells by Chlorella exudate. (A) Cells incubated in the presence of exudate (0.4 nglcell); (B) in the absence of added exudate.
TOSTESON AND CORPE: ADHESION OF CHLORELLA TO GLASS
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WATER
1
0.0IN
I
Na OH 0.05N
I
O.ION
I
0.50N
FIG.2. Elution of polyanions from DEAE cellulose column (2 x 9 crn) with ten 5-rnl fractions each of distilled water, 0.01 N NaOH, 0.05 N NaOH, 0.1 N NaOH, and 0.5 N NaOH. Open circles and solid lines show analysis of anthrone carbohydrate; closed circles and broken line shows absorption at 280 nrn.
expressed by 260-nm absorption of an extract from a 1-mg sample testifies to the small amount present compared to the amount of protein. The only clear relationship between the various samples was that they all displayed some adhesion-enhancing activity which is exemplified by the data shown in Table 3 with partially purified materials from T,C and Caulobacter. Table 4 compares the concentration of crude and purified preparations that result in 100% increase in Chlorella adhesion. It is reasonably clear that the active material is effective in very low concentrations and is present in a still complex mixture of substances found in the eluates from ion-exchange cellulose.
Discussion The adhesion of Chlorella vulgaris or any other cells to solid surfaces may depend on the ability of the organism to secrete an intercellular adhesive material. The secretion of such material by Chlorella not only affects its adherence to nonspecific solid surfaces but if present in large enough quantity, caused the cells to clump or aggregate (Table 1, Fig. 1). The mechanism of these two expressions of surface interaction may not be identical in every respect but they must be related since both are enhanced by the same active material. The character of the active material is of interest from several points of view, besides the
apparent lack of specificity. Washed Chlorella cells incubated in the light for 2 h can secrete active exudate to the extent of 126 pglcell, (Tosteson and Almodovar 1972). Adhesionenhancing materials from bacteria or other marine sources could conceivably function as "inducers" of adhesive polymer synthesis by Chlorella, stimulate secretion, stabilize the secreted adhesive, or actually substitute for the product synthesized by Chlorella. Aggregation or agglutination of washed Chlorella cells by low concentrations of the various materials is reminiscent of the action of lectins (Sharon and Lis 1972). Zaidi and Tosteson (1972) suggested that in randomly dividing populations of Chlorella only a fraction of the cells were capable of adhesion, the property being related to a specific phase of the cell cycle. Tosteson and Almodovar (1972) found that when a washed cell suspension was supplemented with 10&200 pg of exudate per cell, more adhesiveness was observed than when the same amount of exudate was added to an unwashed suspension. So the physiological state of the cells may be more important in the expression of these surface reactions, than the total amount of exudate per cell. Corpe (1970) showed that the development of of a bacterial film on solid surfaces always preceded the attachment of microalgae. Slides coated with bacterial exudate stimulated aggregation of
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CAN. J. MICROBIOL. VOL. 21, 1975
,a.3 ;a;a; a 4-
a oaaa Z
??9? 1
000-
oeo
TOSTESON AND CORPE: ADHES I O N O F CHLORELLA TO GLASS
TABLE 3
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The effect of purified preparations on the adhesion of Chlorella cells
% increase in Chlorella adhesion* Extract concn., ~g/cell 0.002 0.004 0.008 0.01 0.02 0.04 0.05 0.1 0.2 0.4 0.6 0.8 I 2 4 5 8 50 250
T6C Crude
Purified
Crude
Purified
- 19 8 - 17 46
8 59 149 60 99 125 290
-8 158
124 225 148 171 3 10 608
26
1129
91 105 99
254 367
58 210
*Percentage increase < 0.01.
control, p
Caulobacter
+
43% is significantly diKerent
from
the
TABLE 4 Estimated concentration of extracted materials resulting in 100% increase in C/~lorellaadhesion: crude preparations compared to purified fractions 100% adhesion increase concn., pg/cell Preparation
Crude
Purified fraction
T6C polymer Caulobacter polymer AgDp extract of seawater particulate material
0.13 8.0
0.0075 0.07
0.26
0.062
bacteria and colonization of microalgae. Sieburth (1967) also described a substance produced by a marine pseudonomad that agglutinated cells of an Arthrobacter species. It is not surprising then that active material occurred in seawater both in a particulate and in a soluble form. Active substances may be complexed with various kinds of material which could confer improved stability and even improve their resistance to biodegradation. The distribution and stability of these materials in the sea and their function in the ecology of marine life is being explored in greater detaiL3 3Tosteson, Hale, Zaidi, and Atwood. In preparation.
1031
Acknowledgments The authors are indebted to B. R. Zaidi and D. R. Hale for assistance and advice in various aspects of the work. The technical assistance of Linda Matsuuchi and Eva Gomolinski is greatfully acknowledged. This investigation was supported in part by a grant from the National Science Foundation (W.A.C.) and from the Office of Naval Research, NR 306-849 (T.R.T.). CORPE,W. A. 1970. An acid polysaccharide produced by a primary film forming marine bacterium. Dev. Ind. Microbiol. ll: 402-412. CORPE,W. A. 19720. Microfouling: the role of primary film forming bacteria. In Third international congress on marine corrosion and fouling. Edited by R. F. Acker et a l . Northwestern Univ. Press, Evanston, Ill. pp. 598609. CORPE,W. A. 1972b. Isolation and study of polysaccharide rich material from heavily fouled test panel scrapings. ONR Tech. Rep. 8-1 1-72. ECHLIN, P., and E. D. DELAMATER. 1962. A cytological and chemical analysis of the bacterial nucleus. 11. A study of the cytochemical changes involved during the isolation of bacterial nuclear material by means of bile salts and high frequency sound. Exp. Cell Res. 26: 229-252. KABAT,E. A., and M. M. MAYER.1961. Experimental immunochemistry. 2nd ed. Charles C. Thomas, Publishers, Springfield, 111. LOWRY, 0. H., N. J. ROSENBROUGH, A. L. FARR,A N D R. J . RANDALL. 1951. Protein measurement with the Folin phenol reagent. J . Biol. Chem. 193: 265-275. NEISH,A. C. 1952. Analytical methods for bacterial fermentations. National Research Council of Canada, N.R.C. No. 2952, Saskatoon, Saskatchewan, Canada. Rep. 46-8-3. NEUKOM, H., and W. KUENDIG. 1965. Fractionation on diethylaminoethyl-cellulose columns. In Methods in carbohydrate chemistry. Vol. 5. Edited by R. L. Whistler. Academic Press, Inc., New York. Chap. 5. pp. 14-17. SCOTT,J . E. 1965. Fractionation by precipitation with quaternary ammonium salts. In Methods in carbohydrate chemistry. Vol. 5. Edited by R. L. Whistler. Academic Press Inc., New York. Chap. 11. pp. 38-44. SHARON, N., and H. Lis. 1972. Lectins: cell aggregating and sugar specific proteins.Science, 177: 949-959. SIEBURTH, J. McN. 1967. Inhibition and agglutination of arthrobacters by pseudomonads. J . Bacterial. 93: 1911-1916. TOSTESON,T . R., and L. M. ALMODOVAR. 1972. The adhesive properties of ChloreNa vitlgaris and the enhancement of the adhesion by substances found in ambient seawater. ONR Tech. Rep. 2. NR 306-849. TOSTESON,T. R., B. R. ZAIDI,and D. R. HALE. 1973. Surface active organics in the Caribbean Sea. Proc. 10th Meet. Assoc. of Island Marine Laboratories of the Caribbean. (Abstr.) p. 12. VANBAALEN, C. 1967. Further observations on the growth of single celIs of coccoid blue-green algae. J. of Phycol. 3(3): 154-157. ZAIDI,B. R., and T. R. TOSTESON.1972. The differential adhesion of Chlorella cells during the life cycle. Proc. VII Int. Seaweed Symp. pp. 323-328.
Enhancement of adhesion of the marine Chlorella vulgaris to glass1 T. R. TOSTESON Department of Marine Sciences, University of Plrerto Rico, Mayaguez, Plrerto Rico 00709 AND
W . A. CORPE Departrnent of Biological Sciences, Barnard College, Columbia University, New York, Nerv York 10027 Accepted February 12, 1975 TOSTESON, T. R., and W. A. CORPE.1975. Enhancement of adhesion of the marine Chlorella v~rlgaristo glass. Can. J. Microbiol. 21: 1025-1031. The adhesion of washed cells of a marine Chlorella vrrlgaris to solid surfaces was enhanced by non-diffusible material recovered from Chlorella exudate, marine bacterial cultures, natural seawater, and fouled marine surfaces. Materials isolated from certain bacterial cultures and from particulate materials filtered from seawater were three orders of magnitude more active than Chlorella exudate per unit weight. Active polymer materials from several sources were chromatographed on DEAE cellulose. The major fraction eluted with dilute base contained both protein and carbohydrate and enhanced adhesion more than the unchromatographed material. TOSTESON,T. R., et W. A. CORPE. 1975. Enhancement of adhesion of the marine Chlorella vulgaris to glass. Can. J. Microbiol. 21: 1025-103 1. L'adhesion B des surfaces solides de cellules delavees d'une Clllorella vulgaris marine est augmentee par I'emploi de substances non diffusibles obtenues d'exsudats de Chlorella, de cultures de batteries marines, de I'eau de mer naturelle et de surfaces agglutinees de matkriaux de mer. Les substances isolees de certaines cultures bacteriennes ou des particules obtenues par filtration de I'eau de mer s'averent trois fois plus efficaces par unite de poids que celles obtenues d'exsudats de Chlorella. Du materiel polymere actif des diverses sources fut chromatographit surcellulose DEAE. La fraction majeure obtenue, mise en presence d'une base diluee, contient a la fois des proteines et des hydrates de carbone et augmente I'adhesion plus que le materiel non soumis a la chromatographie. [Traduit par le journal]
Introduction The adhesion of marine Chlorella vulgaris cells to surfaces and the aggregation Of sing1e cells into large clumps is apparently mediated by a non-diffus;b]e materia( b n the cell surface ( T and ~ ~ ~l 19721.~~ ~ d ~ h en-~~ hancing has been found in non-diffusible fractions isolated from seawater and marine bacterial cultures. A comparison of adhesion enhancing activity of these products with that of Charella exudate is the subject of the present paper. Materials and Methods Chlorella Adhesion Assay System Chlorella ulrlgaris (Beijerinck) used in these experiments was '"lated from the sea in the area of the Marine Station a t La Parguera, Puerto Rico; and maintained as axenic, growing, batch cultures in the laboratory on a modified seawater medium, Cg-loy after Van (1967). Cultures were kept in constant illumination (3500 1x1 both a t laboratory temperatures (22-23C) and in a n incubator at 29C. 'Received September 23, 1974.
The assay system was that of Zaidi and Tosteson (1972). Washed cells of Cl~lorellawere introduced into glass tubes (3 x in. i.d.) and the open end sealed shut. The sealed tubes were placed on a horizontal rotating wheel and spun at about 72 rom for the duration of the e x ~ e r i m e n (5 t h). under constant illumination. of Chlorella cells in ~ ~The ifinal d ~ ~concentration ~ ~~ ~ ~ the individual tubes was 4 x lo7 cells in a final volume of 4 ml. At the end of the experiment the tubes were opened and the cell suspension discarded. The emptied tubes were rinsed by immersion sequentially into three 40-ml volumes of fresh media. After these washings the glass tubes were immersed into 40 ml of fresh medium and exposed to mild sonication for 5 min. The number of cells released from the glass surface by sonication was counted with the usual haemo~~tometer-type counting chamber. The cells removed from glass tubes by sonication were regarded as having been "adhered" to the surface. The of various materials on the adhesion of Cl~lorellacells to glass surfaces were determined by mixing known amounts of the materials with washed cells. The number of cells adhering to the inner walls of the tube after incubation was compared to the number of adherent cells in the unsupplemented corltrol tubes. The results were expressed in terms of the percentage increase- (or decrease) in the number of adherent cells in the Dresence of the test material as compared t o the control:
+
~
1026
CAN. J. MICROB IOL. VOL. 21. 1975
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% increase / adherent cells in \ / adherent cells \ - \experimental tube) - \in control tube) x 100. adherent cells in control tube The increase in the percentage of adherent cells reflects the adhesion enhancement activity (AE) of the materials under test. In the data t o be presented here, a total of 268 tubes were analyzed. In the control experiments ( N = 60) an average of 24 x lo4 cells adhered t o glass during the 5-h time period. The standard deviation was 2.97 x lo4 or 12.4% of the mean value; thus 3.5 x standard deviation was arbitrarily selected from statistical tables to represent a variation of 43.4z around this mean value. Since this degree of variation has a probability well below 0.01, experimental values falling outside of this variation are regarded as significantly different from the control. In the experimental tubes supplemented with various materials cell counts were done in duplicate when the adhesionenhancing activity was apparent by visual comparison with the control. Sources and Preparation of Material Eval~tated for Adhesion-enlrancement Activity Chlorella Exudates Chlorella cells grown in Cg-10 seawater medium were centrifuged at 5000 x g, washed in distilled water, resuspended in seawater medium, and incubated for 2 h in the light. The cells were again centrifuged and washed with distilled water two times. The washings were dialyzed against distilled water and freeze-dried. The white powder described hereafter as Chlorella exudate was used in the experiments to be reported. The cells showed no evidence of damage during the brief wash with distilled water. Bacterial Polymers Polyanionic carbohydrate materials were recovered from bacterial-culture filtrates and cell washings as previously described (Corpe 1972~).The dialyzed, desalted products contained non-diffusible, alcohol-insoluble material with both carbohydrate and protein components. The material was recovered from cultures of the following periphytic marine bacteria: Pseudomonos atlantica, strain T,C; Pseudomonas spp., Mag; Ca~tlobacter halobacteroides Poindexter (ATCC2 15269); and Saprospira grandis Gross (ATCC 23 116). Extracts of Marine Material Materials that had been scraped from wooden test panels exposed in the sea at various depths and locations for from 1-6 months were supplied by John DePalma of the United States Naval Oceanographic Office, Washington, D.C. The materials were described in an earlier report (Corpe 1972b). The samples identified as Hawaii (No. 1657) will be described in this paper. Insoluble material was recovered from large volumes of seawater pumped through Fulflow Honeycomb filter tubes (Commercial Filters Corp., Lebanon, Indiana) at the New Jersey shore, south of Sandy Hook State Park. Material collected in the filter included microorganisms grown at the expense of filtered material. Water-soluble 2ATCC, American Type Culture Collection.
substances were leached out with sterile seawater and steamed in an autoclave. The extract was desalted by dialysis against dilute HCI, exhaustively against distilled water t o remove chloride, and lyophilized to give a fibrous mass, light brown in color. The sample used in the present work is identified as AGDp. Recovery of Seawater-soluble Materials Water samples obtained at stations in the Caribbean were stored under refrigeration, transferred to the laboratory, and filtered through a 0.45-micron filter t o remove suspended materials. The water was reduced in volume by the addition of dry Sephadex gel (G-50). Active material was excluded and retained when dialyzed against distilled water. The desalted material was freeze-dried to a fine, light powder (Tosteson et a / . 1973) and is identified as "SW sol". Anion Exchange Clzromatography of'Exudates Lyophilized samples of a few of the recovered materials were dissolved in distilled water. Any insoluble residues, if present, were removed by centrifugation and the soluble material seated on the top of a column of diethylaminoethyl (DEAE) cellulose in the -OH form (Neukom and Kuendig 1965). Columns were eluted with ten 5-ml portions of distilled water, 0.05,0.1, and 0.5 N NaOH in that order; the fractions were examined for their absorption at 278 nm; and were analyzed for carbohydrate and protein as described below. Fractions containing carbohydrate and (or) protein were neutralized, dialyzed, lyophilized, and tested for adhesion-enhancing activity. Chemical Analyses The anthrone method for Carbohydrate determination as described by Neish (1952) was used. The standard curve was prepared using glucose. Uronic acid content of polysaccharides was estimated by the Dische carbazole -H2S04 method (Kabat and Mayer 1961). Glucuronic acid was used as the standard. Neutralized hydrolysates (in HZSO,, 6 h 100°C) containing amino sugar. were analyzed by the Rondel and Morgan method described by Kabat and Mayer (1961). Protein was determined using the procedure described by Lowry et al. (1951). Bovine serum albumin was the standard. Nucleic acids were analyzed using the hot perchloric acid extraction method described by Echlin and DeLamater (1962). Results were reported as 260 nm absorption per 3 millilitres containing 1 mg of sample.
Results The data presented in Table 1 show the percentage increase in the adhesion of cells of Chlorella uulgaris with an increasing concentration of Chlorella exudate. When theconcentration reached 0.2 ng/cell the percentage increase of cells adhering to glass as compared to the control, dropped. This effect was brought about by an increase in cell-cell adhesions at high exudate concentration. Large clumps of cells were found in the cell suspension when it was removed from the tube (Fig. 1). The large aggregates were easily removed from the glass surface by rinsing
-
1027
TOSTESON AND CORPE: ADHESION O F CHLORELLA TO GLASS
TABLE 1
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Effect of extracted preparations and exudates on the adhesion of Chlorella Exudate and extract concentrations, pg/cell 0.002 0.01 0.02 0.04 0.1 0.2 0.4 0.6 1 2 4
increase in Chlorella adhesion* produced by preparations from: Chlorella exudate
Hawaii No. 1657 Caulobacter Saprospira
T,C
Ma8
AgDp
SW,
- 19 8
- 17 46 25
- 19
124
29 63
148 171 310 608
150
119
26 58
-6
283 129
14
'Percentage increases ? 43% are significantly different from the control, p < 0.01
the tube resulting in a significant decrease in cell to glass adhesion when compared to the untreated cell control. Table 1 also shows the activity of various exudates and extracts from bacterial cultures and seawater compared with Chlorella exudate on a weight basis. The least active of these preparations was bacterial polymer Ma8. The Chlorella exudate and the preparations from Saprospira culture supernatant fluid were essentially an order of magnitude more active than Ma8. Activity was also observed in several other bacterial cultures and in extracts of scrapings from a heavily fouled wooden panel that had been submerged in the sea (Hawaii, No. 1657), but by far the most active of the crude preparations were bacterial polymer T6C and the extract of the seawater-insoluble material (AgDp). These preparations were essentially four orders of magnitude more active than bacterial polymer Ma8, in
terms of the concentration at which they doubled the number of adhering Chlorella cells. An elution curve of the polyanionic material (T6C) from DEAE cellulose by dilute base is shown in Fig. 2. The carbohydrate (Anthrone) and 280-nm absorbing material did not peak in precisely the same fraction, which was typical of all of the active materials. When the protein content of each fraction was determined with Folin phenol reagents, the resulting curve was essentially identical with the 280-nm absorption curve. Most of the material with adhesion-enhancing activity from each sample was found in a single peak. Fractions under the major carbohydrate peak were combined, neutralized, dialyzed to remove salt, and lyophilized. The quantitative and qualitative differences in the composition of the samples were marked (Table 2). The protein and nucleic acid contents were variable and not correlated with activity. Nucleic acid content
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1028
FIG.1 . The enhancement of aggregation of washed Chlorellacells by Chlorella exudate. (A) Cells incubated in the presence of exudate (0.4 nglcell); (B) in the absence of added exudate.
TOSTESON AND CORPE: ADHESION OF CHLORELLA TO GLASS
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WATER
1
0.0IN
I
Na OH 0.05N
I
O.ION
I
0.50N
FIG.2. Elution of polyanions from DEAE cellulose column (2 x 9 crn) with ten 5-rnl fractions each of distilled water, 0.01 N NaOH, 0.05 N NaOH, 0.1 N NaOH, and 0.5 N NaOH. Open circles and solid lines show analysis of anthrone carbohydrate; closed circles and broken line shows absorption at 280 nrn.
expressed by 260-nm absorption of an extract from a 1-mg sample testifies to the small amount present compared to the amount of protein. The only clear relationship between the various samples was that they all displayed some adhesion-enhancing activity which is exemplified by the data shown in Table 3 with partially purified materials from T,C and Caulobacter. Table 4 compares the concentration of crude and purified preparations that result in 100% increase in Chlorella adhesion. It is reasonably clear that the active material is effective in very low concentrations and is present in a still complex mixture of substances found in the eluates from ion-exchange cellulose.
Discussion The adhesion of Chlorella vulgaris or any other cells to solid surfaces may depend on the ability of the organism to secrete an intercellular adhesive material. The secretion of such material by Chlorella not only affects its adherence to nonspecific solid surfaces but if present in large enough quantity, caused the cells to clump or aggregate (Table 1, Fig. 1). The mechanism of these two expressions of surface interaction may not be identical in every respect but they must be related since both are enhanced by the same active material. The character of the active material is of interest from several points of view, besides the
apparent lack of specificity. Washed Chlorella cells incubated in the light for 2 h can secrete active exudate to the extent of 126 pglcell, (Tosteson and Almodovar 1972). Adhesionenhancing materials from bacteria or other marine sources could conceivably function as "inducers" of adhesive polymer synthesis by Chlorella, stimulate secretion, stabilize the secreted adhesive, or actually substitute for the product synthesized by Chlorella. Aggregation or agglutination of washed Chlorella cells by low concentrations of the various materials is reminiscent of the action of lectins (Sharon and Lis 1972). Zaidi and Tosteson (1972) suggested that in randomly dividing populations of Chlorella only a fraction of the cells were capable of adhesion, the property being related to a specific phase of the cell cycle. Tosteson and Almodovar (1972) found that when a washed cell suspension was supplemented with 10&200 pg of exudate per cell, more adhesiveness was observed than when the same amount of exudate was added to an unwashed suspension. So the physiological state of the cells may be more important in the expression of these surface reactions, than the total amount of exudate per cell. Corpe (1970) showed that the development of of a bacterial film on solid surfaces always preceded the attachment of microalgae. Slides coated with bacterial exudate stimulated aggregation of
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CAN. J. MICROBIOL. VOL. 21, 1975
,a.3 ;a;a; a 4-
a oaaa Z
??9? 1
000-
oeo
TOSTESON AND CORPE: ADHES I O N O F CHLORELLA TO GLASS
TABLE 3
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The effect of purified preparations on the adhesion of Chlorella cells
% increase in Chlorella adhesion* Extract concn., ~g/cell 0.002 0.004 0.008 0.01 0.02 0.04 0.05 0.1 0.2 0.4 0.6 0.8 I 2 4 5 8 50 250
T6C Crude
Purified
Crude
Purified
- 19 8 - 17 46
8 59 149 60 99 125 290
-8 158
124 225 148 171 3 10 608
26
1129
91 105 99
254 367
58 210
*Percentage increase < 0.01.
control, p
Caulobacter
+
43% is significantly diKerent
from
the
TABLE 4 Estimated concentration of extracted materials resulting in 100% increase in C/~lorellaadhesion: crude preparations compared to purified fractions 100% adhesion increase concn., pg/cell Preparation
Crude
Purified fraction
T6C polymer Caulobacter polymer AgDp extract of seawater particulate material
0.13 8.0
0.0075 0.07
0.26
0.062
bacteria and colonization of microalgae. Sieburth (1967) also described a substance produced by a marine pseudonomad that agglutinated cells of an Arthrobacter species. It is not surprising then that active material occurred in seawater both in a particulate and in a soluble form. Active substances may be complexed with various kinds of material which could confer improved stability and even improve their resistance to biodegradation. The distribution and stability of these materials in the sea and their function in the ecology of marine life is being explored in greater detaiL3 3Tosteson, Hale, Zaidi, and Atwood. In preparation.
1031
Acknowledgments The authors are indebted to B. R. Zaidi and D. R. Hale for assistance and advice in various aspects of the work. The technical assistance of Linda Matsuuchi and Eva Gomolinski is greatfully acknowledged. This investigation was supported in part by a grant from the National Science Foundation (W.A.C.) and from the Office of Naval Research, NR 306-849 (T.R.T.). CORPE,W. A. 1970. An acid polysaccharide produced by a primary film forming marine bacterium. Dev. Ind. Microbiol. ll: 402-412. CORPE,W. A. 19720. Microfouling: the role of primary film forming bacteria. In Third international congress on marine corrosion and fouling. Edited by R. F. Acker et a l . Northwestern Univ. Press, Evanston, Ill. pp. 598609. CORPE,W. A. 1972b. Isolation and study of polysaccharide rich material from heavily fouled test panel scrapings. ONR Tech. Rep. 8-1 1-72. ECHLIN, P., and E. D. DELAMATER. 1962. A cytological and chemical analysis of the bacterial nucleus. 11. A study of the cytochemical changes involved during the isolation of bacterial nuclear material by means of bile salts and high frequency sound. Exp. Cell Res. 26: 229-252. KABAT,E. A., and M. M. MAYER.1961. Experimental immunochemistry. 2nd ed. Charles C. Thomas, Publishers, Springfield, 111. LOWRY, 0. H., N. J. ROSENBROUGH, A. L. FARR,A N D R. J . RANDALL. 1951. Protein measurement with the Folin phenol reagent. J . Biol. Chem. 193: 265-275. NEISH,A. C. 1952. Analytical methods for bacterial fermentations. National Research Council of Canada, N.R.C. No. 2952, Saskatoon, Saskatchewan, Canada. Rep. 46-8-3. NEUKOM, H., and W. KUENDIG. 1965. Fractionation on diethylaminoethyl-cellulose columns. In Methods in carbohydrate chemistry. Vol. 5. Edited by R. L. Whistler. Academic Press, Inc., New York. Chap. 5. pp. 14-17. SCOTT,J . E. 1965. Fractionation by precipitation with quaternary ammonium salts. In Methods in carbohydrate chemistry. Vol. 5. Edited by R. L. Whistler. Academic Press Inc., New York. Chap. 11. pp. 38-44. SHARON, N., and H. Lis. 1972. Lectins: cell aggregating and sugar specific proteins.Science, 177: 949-959. SIEBURTH, J. McN. 1967. Inhibition and agglutination of arthrobacters by pseudomonads. J . Bacterial. 93: 1911-1916. TOSTESON,T . R., and L. M. ALMODOVAR. 1972. The adhesive properties of ChloreNa vitlgaris and the enhancement of the adhesion by substances found in ambient seawater. ONR Tech. Rep. 2. NR 306-849. TOSTESON,T. R., B. R. ZAIDI,and D. R. HALE. 1973. Surface active organics in the Caribbean Sea. Proc. 10th Meet. Assoc. of Island Marine Laboratories of the Caribbean. (Abstr.) p. 12. VANBAALEN, C. 1967. Further observations on the growth of single celIs of coccoid blue-green algae. J. of Phycol. 3(3): 154-157. ZAIDI,B. R., and T. R. TOSTESON.1972. The differential adhesion of Chlorella cells during the life cycle. Proc. VII Int. Seaweed Symp. pp. 323-328.
