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The agar-digesting bacteria of the Anclote estuary

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Title:
The agar-digesting bacteria of the Anclote estuary
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v, 61 leaves : ill. ; 29 cm.
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English
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Ford, Earl
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University of South Florida
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Tampa, Florida
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Bacteria -- Florida -- Anclote estuary   ( lcsh )
Microbial ecology -- Florida -- Anclote estuary   ( lcsh )
Dissertations, Academic -- Marine Science -- Masters -- USF   ( fts )

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General Note:
Thesis (M.S.)--University of South Florida, 1977. Bibliography: leaves 56-61.

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University of South Florida
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Universtity of South Florida
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All applicable rights reserved by the source institution and holding location.
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aleph - 028065145
oclc - 05427337
usfldc doi - F51-00013
usfldc handle - f51.13
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SFS0040004:00001


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THE AGAR-DIGESTING BACTERIA OF THE ANCLOTE ESTUARY by Earl Ford A thesis submitted in partial fulfillment of the for the degree of Master of Science in the Department of Marine Science in The University of South Florida December, "i977 Thesis supervisor: Professor Dr. Harold J. Humm

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Graduate Council University of South Florida Tampa, Florida CERTIFICATE OF APPROVAL NASTER'S THESIS This is to certify that the Master's Thesis of Earl Spencer Ford with a major in Marine Science has been approved by the Examining Committee on i'iovember 22, 1977 as satisfactory for the thesis requirement for the Master of Science degree. Thesis Committee: Major Professor: Harold J. Humm, Ph.D. Hember: Thomas L. Hopkins, Pi.1.D. Hember: Norman J. Blake, Ph.D.

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ACKNOWLEDGEMENTS The writer thanks Paul Behrens, a fellow graduate student at USF, for providing K-carrageenan, extracted from Hypnea musciformis. To Dr. Humm, he extends his gratitude for supporting him during his years at USF. To Susan Zabranskey, who saved him endless hours with her typing,his sincere thanks. ii

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List of Tables List of Figures Introduction Literature Review TABLE OF CONTENTS Area Description Materials and Methods Results Discussion Appendices Appendix A Appendix B Bibliography iii PAGE iv v 1 3 13 16 26 4.5 .51 .52 .54 .5.5

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iv LIST OF TABLES Table No. 1. 2. 3 4. 5 Previously reported agarolytic bacteria. 4 Composition of media used in the isolation and main-18 tenance of agarolytic bacteria. Counts of total bacterial population in the Anclote 27 estuary from 1974 1975. (expressed in number/ml for surface water; number/gram for sediment). Population counts of agarolytic bacteria in the An29 clote estuary during 1974 1975. Counts expressed as no. per ml for surface water and as no. uer gram for bottom sediment. For station location refer to Figure 1. The percentage of agarolytic bacteria of the total 32 population in the Anclote estuary during 1975 1975. 6. Colony and cellular characteristics of the agarolytic 33 bacteria isolated from the Anclote estuary. 7 Growth characteristics of agar-digesters on agar 35 slants and in nutrient broth 8. Nutritional and biochemical characteristics of the 37 agarolytic bacteria from the Anclote estuary. 9 The action of some antibiotics on the agar-digesters 42 from the Anclote estuary. 10. Salinity and temperature tolerTn.ces of the agar-44 digesting bacteria of the Anclote estuary.

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LIST OF FIGURES Figure No. 1. 2. Sampling station locations in the Anclote estuary near Tarpon Springs, Photographs of the agar-digesters isolated from the Anclote estuary. v Page 14 54

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THE AGAR-DIGESTING BACTERIA OF THE ANCLOTE ESTUARY by Earl Ford An Abstract Of a thesis submitted in partial fulfillment of the requirements for the degree of Master Science in the Department of Marine Science in the University of South Florida December, 1977 Thesis supervisor: Professor Harold J. Humm

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ABSTRACT In connection with a multidisciplinary environmental analysis of the Anclote estuary near Tarpon Springs, Florida Gulf coast, prior to the construction of a fossil fuel electric generating plant, a quantitative and qualitative study of marine agar-digesting bacteria was conducted from February, 1974, to September, 1975. Water samples obtained from 15 localities gave counts ranging, with season, from 1100 to over 100,000 aerobic, heterotrophic bacteria per ml, and from 2.5 to 182 agar-digesters per ml. During cooler months, the "total" counts were mostly 1500 to 5000; during warmer months, 10,000 to 100,000. Agar-digester counts varied little with season or with locality, although different species were predominant at different seasons. Agar-digesters constituted from 1% to 5.5% of the total population. Of 24 pure cultures of agar-digesting bacteria isolated and studied in detail, 11 species were recognized belonging to the genera Cytophaga, Flavobacterium, Pseudomonas, and Vibrio. All were aerobic, non-spore-forming, gram-negative rods. They exhibited a wide range of manner and rate of hydrolysis of agar and k-carrageenan, the two algal polysaccharides on which they were tested. About half were able to utilize an ammonium salt as a sole source of nitrogen. Morphological and biochemical characteristics of each species were recorded. Abstract approved: ______________________________ thesis supervisor Professor, Department of Marine Science December 28, 1977

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1 INTRODUCTION The importance of bacteria in the marine environment is based primarily on their role as decomposers of organic matter and transformers of inorganic compounds, especially in the nitrogen and sulfur cycles. Since marine bacteria have been of little interest to medical bacteriologists because of their apparent lack of pathogeny, they have been studied far less than those of land and freshwater. There are two noteworthy physiological groups of bacteria that are far more abundant in the sea than elsewhere, those that are bioluminescent, and those that hydrolyze certain complex cell wall polysaccharides of marine algae such as agar, carrageenan, and algin. The latter group is the subject of this study. While agar-digesters are pandemically found, they are rarely reported from land or freshwater sources. In the sea, however, they are abundant and of great variety. This may be attributed to agar production occurring only in the marine environment, as far as is presently known, and only by certain species of red algae. Little is known of the agar-digesters of the eastern Gulf of Mexico. In an effort to contribute to the knowledge of these microorganisms, an investigation was conducted to

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study the agarolytic bacteria in the subtropical Anclote estuary near Tarpon Springs, Florida. The study is concerned with the abundance and characteristics of those isolated from surface waters and sediments. 2 The study was undertaken at a time when the construction of a power plant was near completion. In this context the work may serve as an aid in evaluation of its environmental impact after the initiation of plant operations. The writer was the recipient of a half-time research assistantship from funds supplied by the Florida Power Cor poration of St. Petersburg during the period of field work.

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3 LITERATURE REVIEW Table 1 lists all the agar-digesting bacteria described to date with reference, habitat, and source. Many papers dealing primarily with the reporting of agar-digesters are summarized in this fashion and are not included in the disquisition below. Appendix 1 contains a discussion of the structure of agar, and possible modes of the breakdown of this polysaccharide by agarolytic bacteria. Twenty years after the use of agar in preparation of bacterial media began, the first agar-digester was isolated, described, and named Bacillus gelaticus (now Pseudomonas gelatica) by Gran in 1902. This organism was obtained from waters off the coasts of Norway and Holland. Waksman and Bavendamm (1931) and Bavendamm (1931) were the first to report population counts of agarolytic bacteria finding 50,000 to 200,000 per gram of marine mud in the Bahama islands. In addition they studied the action of Pseudomonas gelatica on agar, which they found to be rapidly hydrolyzed with the end products being used as energy sour-ces. Zobel! and Allen (1935) reported that Flavobacterium amocontactum lost its ability to liquefy agar upon prolonged cultivation. This phenomenon was again reported by Zobel!

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Table 1 Previously reported agarolytic bacteria Acetobacter diversum pot ens singulare Achromobacter nenckii Agarbacterium amocontactum aurantiacum boreale bufo ceramicola delesseriae mesentericum past ina tor polysiphoniae reducans rhodomelae uliginosum sp. sp. sp. sp. Alginomonas alginivora fucicola Bacillus agar-exedens Bacterium betae-viscosum Beneckea sp. REFERENCE Humm 1946 Humm 1946 Humm 1946 Biernacki 1911 ZoBell and Allen 1934 Angst 1929 Lundestad 1928 Angst 1929 Lundestad 1928 Lundestad 1928 Angst 1929 Goresline 1933 Lundestad 1928 Angst 1929 Lundestad 1928 ZoBell and Uphamm 1944 Buck 1960 Fujita and Zenitani 1971 Kimura 1961 Swartz and Gorden 1959 Waksman, Carey, and Allen 1934 Waksman, Carey, and Allen 1934 Wieringa 1941 Panek 1905 Fujita and Zenitani 1971 HABITAT marine marine marine terrestrial marine marine marine marine marine marine marine terrestrial marine marine marine marine marine marine marine terrestrial marine marine terrestrial terrestrial marine SOURCE seawater, algae sediment seawater dried grapes seawater algae seawater algae seawater seawater. algae creamery wastes seawater seawater, algae seawater seawater, sediment seawater, sediment, algae algae seawater contaminant of blood-agar plate seawater, sediment, algae seawater soil, manure red beets algae

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Table 1 (Cont'd) Cytophaga diffluens flevensis krzemieniewskae sensitiva sp. Chondrococcus blasticus Flavobacterium sps. Nocardia atlantica marina Pseudomonas atlantica beaufortensis coenobios corallina droebachense elegans elongata floridana gelatica gelidicola hypothermis inertia iridescens kyoto ens is REFERENCE HABITAT Stanier 1941 marine Van der Meulen,Harder, fresh water and Veldkamp 1974 Stanier 1941 marine Humm 1946 marine Turvey and Christison marine 1967 Beebe 1941 terrestrial Chan and McManus 1969 marine Humm and Shepard 1946 marine Humm and Shepard 1946 marine Humm 1946 marine Humm 1946 marine ZeBell and Upham 1944 marine Humm 1946 marine Lundestad 1928 marine Araki and Arai 1954 marine Humm 1946 marine Humm 1946 marine Gran 1902 marine Kadota 1951 marine ZeBell and Upham 1944 marine Humm 1946 marine Stanier 1941 marine Araki and Arai 1954 terrestrial? SOURCE seawater lakewater seawater algae seawater soil seawater, algae algae sediment seawater, algae, sediment seawater, algae, sediment seawater, film of marine fouling organisms algae seawater, algae seawater sediment algae, sediment seawater seawater, algae sediment sediment seawater, algae, sediment agar "!anufacturing

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Table 1 (Cont'd) Pseudomonas lacunogenes marinopersica perfectomarinus periphyta roseola segnis stereotropis sp. sp. sp. sp. sp. sp. Streptomyces coelicolor Vibrio agarliquefaciens agarlyticus andoii avidus bei .i erinckii fortis freguens fuscus granii notus purpurens stanierii turbid us sp. REFERENCE Goresline 19JJ ZoBell and Upham 1944 ZoBell and Upham 1944 ZoBell and Upham 1944 Hurnm 1946 Goresline 1931 ZoBell and Upham 1944 Boffi 1969 Buck 1960 v Hofsten and Malmqvist 1975 Kimura 1961 Mitchell and Neva 1965 Nichols 1933 Stanier 1942 Gray and Chalmers 1924 Humm 1946 Cataldi 1940 Aoi and Orikura 1928 Humm 1946 Stanier 1941 Humm 1946 Humm 19'+6 Stanier 1941 Lundestad 1928 Humm 1946 Kadota 1951 Humm 1946 Humm 1946 Chan and M cManu s 1969 HABITAT terrestrial? marine marine marine marine terrestrial marine marine marine terrestrial marine marine terrestrial terrestrial terrestrial marine terrestrial terrestrial marine marine marine marine marine marine marine marine marine marine marine SOURCE creamery wastes sediment sediment film of marine fouling organisms sediment creamery wastes seawater seawater seawater, algae sewage seawater seawater soil soil soil sediment sludge manure sediment seawater, algae algae algae seawater seawater seawater, sediment seawater, intestine of sea-slug algae algae seawater, algae

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and Upham (1944) with several others isolated from the west coast. 7 Stanier (1941) studied the agar-digesting bacteria from the coast of California. Five new species were characterized with respect to their physiology and nutritional requirements. A comparison of agarolytic bacteria from marine and terrestrial sources showed marine agar-digesters not to have been found on land and vice versa up to that time. Humm (1946) in a detailed study, isolated twenty species of agarolytic bacteria, seventeen of which were new, from seawater, bottom sediments, and marine algae from the coasts of North Carolina and Florida. He studied their nutrition especially with respect to the utilization of carbon and nitrogen sources. Population counts ranging from 2 143 agarolytic bacteria per ml of seawater and from 2 million to 20 million per gram of intertidal sand were reported. If the study of agar-digesters up to this time had been largely concerned with their isolation and characterization, beginning in the 1950's their biochemistry commanded greater attention commencing with the agarases that these bacteria produced. Working with Vibrio agar-liguefaciens, Ishimatsu (1952, 1953 a, b) showed its enzyme to be adaptive. This enzyme showed an optimum pH of 6 -7 for liquefaction of agar with activity markedly reduced when the pH was below 5. Optimum temperature occurred near 40C, and the enzyme was inhibited below 30C and above 50C. Kadota (1951, 1953) obtained the enzyme of Vibrio purpurens and showed galactose

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to be the major end product with minor quantities of two unidentified sugars also produced. He also considered the effect of pH, temperature, and NaCl concentration on the activity of the enzyme. Continuing studies with y.agar-liguefaciens, Ishimatsu and Kibesaki (1955) thought that galactosido-3, 6-anhydroL-galactose was a component of agar based on its enzymic hydrolysis. Ishimatsu, Kibesaki, and Minamii (1956) studied the mechanism of agar hydrolysis and concluded that agar is first split into compounds of fairly high molecular weight and then split into compounds of lower molecular weight. Araki and Arai (1956, 1957) showed that the enzymic hydrolysis of agar by Pseudomonas kyotoensis gave neoagarotetrose which upon further enzymic hydrolysis produced neoagarobiose. The results of the acid hydrolysis of this disaccharide indicated that it was constructed of 3-o-3', 6'-anhydro-L-galactopyranosyl-D-galactose with a'-glycoside linkages. Yaphe (1957) used the extracellular hydrolase of Pseudomonas atlantica to break down agar obtained from various algae in order to compare the end products of these reactions. Ducksworth and Yaphe (1971) using the agarase of the same bacterium broke down the major components of agar in order to characterize the products of hydrolysis. They concluded that 4, 6-0-(1-carboxyethylidene)-D-galactose replaces the basic repeating unit of D-galactose in low sulfur agar molecules. 8

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9 Swartz and Gorden (1959) isolated an agar-digester (probably an Agarbacterium) from a blood-agar plate, studied its adaptive agarase and showed the release of oligosaccharide units but not free galactose from agar. Turvey and Christison (1967 a, b) prepared an enzyme extract from a marine Cytophaga which was more active on agarose than on other polysaccharides. Ducksworth and Turvey (1968, 1969 a, b, c) continued the research of the agarase obtained from the aforementioned bacteriQm on agarose and related polysaccharides, and concluded that the enzyme is most active on linkages positioned away from the ends of the polysaccharide chains and the sulfate hemiester groups. Sampietro and Vattuone de Sampietro (1971) studied the agarolytic system of Agarbacterium pastinator and showed the enzymic extract to have a high specificity for polysaccharides containing 3, 6-anhydro-L-galactose and to split the linkages of agarose. They suggest that the hydrolysis of agarose by Vibrio agar-liguefaciens (Ishimatsu, Kibesaki, and Minamii 1956) occurred at the a(l-3) linkages. Young et al. (1971) postulated the necessity of three enzymes for the complete breakdown of agarose, an extracellular a or agarase, an a or {3 cell wall bound tetrasaccharidase, and an intracellular disaccharidase. This notion was also presented by Van der Meulen and Veldkamp (1974) for Cyto phaga .!) evens is Hofsten and Malmqvist (1975) isolated an agar-digester from sewage (probably a Pseudomonas species). Its agarolytic

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10 enzyme does not attack cross-linked agars. They suggested that living bacteria are more effective in degrading agar gels possibly due to their ability to move on the gel matrix and align the enzyme molecules in a manner assisting the process. From the coast of Connecticut Buck (1960) isolated thirty agar-digesters from open waters, sediment, and the algae Fucus and Ulva, while comparing methods for enumeration of marine bacteria. He studied them from a physiological standpoint. None of his strains corresponded to any agar-digester described up to that time. In a follow up study Girard, Buck, and Cosenza (1968) examined the nutritional characteristics of 29 agar-digesters (28 of which were from the last mentioned study) belonging to the genera Vibrio, Agarbacterium, Pseudomonas, or Alginomonas. They considered these requirements on the whole as "simple" since most could subsist on galactose and a single amino acid. All but one of their bacteria could synthesize vitamins. Yaphe (1963) attempted to deal with the classification of bacteria capable of using algal polysaccharides. He suggested revisions in the definitions of the genera Alginomonas, Alginobacter, and Agarbacterium. A halophilic marine Pseudomonas was isolated from media enriched with capsules of Flavobacterium by Mitchell and Neva (1965). This bacterium caused complete liquefaction of agar, and was able to use as a sole source of carbon the capsular polysaccharides of Flavobacterium. Other agar-digesters

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11 were also obtained on media similarly enriched with capsules of Azotobacter, Rhizobium, Arthrobacter, and Escherichia coli ']2' Boffi (1969) studied three heterotrophic bacteria, one of which was an agar-digester, with regards to their nutrition, biochemistry, amino acid pools, respiration rates, and growth inhibitors. Studying the distribution, characterization, and nutrition of marine bacteria from the algae Polysiphonia lanosa and Ascophyllum nodosum, Chan and McManus (1969) found five agarolytic isolates. Tilak and Gordon (1971) obtained some agar-dissolving bacilli from soil, leaf manure, and mud, but did not describe or identify them. From the algae Porphyra yezoensis Fujitani and Zenitani (1971) isolated a number of bacteria which attacked algal polysaccharides. Regarding the classification of these bacteria, they felt that neither agarolytic and chitinoclastic activities nor type of flagellation constituted legitimate taxonomic features. Kim and ZoBell (1972) studied the effect of high pressures on several enzymes including agarase from Pseudomonas atlantica. While the enzyme still functioned at pressures and temperatures corresponding to those of the deep ocean, there was a decrease in the rate of agar hydrolysis due to inactivation of the enzyme.

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12 In order to contribute to the body of knowledge concerning agar-digesters, this study was conducted in a previously uninvestigated location. A main directive was the enumeration of agarolytic bacteria, an area relatively unexplored as the literature review demonstrates, thereby unraveling part of the ecological role of these organisms in the environment.

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13 AREA DESCRIPTION About 30 miles north of St. Petersburg, the Anclote River meets the Gulf of Mexico. At this junction a shallow open ended estuary, known as Anclote anchorage, is formed that measures approximately 3 x 3 nautical miles (Figure 1). This Figure also shows the locations of the sampling stations. Water temperatures ranged from 10 34C with the highest recordings occurring during the summer months, June through August, and the lowest in January. Water temperatures in the river mouth generally reflect those of the anchorage. Salinities during 1974-1975 ranged from approximately 23 to 36 ppt in the anchorage, and are largely a function of the occurrence of wet and dry seasons. During 1974 highest salinities were recorded in late spring and the lowest ones in summer and early fall, while in 1975 the highest measurements occurred in summer and the lowest in winter. (Maynard et al. 1974, Ford unpublished data) The river mouth exhibited slightly lower salinities depending on tidal stage and weather conditions. Generally the difference was no more than 6 ppt and salinity was 20 ppt or better.

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0 t 0 1 4 N 15 2 m Figure 1. Sampling station in the Anclote estuary.

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15 Conditions in the mouth of the river have been modified somewhat since a power plant commenced operation in October of 1974 (Behrens 1975). Due to the low river flow and the high water requirements of the power plant, water from the estuary is drawn into the river mouth. Consequently, water quality now resembles that of the anchorage to a greater degree.

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16 MATERIALS AND METHODS In order to avoid contamination all glassware to come into contact with media was thoroughly scrubbed with Alconox, rinsed, soaked for 24 hours in 5% HCl, and rinsed again numerous times, the final rinses with deionized water. Sampling. Sampling of surface water was done in accordance with procedures described by the American Public Health Association (1973). Samples were collected in sterile wide mouthed bottles, and cooled while in transport to the laboratory. Bottom surface sediments were obtained with sterile 2 inch diameter PVC cores. These were also cooled until they were processed. No more than three hours lapsed between the time of collection and the time of plating. Enumeration. Total bacterial counts were made using an extinction-dilution method with seawater nutrient broth composed of 0.5% nutrient broth and 0.01% yeast extract. Tubes were incubated for one week at room temperature, at which time the number showing growth was recorded. The number of bacteria could then be obtained from the Halvorson and Ziegler (1934) tables. Enumeration of the agarolytic bacteria was performed by the pour plate method.(Humm 1946) Aliquots of 0.2 ml of raw water and 0.1 and 1.0 ml of a gram of sediment that had

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17 been dilutes 100 x were placed on the bottom of a Petri dish. Approximately 20 ml of molten medium at 45C were added. The contents of the dishes were swirled around to achieve uniform distribution of the bacteria. The media used was that of Humm (1946) (see Table 2). Plates were incubated at room temperature. Counts were performed approximately two weeks after plating since a sufficient period of time had to elapse for the colonies to become visible. Agar-digesters were recognized by the formation of a depression in the agar. Controls for all tests were run in order to assure the sterility of all media and equip ment.Isolation and maintenance. Strains were subsequently isolated by removing a colony from the plate, streaking it out numerous times to assure the purity of the isolate, and inoculating agar slants, slopny agar broths, and nutrient broth tubes. (see Table 2). Strains were maintained at room temperature and transferred weekly or according to the growth rate of the isolates. Complications arose initially in the isolation of agar-digesters. Apparently, high nutrient concentrations in media are not conducive to their proliferation. By substantially reducing the organic content in the media no further problems were experienced in maintaining the cultures. All incubations were at room temperature unless otherwise stated.

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18 Table 2 Composition of Media Used in the Isolation and Maintenance of Agarolytic Bacteria IV!edium I KNOJ 0.1 % K 2HP04 0 05 % Feso4 trace agar 1 5 % Agar Slants tryptone yeast extract TRIS NH4so4 agar 0 1 % 0.05 % 0.01 % 0.01 % 1 5 % ISOLATION !VIAINTENANCE Nutrient Broth Medium II KNOJ 0.1 % K 2HP04 0 05 % Feso4 trace peptone 0.1 % agar 1 5 % Slopny Agar tryptone yeast extract TRIS NH4so4 agar yeast extract 0.1 % tryptone 0.1 % nutrient broth 0.1 % 0.1 % 0 05 % 0 01 % 0.01 % O J %

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19 Identification. The latest edition of Bergey's Manual of Determination Bacteriology was the main source for the identification of the agarolytic bacteria isolated from the Anclote estuary. However, most agar-digesters are currently of uncertain taxonomic status and therefore characteristics of the isolates were also cross checked against the other descriptions provided by the literature, and where similar features were encountered these names were assigned. Colony and cell characteristics. Following isolation, colony characteristics on nutrient agar, growth on nutrient slants, and in nutrient broth were recorded. Gram stains were performed on 22 hour cultures using Hucker's modification. Cell morphology and motility were observed in a hanging drop from 22 24 hour nutrient broth cultures. If a culture showed motility, it was stained for type of flagellation using the technique described by Leifson (1951). Artificial seawater. Much of the media employed was prepared with artificial seawater. Its basic composition is described by Lyman and Fleming (1940). In addition 0.5 ml of a mixture of salts (Mgso 4 4 g; Feso4 .?H 2o, 0.02 g; MnS04 .4H 20, 0.002 g; deionized water 100 ml) was added to each liter. al. 1968). Salinity. Salinity tolerances were determined by inoculating cultures on nutrient agar plates containing o, 1, 2, J, 4, 6, and ? % NaCl. Growth was observed at 2, 4, ?, and 10 days.

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20 Temperature. Temperature tolerances were determined by inoculating seawater nutrient agar slants with the cultures and by incubating them in water baths at the following temperatures: 5, 15, 25, 35, and 45C. Observations were made every two days for two weeks. Catalase. The presence of catalase was tested for by making a smear of an isolate grown on an agar slant on a glass slide and adding a few drops of 3% hydrogen peroxide. The formation of bubbles was considered positive. (Society of American Bacteriologists 1957). Oxidase. The oxidase reaction was performed by flooding, after 48 hours, inoculated seawater nutrient agar plates with a solution of dimethylparaphenylenediamine. Results were considered positive if the color of the colonies changed to maroon (Kovacs 1956). Hydrogen sulfide. Production of hydrogen sulfide was tested for by suspending sterile strips of filter paper impregnated with lead acetate over an inoculated seawater broth medium. were checked periodically for four weeks. Development of a brown to black color on the filter paper was regarded as positive. The composition of the broth was protoeose peptone 3%, dextrose 0.1% (Humm 1946). This test was also attempted using SIM medium. Results, however were judged unsatisfactory, and this method was therefore abandoned. (Society of American Bacteriologists 1957 ).

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21 Indol. All isolates were grown in 1% seawater tryptone broth. When good growth had developed the production of indol was tested for by adding Kovac's reagent, composed of paradimethylaminobenzaldehyde in a mixture of amyl alcohol and hydrochloric acid to the tubes. The development of a pink color was interpreted as a positive result (Society of American Bacteriologists 1957). Acetyl-methyl-carbinol. Barritt's (1936) method for the production of acetyl-methyl-carbinol was used. Fluorescent pigments. The media of King et al. (1954) were employed to determine the presence of fluorescin and pyocyanin. Antibiotics. The bacteria were tested for their susceptibility to low levels of the antibiotics penicillin, chloramphenicol, erythromycin, streptomycin, and tetracycline. Culture tubes containing 4 ml of seawater nutrient broth were inoculated. Penicillin was added at levels of 2.5 and 5 i.u., and the remaining antibiotics were added at concentrations of 10 mg/ml. One set was used as the control to judge the development or lack of growth against. Results were read at the end of 24 hours (Tunstall and Gowland 1974) Starch. Utilization of starch was undertaken by inoculating seawater nutrient agar plates containing 0.2% starch. After 4 days the plates were flooded with Gram's I 2KI solution to determine the presence or absence of clear zones surrounding the colonies. (Society of American Bacteriologists 1957).

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Cellulose. In order to test for the utilization of cellulose, strips of filter paper were placed in culture tubes 22 so that the ends extended above the surface of the inoculated broth. Composition of the broth was yeast extract 0.2%, peptone 0.5%, K 2HP04 trace. Cultures were checked periodically for two months for damage to the strips. If at the end of the incubation period no noticeable action had occurred, the filter strips were removed, dried, and examined for deterioration (Humm 1946). Chitin. An attempt was made to test for the utilization of chitin by inoculating media composed of precipitated chitin 0.1%, yeast extract 0.1%, and agar 1.5%. However, the ingrowing bacteria made interpretation of the test impossible (Lear 1963). Alginic acid. To a sterile basal medium consisting of 0.2% yeast extract, 0.5% peptone, 0.1% K 2HP04 a trace of Feso4 and 1.5% agar was added powdered alginic acid covered with ethyl alcohol. The mixture was boiled for a few minutes to drive off excess alcohol, cooled to 45C, shaken to bring the particles into suspension, and poured into Petri dishes. Results were judged positive if a clear zone developed around the colonies (Humm 1946). Seawater nutrient plates containing CaC12.2 H 2 o 0.01%, Mgso 4 .7H 2o 0.2%, and Tween 20 or 80 1% were inoculated. Speckled and clouded medium surrounding colonies was interpreted as a positive result (Ullmann and Basius 1974).

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K-carrageenan. Seawater plates of 1.5% unrefined K-carrageenan from Hypnea musciformis with 0.2% KCl were prepared and inoculated. Formation of depressions was considered positive (Humm 1946). Organic acids. Organic acids tested included sodium acetate, sodium citrate, sodium fumerate, sodium gluconate, lactic acid, sodium malate, ammonium oxalate, pyruvic acid, and sodium tartrate. The technique employed was based on that described by Humm (1946) with a slight alteration in the composition of the media. Media consisted of organic acid 0.25%, NH4No3 0.2% in artificial seawater. The pH was adjusted to 7.8. The medium was filter sterilized, added to sterile tubes, 23 and observed 5 days for sterility. Cultures were observed at the end of 2, 4, 10, and 21 days for development of growth. A control set without organic acids was prepared and inocu-lated. Sugars and alcohols. Leifson's (1963) MOF medium was employed to test acid production from the following substances ara-binose, cellobiose, dextrose, fructose, galactose, glycerol, inulin, lactose, maltose, mannitol, mannose, melibiose, raf-finose, rhamnose, ribose, sorbitol, sucrose, trehalose, and xylose. Cultures were observed at 1, 2, 3, 7, and 14 days. Two sets were originally inoculated, one covered with mineral oil to check for fermentation. But after several sets it became apparent that the bacteria would not grow under anaerobic conditions, and subsequent tests omitted

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24 this step. A third set without a sugar or alcohol was inoculated in order to establish the veracity of the observed results. Poly-8-hydroxybutyrate. This compound was tested as a sole source of carbon in a mineral medium free of nitrogen sources at concentrations of 0.01%, 0.1%, and 1%. Utilization was judged by the development of growth. Amino acids. As sole sources of carbon and nitrogen 0.1% of amino acid in artificial seawater was prepared. As a sole source of nitrogen the medium consisted of 0.5% dextrose and 0.1% amino acid in artificial seawater. The pH was adjusted to 7.8. The media were filter sterilized, added aseptically to sterile tubes, and observed for a period of 5 days to assure sterility. After inoculation cultures were observed at 1, 2, 3, 4, 7, 14, and 21 days. Controls without amino acids but with dextrose were also run, (Humm 1946). Amino acids tested included: DL-alanine, L-arginine, L-glutamic acid, glycine, L-histidine, L-leucine, L-lysine, DL-methionine, DL-phenylalanine, L-tyrosine, proline, and DL-tryptophan. Gelatin. Initially media containing 12% gelatin was prepared. This concentration turned out to be too high for most isolates, and therefore it was decided to use Frazier's (1926) method where the gelatin level is 0.4%. Nitrate. Seawater nitrate broth was inoculated. When good growth had developed 1 ml of solution A (sulfanilic acid

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25 8 g, acetic acid 5 H 1000 ml) and 1 ml of solution B (dimethyl-a-naphthalamine 5 g, acetic acid 5 n 1000 ml) were added. The presence of a red color indicated that nitrate had been reduced to nitrite. If no color change was observed, several chips of amalgamated cadmium were added to check if nitrate had been reduced to free nitrogen (Society of American Bacteriologists 1957). Nitrate was also tested as a sole source of nitrogen in a medium composed of 0.5% dextrose and 0.1% KNOJ in artificial seawater (Humm 1946). Ammonia utilization. The media composed of 0.5% dextrose and 0.1% NH4Cl in artificial seawater was inoculated. Obser vations were made regularly over a period of three weeks. Development of turbidity was used as the criterion for a positive result. (Girard et al. 1968). Urea. Urea was tested as a sole source of nitrogen at concentration of 0.1% with dextrose at 0.5% in artificial seawater. The media was filter sterilized. pH was adjusted to 7.8. Observations were made periodically over three weeks. Utilization was determined by the development of turbidity. ( Humm 1946)

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RESULTS Table 3 contains the data collected on the total bacteria population in the surface waters and the sediments of Anclote anchorage. Numbers ranged from 1100 to over 100,000 bacteria per ml in the surface water. The total population varied seasonally. During the cooler months, 26 the period from November to April, counts were lower, generally between 1500 and 5000. In the warmer months from 10,000 to over 100,000 bacteria per ml were found. Two bottom samplings showed total bacterial populations in the surface sediments to range from 50,000 to over 10,000,000 bacteria per gram. The agarolytic bacteria numbered from fewer than 2.5 to 182.5 bacteria per ml. in surface waters (Table 4). Between 10 and 25 agar-digesters would appear to be the norm. Agar-digesters showed no clear seasonality. Their distribution in the estuary was fairly uniform except for three stations located at the intake canal, discharge canal, and 200 yards from the outlet (stations 9, 6, and 5 respectively, Figure 1). The agar-digesting bacteria showed a slight decrease in numbers during the period the outlet station was monitored.

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['Table 3 Counts of Total Bacterial Population in the Anclote Estuary From 1974 1975 C\l (expressed.in number/ml for surface water; number/gram for sediment). SURFACE WATER Station 2/6/74 3/2/75 4/10/74 5/8/74 7/3/74 11/3/74 ll/26h4 1 30,000 5, 750 25,000 7,020 2 10,500 23,000 3 12,000 1,930 4 '4,280 2,000 5 >100,000 2,900 6 6 ,'500 2,000 > 100,000 2,000 4,740 7 3,000 >100,000 3,000 2,750 8 9 13,975 1,350 2,500 >100, 000 10 3,275 1,475 1,000 11 1,160 12 3,200 13 14 15 1,000 >100,000 1,100

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CX) Table 3 (Cont'd) N SURFACE WATER BOTTOM SEDIMENTS Station 4/_16/.Z'2 '2LlLZ'2 6/.12/.Z'2 2L2LZ'2 6f.l2/.Z'2 2L2LZ'2 1 2 >100, 000 116,000 3 4 5 2,630 2,630 >100' 000 100,000 171,000 >10,000,000 6 5,420 9,180 100,000 298,000 >10,000,000 7 2,190 8 9 >100,000 23,000 49.300 93,000 10 11 12 13 14 15 >100,000 197,000

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0\ C\1 Table 4 Population counts of agarolytic bacteria in the Anclote esturay during 1974 -1975. Counts expressed as no. per ml for surface water and as no. per gram for bottom sediment. For station location refer to Figure 1. SURFACE WATER 1974 1975 Station 2-16 2-2 4-24 Z-2 2-16 11-12 4-16 6-12 2-2 1 12.5 82.5 10 2 12.5 0 5 25 5 17 7 2.5 0 3 12.5 10 15 5 5 4 5 0 2.5 22.5 5 10 25 12.5 7 70 7 27.5 45 7 2.5 27.5 15 6 15 167.5 17 12.5 50 10.5 22.5 25 12.5 2.5 27.5 7 7 7 17 22.5 50 7 7 2.5 5 25 2.5 8 12.5 0 10 0 25 7 9 55 42.5 15 32.5 55 11.5 15 22.5 10 7 40 7 10 182.5 45 42.5 65 7 11 0 0 5 17.5 7 5 10 12 25 30 10 2.5 5 10 13 40 22.5 12.5 15 14 50 5 15 12.5 7 5 16 3 17 5 5 12.5 18 12.5

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0 (""\ Table 4 (cont' d) BOTTOM SEDIMENTS Station 1975 5-1 5-23 6-12 9-9 2 500 150 150 5 250 300 6 800 1000 200 450 7 150 300 100 8 100 9 100 100 150 11 50 12 900 100 14 1000 250 15 1000 300

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31 Since most of the effort was concentrated on surface waters, the sampling of agarolytic bacteria in the bottom sediments was limited. Data available indicated the number ranged from 50 1000 per gram with an average number between 100 and 450. The number of agar-digesters in the sediment was from 10 to 100 times greater than the overlying waters. The sampling site closest to the power plant (Sta tion 6) contained the highest number of agar-digesters in a gram of sediment. This may be related to the high content of organic material contained in these samples as judged from a comparison of color and appearance. As a percentage of the total the agar-digesters comprise only a small fraction of both surface waters and bottom sediments. Generally they account for less than 1% though in some samples they accounted for up to 5.5% (Table 5). None of the agar-digesters were limited to a particular substrate in the estuary. While some were isolated from the sediment and some from the water column, all were encountered in both locations at some time. Characterization of the isolates was originally begun with 24 strains. During the course of investigation, however, i t became apparent that duplicates were included. It was therefore concluded that eleven species belonging to the genera Cytophaga, Flavobacterium, Pseudomonas, and Vibrio were present. Their morphological features are presented in Table 6 and their growth characteristics on agar slants and nutrient broth in Table 7 All the isolates proved to

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C\l (""\ Table 5 The percentage of agarolytic bacteria of the total population in the Anclo t e estuary during 1974 -1975. SURFACE WATER BOTTOM SEDIMENTS Station 2-6 3-2 4-24 5-8 7-3 11-13 4-16 5-l 6-12 9-9 6-12 9-9 1 1.43 0.04 2 0.12 0.02 0.003 0.13 0.04 3 0.08 4 1.12 5 0.07 1.99 0.29 0.03 0.02 0.15 6 0.27 o.63 0.05 0.47 0.48 0.30 0.01 o.o7 7 0.75 0.05 0.28 0.11 9 0.39 1.11 1.30 0.06 0.04 0.03 0.20 0.16 10 5 2.88 2.25 15 0.005 0.15

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C"'\ C"'' Table 6 Colony and Cellular Characteristics of the Agarolytic Bacteria Isolated From the Anclote Estuary. Isolate Gram Colony Description Cellular rods, single, short chains, some curved slightly, 3-5 1 2 3 4 5 6 circular, entire, flat, smooth, glistening, cream color with brown pigment developing in center of colony opaque, 3 mm circular, entire, flat, smooth, glistening, white, translucent, 1 mm circular, entire, flat, smooth, glistening, opaque, white with bluish hue, 3 mm X 0.25 J.1. rods, single, pairs, 1 x 0. 3 J.l. rods, curved, single, 4 -4.5 X 0.25 J.1. circular, entire, flat, rods, single, smooth, light yellow, pairs, 1.5 -opaque, 5 mm 3 x 0.3J.I. circular -irregular, entire, flat, smooth, opaque, dark yellow, 2mm circular, entire, convex, smooth, opaque, orange-red -orange, 3 mm, dies rapidly rods, single, 1 -1.5 X 0.4 J.l. rods, single, 2 3 X 0.25 J.l. Motility Flagellation Genus + + t + polar monotrichous polar monotrichous polar monotrichous polar monotrichous polar monotrichous Pseudomonas (elongata) Pseudomonas Vibrio Pseudomonas Pseudomonas Pseudomonas (corallina)

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Table 6 (Cont'd) Isolate Gram Colony Description 7 8 9 10 11 circular, entire -undulate, raised, granular, opaque, oker, 2mm irregular, curled, smooth, opaque, pink, spreader circular -irregular, undulate, striated pattern in colonies, translucent, yellow, 2 3 mm circular, undulate, flat, translucent, 3 mm goldenyellow circular, entire, flat, smooth, glistening, translucent becoming opaque, white producing red-brown pigment, 5 mm Cellular rods, may form chains to 130 4-6x0.2J.t rods, may form chains, 6 10 X 0.4 J.L rods, single, 3 -5 X 0.3 J.L rods, single, some pair, 2 3 X 0.25J.L rods, single, pairs, 1.5 x 0.25 J.L Motility Flagellation Genus + .. none polar monotrichous Pseudomonas (droebachen Cytophaga Flavobacterium Flavobac-Pseudomonas

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35 Table 7 Growth characteristics of agar-digesters on agar slants and in nutrient broth. Isolate 1 2 3 4 5 6 7 8 9 10 11 Agar slant filiform, slowly colored producing brown pigment, produced liquid filiform, white, glistening, produced liquid filiform, thin layer spreading from edges, creamy colored, produced liquid filiform, thin layer spreading from edges, yellow filiform, dark yellow filiform, orange to red-orange, died off rapidly filiform, becoming arborescent, yellow-orange, becoming stringy and tough rapidly spreading, pink, liquefied agar filiform, light yellow filiform, thir layer spreading from edges, oker filiform, red-brown to darkbrown, much liquid Nutrient broth ring, heavy growth flocculent, poor growth pellicle, moderate growth pellicle, light growth thin pellicle, moderate growth thin pellicle, granular, light growth pellicle, granular, light growth slight ring, poor growth flocculent, poor growth pellicle, moderate growth flocculent, poor growth

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36 be aerobic, gram negative rods. Photographs taken at 2000 x are included in Appendix II. One of the strains showed a slight curvature and based on this was thought to be Vibrio. All the rods are extremely thin, less than 0.5 Most were motile by means of single polar flagellum. The fragility of these flagella made staining exceedingly difficult. One strain, Cytophaga, distinguished itself by its gliding movement on agar plates, and by its extremely vigorous attack of agar. Considerable variation occurred in their action on agar, ranging from rapid and virtually total liquefaction to the formation of a slight depression, probably reflecting the difference in their enzyme systems. A similar range of enzymic action was observed on the other algal polysaccharide tested, k-carrageenan. The results of the nutritional investigation are presented in Table 8. Sugars were readily used by most bacteria as shown by the production of acid in the Leifson tests. Dextrose, galactose, and maltose were used by all and mannose by all but one. Only ribose was poorly utilized. No preference for mono-, di-, tri-, or polysaccharides was encountered indicating that they all have the necessary enzymes to break down the more complex sugars. A slightly higher rate of utilization of the hexoses by the agar-digesters as compared to the pentoses occurs amongst the monosaccharides. Cytophaga was the most active in sugar utilization using all, generally within 24 hours, with a consequent

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37 Table 8 Nutritional and biochemical characteristics of the agarolytic bacteria from the Anclote estuary. ISOLATE 1 2 J. 4 2 6 1 8 2. 10 11 -control + + + + + + + + arabinose + + + + + + + + + + + dextrose + + + + + + + + + fructose .. 1' + + + + + + + + 1' galactose 1' + + inulin + + + + + 1' + T lactose + + + + + + + + + + + maltose + + + + + + + + + + mannose + + + + + + + + melibiose + + + 1' + + + raffinose + + + + + + + + rhamnose + + + + ribose + + + + + + + + sucrose + + + + -t + + + + trehalose + + + + + + + + xylose ethanol + + glycerol + + t mannitol + sorbitol poly-B-hydroxybutyrate 0.01% poly-B-hydroxybutyrate 0.01% poly-B-hydroxybutyrate 1.0% cellulose t + + + + + alginate + + + + .. + + + .. + starch + + + + + + t + k-carrageenan

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38 Table 8 (Cont'd) ISOLATE 1. 2 1 4 .2. 6 2 8 .2 10 11 (ll 0 -control s:: 'i arginine Q + + + + + + + + (t) + + + + + + aspartic acid 0 H) T + + + + glutamic acid Q I + + + glycine 'i o' leucine 0 + + ::s + + t + t lysine I ::s methionine p. + T ::s + t + + phenylalanine 1-' c+ proline 'i + + + + 0 aq tryptophan (t) ::s + + + + tyrosine control + + + alanine + arginine (/) aspartic acid 0 s:: glutamic acid 'i + + + + + + + Q (t) glycine 0 leucine H) + + ::s ... lysine 1-' c+ + + + methionine 'i 0 aq -+ + phenylalanine (t) ::s + + + + + + + proline tryptophan + tyrosine

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39 Table 8 (Cont'd) ISOLATE 1 2 1 4 2 6 1 8 2 10 11 control + + acetate citrate formate + + + + + + lactate malate oxalate + + + + + + oxoglutarate + + + pyruvate succinate + tartrate + + + + + + tween 20 + + + + + + tween 80 dextrose in seawater + + ... dextrose + N03 + + + + + + dextrose + NHJ + + + + + + + + nitrate broth + + + urea in dol .. + + + + + gelatin + + + + + + + + ... + + catalase + + + + oxidase + + + + + + H 2 S acetyl-methyl-carbinol fluorescent pigment A fluorescent pigment B

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lowering of the pH of the medium. Pseudomonas (strain 11) was the next most active. While this isolate too produced acid from all the sugars, its action was somewhat slower. The amino acids were the "second most used" group as 40 a source of carbon. Here, glutamic acid and proline were most widely used. Vibrio metabolized the most amino acids, followed by Pseudomonas (strain 1) and Pseudomonas (strain?). Two isolates Pseudomonas (strain 5) and Flavobacterium (strain 9) did not use any. Organic acids and the alcohols proved to be the least readily utilized. In the former group lactic and oxoglutaric acid produced the most positive results, followed by acetic and pyruvic acid. Nine out of eleven bacteria were able to use at least one organic acid. Even when growth occurred in these media, it was poor. Only three bacteria showed acid production from alcohol, Pseudomonas (strain 2), Cytophaga, and Pseudomonas (strain 11). None of these was able to use ethanol, only one sorbitol, and glycerol and mannitol by three. Starch was used by all but one strain, alginate was used by only five, and cellulose not by any. While all species except Cytophaga, grew well on unrefined k-carrageenan, only two isolates (Pseudomonas strains 2 and 11) attacked the gel vigorously. The others except Flavobacterium (strain 9) caused only slight depressions. Poly-B-hydroxybutyrate proved to be a poor source of carbon. No bacteria showed any growth.

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Sources of nitrogen tested included nitrate, ammonia, urea, and twelve amino acids. Over half of the agar-digesters were able to use an inorganic source of nitrogen for growth, ammonia being preferred. Four others grew in the presence of an amino acid, while Flavobacterium (strain 9) did not grow in any of these media indicating more complex requirements. The amino acids proved to be better sources of nitrogen than carbon. Only tryptophan failed to stimulate any growth of those tested. Arginine was the most readily used of all sources of nitrogen tested followed by ammonia, alanine, and aspartic acid. While eight strains showed production of nitrite from nitrate in the nitrate broth medium, only three strains grew when inoculated into the simple media of glucose and nitrate. Gelatin was used by seven strains. Considerable difficulty was initially encountered when gelatin was incorporated into the medium at a concentration of 12%. Many strains apparently would not tolerate this level. 41 None of the microorganisms gave positive results when tested for the production of indol or acetyl methyl carbinol. None produced any fluorescent pigments, or was luminescent, a characteristic often encountered in marine bacteria. Five produced hydrogen sulfide. Of the five antibiotics tested only erythromycin proved effective by preventing growth of all cultures (Table 9). The remaining listed in order of decreasing potency were chloramphenicol, streptomycin, tetracycline, and penicillin

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C\l Table 9 The action of some antibiotics on the agar-digesters from the Anclote estuary ..:t Penicillin strepto%cin Chloramphenicol Erytromycin Tetracyclin i.u. 10 JJ.g rnl 10 11g/ml 10 JJ.g/ml 10 JJ.g/ml Strain 2.5 5.0 1 w t 2 t 3 + 4 + 5 + 6 + 7 w + 8 w + 9 + w 10 w w + 11 w .. w -weakly affected

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43 the latter being totally ineffective both at 2.5 and 5 i.u. Erythromycin, chloramphenicol, and streptomycin all interfere with the operation of the ribosome (Brock 1970). The first two inhibit the function of the 50 s component, while streptomycin affects the 30 s component. Physiologically the agar-digesters reflect the conditions of the estuary in which they live (Table 10). They were all strictly aerobic as have been all previously described species. Their salinity optimum, as indicated by the amount of growth, lay between 20 and 30 ppt. None would grow at 0 ppt but several strains showed growth at 7 ppt especially Pseudomonas (strain 1). With respect to temperature there appears to be a cool water and warm water population. One group thrives at 15 25C, while the other grows better at temperatures from 25 35C. This may reflect a summer and winter population. At 45C all the bacteria died, while at 5C only Pseudomonas (strain 4) grew well. The others showed good growth once returned to room temperature.

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44 Table 10 Salinity and temperature tolerances of the agar-digesting bacteria of the Anclote estuary. SALINITY (percent NaCl) Isolate 0 1 2 3 4 6 7-5 1 0 ++ +++ +++ ++ ++ + 2 0 +++ t++ +++ ++ + + 3 0 +++ ++ ++ ++ 4 0 +++ +-+ + T T 5 0 ++ ++ +++ ++ + + 6 0 0 +-+ + T T 0 7 0 + ++ + 0 0 0 8 0 t +++ t++ + 0 0 9 0 0 ... 0 0 0 0 10 0 ... + 0 0 0 11 0 ... ++ ++ + T 0 T -trace TEMPERATURE (oC) Isolate 5 15 25 35 45 1 0 ++ + .... + +++ 0 2 0 ++ +++ ++ 0 3 0 +++ +++ +++ 0 4 + +++ ++ofo ++ 0 5 0 +++ ++ + 0 6 0 ++ +++ ++ 0 7 0 ...... +++ ++ 0 8 0 +++ +++ 0 0 9 0 ...... + +++ 0 0 10 0 +.f.+ +++ 0 0 11 0 +t +++ ++ 0

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45 DISCUSSION The eleven bacteria described in this work conform to the general pattern of marine agarolytic bacteria that has been established by previous research. They are aerobic, gram negative rods belonging to genera known to contain agar-digesting bacteria. Some (Pseudomonas strains 1, 6, and 7) have been previously described while the remaining eight are thought to be undescribed strains and have been tentatively classified to the generic level. These eleven agar-digesters are believed to be a most representative sample of their parent population, being encountered repeatedly during population counts. Few population counts of agar-digesters are found in the literature. Waksman and Bavendamm (1931) reported 50,000 to 200,000 agarolytic bacteria in a gram of mud in mangrove stands in the Bahama islands. Pearse, Humm, and Wharton (1942) found between 200 and 15,000 per gram of sand on beaches near Beaufort, North Carolina. Humm (1946) reported counts ranging from two million to twenty million per gram of muddy wet sand of intertidal beach and from 2-143 agar-digesters per ml of seawater. In the present study agar-digesters numbered from fewer than 2 to 182 per ml of estuarine water and from 100 1000 per gram of bottom sediment at depths of 4 to 12 feet. From the latter two sets

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46 of figures it can be seen that the populations in the water column compare favorably, while those in the sediment reported in this study are somewhat lower. The percentage of the total population of agar-digesters from the water from both studies are in good agreement. Generally they account for less than 1% of the total. While Humm (1946) reported from 2 to 40% agarolytic bacteria in the sediment, those in the sediments of the Anclote estuary constitute a considerably smaller fraction, reflecting percentages of the overlying water. Of the agarolytic bacteria in the water column, one segment is undoubtedly part of a naturally occurring population swept along by water currents, the remainder deriving its origins from the bottom sediments when strong winds suspend that matter in the water column. During such periods the incidence of bacteria, including agar-digesters, increases drastically and subsequently declines as the water column loses its suspended load to the bottom once calmer metereological conditions prevail. The ostensible lack.of a seasonal response in the agar-digesting population is surprising. Two reasons are proposed. Since the population in the water column is relatively small, the sampling that was conducted may have been inadequate to detect seasonal fluctuations. The second explanation involves the existence of a winter and a summer population. The former would predominate during the cooler months of the year, while the latter would be at a maximum

PAGE 55

during the summer months. The nutritional study was concerned primarily with carbon and nitrogen requirements, and is therefore by no 47 means plenary. Since the composition of the media used in the nutritional tests obviously differs from that of the oceanic environment, the-results obtained are only an indication of what might be expected of agarolytic bacteria in their natural environment. They indicate a likely order of utilization amongst the substances tested as is shown by the high use of sugars compared to the other sources tested. Likewise some of the amino acids served as better sources of nitrogen than the inorganic species. While the nutritional requirements of many agar-digesters may be thought of as "simple", others have more complex ones as suggested by Girard et al. (1968). The results of this and other studies point this out. Many are capable of surviving on a single source of carbon and a single source of nitrogen. For some a single amino acid will suffice. McLeod et al. (1954) found the amino acids alanine, aspartic acid, and glutamic acid to be the most important to some marine bacteria. These three amino acids and proline were widely used by the bacteria in this work. These workers ascribed the need for these amino acids to the inability to transaminate keto acids or to a different terminal oxidative pathway. There seems to be a general agreement with Humm (1946), Stanier (1941), and Girard et al. (1968) in amino acid utilization by agar-digesters.

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48 Interestingly no agar-digester to date has been reported to possess bioluminescent properties or vice versa. There-fore at present bioluminescent and agarolytic bacteria have .to be regarded as mutually exclusive groups. This contrasts with other bacteria such as cellulose or chitin digesting bacteria some of which possess agarolytic characteristics. The operation of the power plant which began in Octo ber 1974 has had no measurable effect with respect to numbers on the agar-digesting population that exists in the estuary during the period of this study. The data obtained are insufficient to show any possible changes in the qualitative composition of the agarolytic population. The study of agar-digesters up to this time has dealt with describing species, measuring their population size, examining the enzymic breakdown of agar, and determining their nutrition, biochemistry, and physiology. Their ecological role remains largely unknown. It seems likely that Stanier (1941) is correct when he writes of the "undoubted importance of agar-digesters in the cycle of matter in the ocean, where agar and similar polysaccharides form a large part of the carbohydrate constituents of many marine algae, particularly the Rhodophyta." A simplified model of this cycle might look like: 0 2 + H 2o respiratio sugars sugari cell agarolytic bacteria wall algae others polysaccharides

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From available population counts the site of largest activity appears to be the bottom sediments especially in the near shore areas. In the open ocean the reverse may pccur where as Waksman and Bavendamm (1921) suggest a close relation exists between agar-digesters, nitrogen fixers, and algae. The Sargasso Sea might be one such area. Another site of potential activity is the surface of algae. Chan and McManus (1969) showed bacteria on algae to be 100 to 1000 times more concentrated than in the surrounding seawater. Although they did not quantitatively study agar-digesters, it would seem reasonable to assume that they would be similarly concentrated. Agarolytic bacteria are not the only organisms capable of recycling agar. Some fungi, echinoids, and fish are known to have the necessary enzymes to accomplish this task. In order to judge the true importance of agar-digest ing bacteria in the marine environment, data are needed regarding the portion of agar available to them that they use. Unfortunately the information in the literature is insufficient to provide an answer since little is known of the rate at.which an agar-digester breaks down the polysaccharide. It should be pointed out that agar-digesting is not their only function although it may be their most important one. They may function in other cycles as well such as the nitrogen and sulfur cycles since they have the necessary biochemical abilities.

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50 That agarolytic bacteria possess a gamut of enzymes is evidenced by their biochemical activities such as starch hydrolysis, carrageenan utilization, etc. When the bacteria were .cultured on media containing relatively high levels of nutrients in addition to agar it appeared that the latter was not as readily digested judging by the depth of depressions formed and the quantity of liquefaction. It may be inferred that agar is not an essential part of their food requirement and that other nutrients when present in abundance can provide the necessary energy sources. No doubt their ability to utilize a wide range of energy sources provides them with the ability to subsist under many different nutrient conditions.

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51 APPENDICES

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52 APPENDIX A Agar The definition of agar has undergone several revisions as more became known about this complex polysaccharide. One problem is that its composition is species dependent. Duckworth and Yaphe (1971 a, b) have redefined agar (Difco Bacto agar) as containing agarose, pyruvated agarose, and a sulphated galactan. The structure of the former may be depicted as: a(1-3) 0 0 D L D L The complete degradation of agar is accomplished by hydrolysis of both the a(l3) and 4) linkages to release D-galactose and 3, 6-anhydro-L-galactose units. While agar-digesters may differ in their approach to the breakdown of agar, depending on whether the a(l -3) or 0 -4) links are initially cleaved, a set of three enzymes,

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an extracellular agarase, a bacterial cell bound tetrasaccharidase, and an intracellular disaccharidase, is required for complete hydrolysis (Young et 1971). 53

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Isolate 1. Pseudomonas (thought to be elongata) Isolate 4. Pseudomonas Isolate 7. Pseudomonas (thought to be droebachense) Isolate 10. Flavobacterium ,, Isolate 2. Pseudomonas Isolate 5. Pseudomonas Isolate 8. Cytophaga Isolate 11. Pseudomonas Isolate 3. Vibrio Isolate 6. Pseudomonas (thought to be corallina) Isolate 9. Flavobacterium 54

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.5.5 BIBLIOGRAPHY

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BIBLIOGRAPHY American Public Health Association, Inc. 1955. Standard Methods for the Examination of Water and Wastewater, 13th Edition, New York, N.Y. 56 Angst, E.C. 1929. Some new agar-digesting bacteria. Publ. Puget Sound Biol. Sta. 7: 49 -63. Aoi, K., and Orikura, J. 1928. On the decomposition of agar, xylan, etc., and the sugars related to these hemicelluloses by Vibrio andoi n. sp. Centrabl. Bakt., Abt. II, 74: 321 333 Araki, c., and Arai, K. 1954. studies on agar-digesting bacteria and their enzymatic activities. Mem. Fac. Ind. Arts, Kyoto Tech. Univ., 3B: 7-23. Araki, C., and Arai, K. 1956. The chemical constitution of agar-agar. XVIII. Isolation of a new crystalline disaccharide by enzymic hydrolysis of agar-agar. Bull. Chern. Soc Japan 29: 339 -345. Araki, C., and Arai, K. 1957. Studies on the chemical constitution of agar-agar. XX. Isolation of a tetrasaccharide by enzymic hydrolysis of agar-agar. Bull. Chern. Soc Japan 30: 287 293 Barritt, M.M. 1936. The intensification of the Voges-Pros Kauer reaction by the addition of a -naphthol. J. Path. Bact. 42a441. Bavendamm, w. 1931. Die Zersetzung von Hemizellulosen, besonders von Agar-agar, durch das Meeresbakterium Bacillus gelaticus Gran. Ber. deut. Bot. Gesell. 49: 288 -289. Beebe, J.M. 1941. Studies on the Myxobacteria. I. Distribution in central Iowa with description of a new spew cies. II. The role of myxobacteria as bacterial parasites. Iowa State Coll. Jour. Sci. 15(3)a 307-337

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Behrens, P. 1975. Physical Data. Part I: Effect of Power Plant Initialization on Temperature.and Salinity Distributions in the Anclote Anchorage. p. 9 -35. In: Maynard, V.I. and Mayer, G.F. (Eds). 1975. Anclote Environmental Project Report 1975. Dept. Mar. Sci., u. South Fla., St. Petersburg, Fla. 57 Biernacki, w. 1911. Bacterium Nenckii Biernacki, ein neuer Agar verflussigender Centrabl. Bakt. Abt. II, 29: 166 -169. Boffi, v. 1969. Biochemical patterns of some heterotrophic marine bacteria grown in defined media. J. Gen. Micro bial. 55: 227 -242. Brock, T.D. 1970. Biology of Microorganisms. 737 p. Prentice-Hall, Inc., Englewood Cliffs, N.J. Buchanan, R.E., and Gibbons, N.E. (Coeditors). 1974. Bergey's Manual of Determinative Bacteriology. 8th Ed. The Williams & Wilkins Company, Baltimore. Buck, J.D. 1960. A Study of the Enumeration of Aerobic Marine Bacteria and of Stenohaline Agarolytic Isolates. M.S. Thesis, University of Connecticut, Storrs, Connec ticut. Cataldi, M.S. 1940. Una nueva bacteria licuante del agar. Rev. Inst. Bact. (Buenos Aires) 9: 366377' Chan, E.C.s., and McManus, E.A. 1969. Distribution, characterization, and nutrition of marine microorganisms from the algae lanosa and Ascophyllum nodosum. Can. J. Microbial. 15: 409 -420. Duckworth, M., and Turvey, J.R. 1968. agarase from a Cytophaga species. The extracellular Biochem. J. 109: 6 p. Duckworth, M., and Turvey, J.R. 1969 a. An extracellular agarase from a Cytophaga species. Biochem. J. 113: 139 -142. Duckworth, M., and Turvey, J.R. 1969. B. The action of a bacterial agarase on agarose, porphyran, and alkali treated porphyran. Biochem. J. 113: 687 -692. Duckworth, M., and Turvey, J.R. 1969 c. The specificity of an agarase from a Cytophaga species. Biochem. J. 113: 693 Duckworth, M., and Yaphe, W. 1971. The structure of agar. Part I. Fractionation of a complex mixture of polysaccharides. Carbohyd. Res. 16: 189 197

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