Identification and characterization of lipolytic and proteolytic bacteria isolated from marine sediments

Marine Pollution Bulletin collection of sediment samples. This work was supported by the Nuffield Foundation, The Natural Environment Research Council (GR/3/655 and GR/3/2420), and the National Aeronautics and Space Administration (NGL 05/003/003). We also thank Mrs J. Pillinger and Mrs A. Gowar of the Organic Geochemistry Unit, for technical assistance. Dutka, B. J., Chau, A. S. Y. & Coburn, J. (1974). Relationship between bacterial indicators of water pollution and faecal sterols, WaterRes., 8, 1047. Eglinton, G., Maxwell, J. R. & Philip, R. P. (1974). Organic geochemistry of sediments from contemporary aquatic environments. Adv. Org. Geochem. 1973, (Eds. B. Tissot and F. Bienner), p. 941. Paris: Editions Technip. Eneroth, P., Hellstrom, K. & Ryhage, R. (1964). Identification and quantification of neutral fecal steroids by gas-liquid chromatography and mass spectrometry: studies of human excretion during two dietary regimens. J. LipidRes., 5,254. Halcrow, W., Mackay, D. W. & Thornton, 1. (1973). The distribution of trace metals and fauna in the Firth of Clyde in relation to the disposal of sewage sludge. J. Mar. Biol. Ass. U.K., 53, 721. Halcrow, W., Mackay, D. W. & Bogan, J. (1974). PCB levels in Clyde marine sediments and fauna. Mar. Pollut. 5(9), 134. Hanes, N. B. & Fragala, R. (1967). Effect of seawater concentrations on survival of indicator bacteria. J. Water Pollut. Control Fed., 39, 97. Kirchmer, C. J. (1971). 5 Beta-Cholestan-3-Beta-ol: An indicator of fecal pollution, University of Florida, Ph.D. Thesis. Mackay, D. W. & Topping, G. (1970). Preliminary report on the effects of sludge disposal at sea. Effluent and Water Treatment Journal. Mackay, D. W., Halcrow, W. & Thornton, I. (1972). Sludge dumping in the Firth of Clyde. Mar. Pollut. Bull., 3(1), 7. Martin, W. J., Ravi Subbiah, M. T., Kottke, B. A., Birk, C. C. &

Naylor, M. C. (1973). Nature of fecal sterols and intestinal bacterial flora. Lipids, 8, 208. Murtaugh, J. A. & Bunch,R. L. (1967). Sterols as a measure of faecal pollution. J. WaterPollut. ControlFed., 39, 404. Natural Environment Research Council (1975). Estuaries research. Report of the NERC Working Party on Estuaries Research Publication Series 'B' No. 9. Newell, B. S. (1967). The determination of ammonia in seawater. £ Mar. Biol. Assoc. U.K., 47, 271. Porter, E. (1973). Pollution in four industrialised estuaries. Four case studies undertaken for the Royal Commission on Environmental Pollution. London: H.M.S.O. Rosenfeld, R. S., Fukushima, D. K., Hellman, L. & Gallagher, T. F. (1954). The transformation of cholesterol to coprostanol. J. Biol. Chem., 211,301. Rosenfeld, R. S. & Gallagher, T. F. (1964). Further studies of the biotransformation of cholesterol to coprostanol. Steroids, 4, 515. Rosenfeld, R. S., Paul, 1. & Yamauchi, T. (1967). Sterol esterification in feces. Arch. Biochem. Biophys. 122, 653. Rosenfeld, R. S. & Hellman, L. (1971). Reduction and esterification of cholesterol and sitosterol by homogenates of feces. J. Lipid Res., 12, 192. Tabak, H. H., Bloomhuff, R. N. & Bunch, R. L. (1972). Coprostanol-a positive tracer of fecal pollution. Developments in Ind. Microbiol., 13,296. Talbot, J. W. (1970). The influence of tides, waves and other factors on diffusion rates in marine and coastal situations. UN-FAU Technical Conference on Marine Pollution and its Effect on Living Resources and Fishing. Paper No. FIR:MP/70/E-43. White, K. E. (1972). The use of radio isotopes for the measurement of flow and dispersion characteristics. Conference on Measurement Techniques in Air and Water Pollution, organized by the Institute of Mechanical Engineers, 1 Birdcage Walk, London, on Wednesday 12 January, 1972.

Identification and Characterization of Lipolytic and Proteolytic Bacteria Isolated from Marine Sediments M A U R E E N F. N I T K O W S K I , S H E A R O N D U D L E Y a n d J O H N T. G R A I K O S K I N a t i o n a i M a r i n e Fisheries Service, N o r t h e a s t Fisheries Center, M i l f o r d Laboratory, Milford, C T 06460, U . S . A .

Lipolytic and proteolytic bacteria were isolated from sediments at two sampling stations in the New York Bight Apex and one sampling station each in Sandy H o o k Bay and Great Bay, New Jersey. The stations in the Bight Apex and Sandy H o o k Bay have received industrial wastes and sewage for several decades, while Great Bay has received little of such materials. Proteolytic counts were 2-4 times higher and lipolytic counts generally 4 times higher in the polluted areas. Of the isolates taken from casein and lipid plates, 76070 were Gram-negative rods; 80°70 of the latter were identified as Vibrio and Pseudomonas. The vibrios comprised more than 60070 of the isolates from Station 4 (Great Bay) and Station 1 (Bight Apex), and were tested for their ability to break down casein, lipid, starch, and chitin. From Station 1, 75°70 of the Vibrio were active in degrading one or more substrates in addition to the substrate of the initial isolation medium; from Station 4, 52070 of the Vibrio were active. This s t u d y was u n d e r t a k e n to e n u m e r a t e a n d ch ar acterize lipolytic a n d p r o t e o l y t i c b a c t e r i a f o u n d in the sediments o f diverse m a r i n e e n v i r o n m e n t s . T h e areas 276

studied were: (1) the N e w Y o r k Bight A p e x w h i c h is the region c i r c u m s c r i b e d by a line d r a w n s o u t h f r o m A t l a n t i c Beach, N e w Y o r k , to its i n t e r s e c t i o n with a line d r a w n east f r o m M a n a s q u a n , N e w Jersey; (2) S a n d y H o o k Bay, N ew Jersey; a n d (3) G r e a t Bay, N e w Jersey. T h e stations which were s a m p l e d in the N e w Y o r k Bight A p e x a n d S a n d y H o o k Bay are s h o w n in Fig. 1. S t a t i o n 1 is in the acid disposal area w h i c h has received the c o r r o s i v e wastes f r o m industrial processes at an a v e r a g e rate o f 2.3 mi l l i on m 3 a n n u a l l y f r o m 1960 to 1974 ( E n v i r o n m e n t a l I m p a c t S t a t e m e n t on the O c e a n D u m p i n g o f Sewage Sludge in the N e w Y o r k Bight, D r a f t , 1976, U . S . E n v i r o n m e n t a l P r o t e c t i o n A g e n c y , N e w Y o r k , N . Y . ) . S t a t i o n 2 is situated on the p e r i p h e r y o f the sewage sludge d u m p , directly in line with the e f f l u e n t f r o m L o w e r Bay, S a n d y H o o k Bay, an d the R a r i t a n River. S t a t i o n 3 is l o c a t e d in S a n d y H o o k Bay w h i c h receives p o l l u t a n t s f r o m m u l t i p l e sources a l o n g the N e w J e r s e y coast a n d is s e p a r a t e d f r o m the Bight A p e x by a n a r r o w peninsula. T h e bay has been designated as o n e o f the centres o f the f i n - r o t s y n d r o m e which occurs in m a r i n e fish in the N e w Y o r k Bight ( M a h o n e y et ai., 1973). S t a t i o n 4 is l o cat ed in G r e a t Bay, N e w Jersey, at 3 9 ° 3 0 ' N 7 3 ° 2 1 ' W , a n d receives very little

Volume 8/Number 12/December 1977

40o31 , totion

3

•

Station

2

25'

• Station

I

l

-~ 4 0 ° 13'

I 74°01 '

Fig.

56'

i

I

l i 50'

~

l 44'

i

i

I 73 °38'

1 Location of sampling sites in Sandy Hook Bay (Station 3) and the New York Bight Apex (Stations l and 2).

industrial or sewage waste from the sparsely populated land surrounding it. In comparison the stations in the Bight Apex and Sandy Hook Bay have received industrial and sewage wastes for several decades. Bacteria which decompose lipid and protein were selected as the target group because their numbers may reflect the organic enrichment of an area. Generally, bacterial counts increase with available organic nutrients; such nutrients can result from plankton blooms, dumping practices and land run-off, or a combination of these factors. In the open oceans, lipolytic and proteolytic bacteria are present in the surface film during organically productive periods when total counts range from 10 ~ to 103/ml (Sieburth, 1971). A direct correspondence was found in the water column of Long Island Sound between the numbers of protein and lipid digesters and changes in the plankton mass (Murchelano & Brown, 1970). This situation involves nutrients introduced from land which, in turn, support more plankton than would the open ocean. In a study of a freshwater stream, lipolytic bacterial counts of 105/ml were recorded below a sewage outfall, while the counts upstream from the outfall were always 100-fold lower (Blaise & Armstrong, 1973). These high lipolytic bacterial counts were sustained by the organic nutrients which were constantly being added to the system.

Materials and Methods Sediment samples were collected in August and September of 1973 and in September and October of 1975, using either a Smith-McIntyre or Ekman dredge. The top centimetre of sediment was removed with a sterile tongue depressor and placed in a sterile 8 oz French square bottle which was immediately packed in ice. All samples were tested within 24 h of collection.

Dilutions of the sediments were made on a volume-tovolume basis. The initial dilution was 100 ml of sediment to 100 ml of 0.1070 peptone-seawater diluent. Aliquots of 0.1 ml from serial dilutions were streaked in triplicate onto the appropriate media at suitable dilutions. The lipolytic medium was based on that developed by Eisenberg (1939) with the NaC1 concentration modified to 3.0070 and Bacto-lipase (Difco) as the lipid source. The base medium consisted of 1.5070 trypticase (Baltimore Biological Laboratories), 0.5070 phytone (Baltimore Biological Laboratories), 0.5070 yeast extract, 3.0070 NaCl, and 1.5070 agar in deionized-distilled water. After sterilization this solution was tempered to 50°C in a water bath, and Nile blue sulfate (Allied Chemical) and lipase added to give a final concentration of 0.001 070 (stock solution was 0.2 070Nile blue sulfate in deionized-distilled water) and 3.0070, respectively. The medium was swirled gently to insure mixing as it was poured into plates; plates were dried at room temperature for 24 h before inoculation. Inoculated plates were incubated at 20°C for 5 days, and colony counts were made at 3 and 5 days of incubation. Colonies were recorded as lipolytic if the fat droplets beneath or around the colony had changed from red to blue, indicating that the fat was no longer in a neutral state, and the surrounding zone of medium had become transparent. Jones & Richards (1952) have attributed toxicity to Nile blue sulphate for some Grampositive cocci; therefore, it was expected that the dye would act somewhat selectively for rods. The proteolytic medium consisted of 0.3070 beef extract, 0.5070 peptone, 3.0070 NaCl, and 1.5070 agar in deionized-distilled water. Casein was provided by adding a 1:1 volume-to-volume solution of 'Pet' (Pet Foods Division) evaporated, skimmed milk diluted aseptically in sterile deionized-distilled water; the final concentration of milk was 1.5070. To prevent the milk from precipitating, the base solution and milk were both tempered to 50°C before mixing. The mixture was swirled gently and poured into plates which were subsequently dried at room temperature for 24 h. After inoculation the plates were incubated at 20°C for 2 days when colony counts were made. A colony was judged to be proteolytic if surrounded by a clear halo of hydrolysed casein. In a study of proteolytic bacteria isolated from fish, Kazanas (1968) found that the isolates digested the protein of a fish-extract medium and casein as well; it would therefore seem that casein hydrolysis is a valid test of bacterial proteolysis in isolates from sources other than dairy products. Following the method described by Sizemore & Stevenson (1970), double-layered plates having a seawater-agar overlay were used to test whether increased lipolytic and proteolytic counts could be achieved. The base layer of the proteolytic medium consisted of sterile skimmed milk added aseptically to a sterile 1.5 070agar solution to yield a final concentration of 1.5070 skimmed milk. The base layer of the lipolytic medium was composed of sterile 1.5070 agar solution to which was added lipase (final concentration of 3.0070) and sterile Nile blue sulphate solution (final concentration of 0.001070). When the base layers of both media had solidified, an overlay of 0.05070 peptone, 0.05070 yeast extract, and 1.5070 agar in aged (minimum of 30 days), 277

Marine Pollution Bulletin filtered seawater (2 /tm filter) was poured on top. Sediment samples collected at Stations l, 2, and 3 were streaked onto these layered media and incubated at 20°C. The total number of colonies and the numbers of colonies which exhibited proteolysis or lipolysis were determined after 5 and 7 days of incubation. Isolated colonies on the non-layered plates which demonstrated lipolytic or proteolytic activity were picked to give as great a variety o f colonial types as possible and an equal sampling number f r o m each plate. Colonies were inoculated into trypticase soy broth (TSB) which had a final concentration of 3.0°70 NaC1, and incubated for 48 h at 20°C. A loopful of inoculum f r o m the broth culture was then streaked onto a plate which corresponded to the base medium (i.e. the nutrient medium without the test substrate) which had been used in the isolation; incubation was for 24 h at 20°C. Wellisolated colonies were picked with a sterile inoculating needle, tested for their G r a m reaction ( A W W A et al., 1971), and inoculated into long-term preservation medium (Bacteriological Analytical Manual for Foods, 1972). No isolates were taken from the double-layered plates. Prior to their characterization, the cultures in preservation medium were inoculated into TSB (NaCl concentration of 3.0%) and incubated at 20°C until the medium became turbid (24-48 h); a loopful of the broth culture was then streaked onto trypticase soy agar (TSA) plates (NaCl concentration of 3.0%) and incubated at 20°C for 24 h. A colony f r o m the plate was subsequently inoculated into TSB (3.0°/0 NaCl) and onto TSA (3.00/0 NaC1) slants which were incubated at 20°C for 24 h. All lipolytic or proteolytic, Gram-negative rods were classified according to the key proposed by Shewan et al. (1960); that is, inocula from the broth and slant were used to test bacterial motility, oxidase reaction, oxidation/ fermentation of glucose, sensitivity to pteridine 0/129 and sensitivity to 2.5 U of penicillin. The isolates identified as Vibrio were patched onto starch, chitin, and either lipid or casein plates to test for additional substrate-degrading activities. The starch agar was that of Vanderzant & Nickelson (1972), except that the sodium chloride concentration was reduced from 7.0% to 5.0%. The chitin plates were prepared according to the procedure of Burman (1967), using practical grade chitin. The starch and chitin plates were incubated at 20°C and checked daily f r o m day 2 to day 7 for growth and clear zones of degradation around the colonized area. The casein and lipid plates were incubated and read as before.

TABLE 2

Proteolytic and lipolyticcounts according to sampling site and layered vs non-layered plating medium. Proteolytic count ( × 103/ml) Sampling area Sewage Sludge Sandy Hook Bay Acid dump

Lipolytic count ( × 10J/ml)

Layered

Nonlayered

Layered

Nonlayered

140 700 40

120 1200 26

100 600 18

44 190 I1

Results and Discussion

Lipolytic and proteolytic counts recorded in 1973 were higher for all sampling sites than were those taken in 1975 (Table l). Counts from the New York Bight Apex stations and Sandy H o o k Bay were 3 or 4 times higher than those from Great Bay. Additionally, Great Bay counts remained stable compared to the fluctuation at the acid dump which perhaps is related to the continually changing character of the dumpsite. Increased proteolytic counts were not found by using layered plates; however, the layered method did yield a two- or three-fold increase in lipolytic counts (Table 2). This difference might be attributed to the toxic effect of the dye on cocci when the sample was streaked directly onto the indicator medium. It was also noted that total colony counts (as opposed to lipolytic or proteolytic counts) were 2 logs higher on the layered plates and in line with total viable counts which had been determined at these stations (unpublished data). Since this 2-log increase was not reflected by a proportionate increase in lipolytic or proteolytic counts, it was concluded that the counts on the non-layered plates were a fairly accurate estimate of the lipolytic and proteolytic bacteria in the marine sediments. O f the total number of isolates examined in this study 15070 were cocci, 9°70 Gram-positive rods, and 76% Gramnegative rods. Additionally, the Gram-negative rods were composed chiefly of vibrios and pseudomonads for all of the areas sampled (Tables 3,4). Members of the genus Vibrio represented f r o m 57 to 97% of the Gram-negative rods isolated from Station 4 (Great Bay) and Station 1 (acid dump); the average was 70% on casein plates from the 2 areas and 81% on lipid plates. Inasmuch as the acid d u m p has received industrial wastes since 1948, while Great Bay remained virtually TABLE 3

Identification of Gram-negativerods isolated on casein plates. No. isolates

°70 Vibrio

tested

°7o Pseudomonas

go Other

1973

1975

1973

1975

1973

1975

1973

1975

I0 18 13 25

--21 28

20 50 69 04

--90 57

80 28 31 24

--5 43

0 22 0 12

--5 0

Sample source

TABLE 1

Proteolytic and lipolytic counts according to sampling site and date. Proteolytic count (x

Sampling area SewageSludge Sandy Hook Bay Acid dump Great Bay

103/ml)

278

Lipolytic count (x

103/ml)

TABLE 4

Identification of Gram-negativerods isolated on lipid plates.

Aug-Sept 1973

Sept-Oct 1975

Aug-Sept 1973

Sept-Oct 1975

110 120 290 3 !/70*

--160 25

22 24 15 5/26"

--2 4

*Sampledtwice, once each month.

Sewage sludge S a n d y H o o k Bay Acid dump G r e a t Bay

~o Vibrio

N o . isolates tested

Sample source Sewage sludge Sandy Hook Bay Acid dump Great Bay

go

°7o Other

Pseudomonas

1973

1975

1973

1975

1973

1975

1973

1975

22 24 14 43

--9 34

41 46 57 79

--89 97

50 54 36 7

--0 3

9 0 7 14

--II 0

Volume 8/Number 12/December 1977 TABLE 5

Substrate utilization by Vibrio isolated on casein plates. Sample source Acid dump Great Bay

No. isolates tested

% isolates lipolytic

07oisolates chitinolytic

07oisolates starch hydrolysis

28 32

95 70

70 45

90 55

TABLE 6 Substrate utilization by Vibrio isolated on lipid plates.

Sample source Acid dump Great Bay

No. isolates tested

070 isolates lipolytic

070 isolates chitinolytic

07oisolates starch hydrolysis

16 67

75 50

53 35

70 55

untouched by such materials, the question arises as to whether the high incidence of vibrios found in both areas represents similar populations. When tested for their ability to break down casein, lipid, starch, and chitin, approximately 75°70 of the Vibrio isolates from Station 1 were active in degrading one or more substrates in addition to the substrate of the initial isolation medium; the average for Station 4 Vibrio isolates was 52070 (Tables 5, 6). These results suggest that different vibrios or at least different proportions of Vibrio species are found in disparate marine environments. More isolates should be tested to determine whether this trend can be statistically corroborated, and additional characterization of the isolates would be needed to evaluate the taxonomic significance of this finding.

The authors thank William Sprague of Northeastern University for his technical assistance. Use of trade names does not imply endorsement by the National Marine Fisheries Service, NOAA. AWWA, APHA & WPCF, (1971). Standard Methods for the Examination of Water and Wastewater, 13th ed. Washington: American Public Health Assoc. Bacteriological Analytical Manual for Foods, (1972). 3rd ed. Food and Drug Administration, Washington, D.C. Blaise, C. R. & Armstrong, J. B. (1973), Lipolytic bacteria in the Ottawa River. Appl. MicrobioL, 26, 733-740. Burman, N. P. (1967). Recent advances in the bacteriological examination of water, pp. 205-206. In Progress in Microbiological Techniques, C. H. Collins (ed.). New York: Plenum Press. Eisenberg, G. M. (1939). A Nile blue culture medium for lipolytic microorganisms. Stain Technol., 40, 63-67. Jones, A. & Richards, T. (1952). Night blue and Victoria blue as indicators in lipolysis media. Proc. Soc. Bacteriol., 15, 82-93. Kazanas, N. (1968). Proteolytic activity of microorganisms isolated from freshwater fish. Appl. MicrobioL, 16, 128-132. Mahoney, J. B., Midlige, F. H. & Deuel, D. G. (1973). A fin rot disease of marine and euryhaline fishes in the New York Bight. Trans. Am. Fish. Soc., 102,596-605. Murchelano, R. A. & Brown, C. (1970). Heterotrophic bacteria in Long Island Sound. Mar. BioL, 7, 1-6. Shewan, J. M., Hobbs, G. & Hodgkiss, W. (1960). A determinative scheme for the identification of certain genera of gram-negative bacteria, with special reference to the Pseudomonadaceae. J. appL BacterioL, 23(3), 379-390. Sieburth, J. MeN. (1971). Distribution and activity of oceanic bacteria. Deep-Sea Res., 18, 1111-1121. Sizemore, R. K. & Stevenson, L. H. (1970). Method for detecting and isolating proteolytic marine bacteria. AppL MicrobioL, 20, 991-992. U.S. Environmental Protection Agency, (1976). Environmental Impact Statement on the Ocean Dumping of Sewage Sludge in the New York Bight. Draft Report, 341 pp. Vanderzant, C. & Nickelson, R. (1972). Procedure for isolation and enumeration of Vibrio parahaemolyticus. Appl. MicrobioL, 23, 26-33.

Distribution of Viral and Bacterial Pathogens in a Coastal Canal Community CHARLES P. GERBA, SAGAR M. GOYAL, ERIC M. SMITH and JOSEPH L. MELNICK

Department of Virology and Epidemioiogy, Baylor College of Medicine, Houston, TX 77030, U.S.A. Significant concentrations of human enteric viruses and bacteria were found to be present in the water and sediment of a coastal canal community into which secondarily treated sewage was being discharged. Canal communities are becoming increasingly popular along the southern coastline of the United States. These communities are designed so that each home has canal frontage which allows owners to have direct water access for their boats, as well as for other recreational activities. Many of the communities, especially along the Texas coast, are designed primarily for resort purposes and are occupied only on weekends and during the summer, but others are designed as permanent family residences. The growth of these communities has been rapid and largely unregulated, and their effect on water quality has yet to be thoroughly ascertained. Pollution of such canal systems would be especially undesirable because of their heavy usage for such recreational activities as bathing,

boating, skiing, fishing, and skin and scuba diving. This study was conducted to determine the occurrence and distribution of bacterial and viral pathogens in a coastal canal community located along the upper Texas Gulf coast into which secondarily treated sewage was being discharged. Materials and Methods Figure 1 shows the location and design of the study site. The canals are approximately 12 yr old and 400 m in length. The homes are served by a collection system and a small activated sludge sewage treatment plant which discharges unchlorinated effluent at the location shown in Fig. 1. The lots are small and the homes are closely packed. The canals range from 2-4.7 m deep and some have had a history of fish kills. The bottom sediment varies from organic muck at some locations to sand and shell at others. Water and sediment samples were taken at the stations indicated in Fig. 1. Water samples for 279