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Substrate specificity of heterotrophic bacteria in the water and sediment of a carp culture pond
Aquaculture, 64 (1987) 39-46 Elsevier Science Publishers B.V., Amsterdam
39
-
Printed
in The Netherlands
Substrate Specificity of Heterotrophic Bacteria in the Water and Sediment of a Carp Culture Pond HARUO SUGITA, KEN OSHIMA,
TOSHINORI
Department of Fisheries, College Of&ricdtuFe Shimouma 3, Setagaya, Tokyo 154 (Japan) (Accepted
8 December
FUSHINO
and YOSHIAKI
DEGUCHI
and Veterinary Medicine, Nihon University,
1986)
ABSTRACT Sugita, H., Oshima, K., Fushino, T. and Deguchi, Y., 1987. Substrate specificity of heterotrophic bacteria in the water and sediment of a carp culture pond. Aquacu/ture, 64: 39-46. A total of 489 strains of bacteria were isolated from the water and sediment of a carp culture pond, and their ability to decompose five types of organic matter was examined. Casein, tributyrin and starch were hydrolyzed by 26-65% of the isolated bacteria while chitin and cellulose were refractory and decomposed by only 1.2-8.8% of the isolates. The decomposers of casein, tributyrin and starch were present in the water and sediment throughout the year and Vibrionaceae were the major decomposers. Chitinolytic and cellulolytic bacteria were detected during defined periods.
INTRODUCTION
It is well known that heterotrophic bacteria assimilate materials directly from the abiotic portion of an ecosystem or from materials released by excretion or death of organisms in the aquatic ecosystems. These materials are utilized as energy sources and building blocks for bacterial growth. Since ZoBell (1946) pointed out the importance of heterotrophic bacteria in the marine ecosystem (Boffi, 1969)) many studies concerning the ecology and physiology of heterotrophic bacteria have been performed in natural waters (e.g., Colwell and Morita, 1974; Skinner and Shewan, 1977; Klug and Reddy, 1984). However, there have been few studies on the quantitative and qualitative aspects of microbiology in fish culture ponds. Kawai et al. (1975) and Ram et al. (1982 ) reported the ecology of bacteria which possess a special function in culture environments; however, no information regarding the taxonomic aspect of bacteria was given. Previously we dealt with seasonal changes of microflora of heterotrophs in the water and sediment of culture ponds rearing carp (Cyprinus carpio) and goldfish ( Carassius aurutus) , and reported that aerobic Gramnegative bacteria including Pseudomonas, Vibrio-Aeromonas group, Flavobac-
0044-8486/87/$03.50
0 1987 Elsevier Science Publishers
B.V.
terium, Acinetobacter, Moraxella and Enterobacteriaceae, and anaerobic Bacteroidaceae predominated in the water of culture ponds (Sugita et al., 1985a). In addition to these bacterial groups, Bacillus, Micrococcus and Clostridium were also major components in the sediment of culture ponds. In the present paper, we describe the biochemical properties of bacteria and the seasonal changes of decomposers of various organic substances in the water and sediment of the carp rearing pond. MATERIALS AND METHODS
The carp ( Cyprinus carpio) rearing pond is ca. 2500 m2 and located in Tokyo Metropolitan Fisheries Experimental Station, Mizumoto, Tokyo. About 5600 kg of carp were maintained and fed artificial diets, mainly pellet diets (Nihon Haigo Shiryo) which contained > 39.0% crude protein, < 15.0% ash, < 5% crude fiber, > 4% oil, > 1.4% calcium, and > 1.3% phosphorus. The water and sediment were collected on 20 May, 16 July, 27 September, 11 November 1982, and 19 January and 4 March 1983 (Sugita et al., 1985a). Each diluted sample was plated onto l/20 PYBGF agar, PYBGF agar (Sugita et al., 1985b), PEA blood agar (BBL),MacConkey agar (Eiken) , EG blood agar (Nissui) , NBGT-1/3S blood agar (Sakata et al., 1981) , modified FM agar (Nissui) , Bacteroides agar (Nissui) and FM-CW blood agar (Eiken) . The first four media were aerobically incubated, and the last five agars anaerobically (Sugita et al., 1985a). After incubation, the bacterial colonies were divided into types according to colonial characteristics, and then three representatives of each colony type were picked up and purified. Methods for characterization and identification of aerobic and anaerobic bacteria have been described previously (Sugita et al., 1985a). A total of 489 strains, of which 120 were isolated from the water and 369 from the sediment, were studied. The substrate specificity tests performed in the present study were the hydrolysis of chitin, casein, tributyrin, starch and Avicel-cellulose. These five organic substrates were chosen as representatives of mucopolysaccharide, protein, lipid, and polysaccharides, respectively. The test media used for detecting these properties of isolates were l/20 PYBGF ( Sugita et al., 1985b) and Trypticase soy agar (BBL) for aerobic bacteria, and EG agar (Nissui) supplemented with 5% Fildes solution, a peptic digestion of blood, for anaerobic bacteria; the organic substrates under study were added separately in the concentrations: 5% precipitated chitin, 2% casein ( NBC), 2% tributyrin ( Wako) , 0.5% soluble starch (Wake), and 0.5% Avicel-cellulose (Asahi Ksaei) . The l/20 PYBGF agar contained 0.05% Trypticase peptone (BBL) ,0.025% Phytone peptone (BBL), 0.01% Lab-lemco powder (Oxoid), 0.01% Bacto-yeast extract (Difco), 0.01% glucose and 1% agar (Oxoid, No. 1); pH 7.5. Each isolate was streaked on the test media, and incubated at 20°C for l-3 weeks,
41 TABLE 1 Environmental
parameters
Parameters Temperature PI-I DO (ppm) COD (ppm)
( ‘C )
in the water of a carp culture pond May
July
Sept.
Nov.
Jan.
March
20.6 8.9 9.7 8.0
23.9 9.4 11.3 9.7
23.8 8.5 10.4 9.2
14.8 7.6 7.7 8.2
5.2 7.8 11.2 5.3
9.7 8.8 12.5 5.5
according to the organic substrate, under aerobic or anaerobic conditions. Anaerobiosis was established using the jar technique of Azuma et al. (1962). The hydrolysis of chitin, casein, tributyrin and cellulose was recognized as clear zones surrounding the growth. Starch hydrolysis was determined by a clearing of the medium around the colonies by flooding the plates with Lug01 iodine solution. After determination of the substrate specificity of isolates, the number of each bacterial genus with positive reactions was calculated into a count per 1 g or ml of material. The maximum counts for each genus among the nine agar media were regarded as accurate viable counts of the corresponding genus capable of hydrolyzing given organic substances. Chemical oxygen demand (COD) was determined by the hot method using potassium permanganate ( Rodier, 1975 ) . Other water quality parameters, temperature, pH and dissolved oxygen, were measured as described by Sugita et al. (1985a). RESULTS
AND DISCUSSION
The water in a carp culture pond had the characteristics given in Table 1. The maximum values. of COD and temperature were observed in July, and there was a positive correlation between the two parameters (r=0.947), using linear regression analysis. A total of 360 strains of aerobes, composed of 11 bacterial genera, and a total of 129 strains of anaerobes, which contained four bacterial genera, were isolated from the water and sediment of a carp culture pond, and examined for the ability to hydrolyze five organic substances ( Table 2 ) . The proportions of the bacterial strains possessing the ability to decompose each substrate varied with the organic substance and bacterial genus tested: mucopolysaccharide (chitin), O-32.2%; protein (casein), O-73.3%; lipid (tributyrin) , O-53.3%; polysaccharide (starch), O-88.9%; and polysaccharide (cellulose), O-5.1%. Generally, Vibrionaceae, coryneforms, Bacillus and Staphylococcus had the ability to decompose a wide range of organic substances. Cellulose was decomposed by only 1.2% of the isolates, which con-
42 TABLE 2 Characteristics culture pond Component
of different
(no. strain)
Acinetobacter (22) Moraxella (6) Pseudomonas (52 ) Enterobacteriaceae (28 ) Vibrionaceae (90) Flavobacterium (30) Coryneforms (39) Bacillus (59) Streptococcus (3 ) Staphylococcus (23) Micrococcus (8 ) Clostridium (26) Bacteroidaceae (92 ) Anaerobic cocci (10) Spirochete (1) Total (489)
genera of bacteria
Hydrolysis
isolated
from the water and sediment
of a carp
( %)
Chitin
Casein
Tributyrin
Starch
Cellulose
0.0 16.7 1.9 10.7 32.2 6.7 5.1 5.1
0.0 0.0
21.3 0.0 44.2 42.9 73.3 46.7 61.5 57.6 33.3 65.2 25.0 50.0 41.3 10.0 0.0
40.9 0.0 30.8 25.0 53.3 26.7 7.7 27.1 0.0 26.1 12.5 19.2 a.7 0.0 0.0
18.2 66.7 46.2 60.7 88.9 50.0 79.5 62.7 0.0 78.3 37.5 ntb nt nt nt
0.0 0.0 0.0 0.0 0.0 0.0 5.1 1.7 0.0 0.0 0.0 0.0 3.3 0.0 0.0
8.8
50.9
26.0
64.7
1.2
0.0 4.3
0.0 3.8
0.0
“Positive % . hnt = not tested.
sisted of coryneforms, Bacillus and Bacteroidaceae isolated from the sediment. Spirochetes could not hydrolyze any organic substances tested. Seasonal changes of bacterial groups which had the ability to decompose each organic substance in the water and sediment of a carp culture pond are given in Tables 3 and 4. Chitinolytic bacteria were detected in November, January, and March, with bacterial densities of 103/ml in the water and 105-106/g in the sediment. Vibrionaceae was the major component. Cellulolytic bacteria were detected in the sediment in May and July, with bacterial densities of 103-104/g, composed of coryneforms, Bacillus and Bacteroidaceae. Proteolytic, lipolytic and amylolytic bacteria (13, 11 and 10 genera, respectively) were detected in all the specimens of water and sediment. The maximum viable counts of the decomposer for the three organic substrates were all observed in September. The major decomposer of these substances was Vibrionaceae. This result seems to be reasonable because this family of bacteria, perhaps genus Aeromonas, has the ability to decompose a variety of organic substances (Table 2 ) , and it was the dominant organism in the water and sediment of freshwater culture ponds (Sugita et al., 1985a). The results obtained in this study show that the chitinolytic and cellulolytic
43 TABLE 3 Seasonal changes of viable counts (log no./ml) of different genera of bacteria decomposing macromolecular substrates in the water of a carp culture pond
various
Substrate
Component
May
July
Sept.
Nov.
Chitin
Moraxella Pseudomonas Enterobacteriaceae Vibrionaceae Flavobacterium Bacillus Clostridium Total
nd nd nd nd nd nd nd nd
nd nd nd nd nd nd nd nd
nd nd nd nd nd nd nd nd
nd nd 2.6 3.6 nd nd
Acinetobacter Pseudomonas Enterobacteriaceae Vibrionaceae Flavobacterium Coryneforms Bacillus Staphylococcus Clostridium Bacteroidaceae Total
nd 1.3 1.6 3.3 2.7 nd 1.3 1.6 1.3 2.3 3.5
nd 1.3 1.3 2.8 nd 1.3 2.7 2.8 nd nd 3.3
3.2
nd nd nd 3.6 2.6 nd nd nd 1.6 2.5 3.7
nd nd nd 2.8 2.3
2.3 2.3 1.6 3.3
1.3
1.3
Acinetobacter Morarella Pseudomonas Enterobacteriaceae Vibrionaceae Flavobacterium Bacillus Staphylococcus Clostridium Total
nd nd nd 1.6 3.3 1.6 nd nd nd 3.3
nd nd nd nd 2.4 nd nd nd nd 2.4
3.2
nd nd
Acinetobacter Moraxella Pseudomonas Enterobacteriaceae Vibrionaceae Coryneforms Bacillus Staphylococcus Total
nd
nd nd
3.2 nd 4.0 1.6 3.7 2.3 2.3 2.8 4.2
Casein
Tributyrin
Starch
‘nd= Not detected.
1.3 1.8 2.1
1.3 1.3
3.3
2.8
1.3
1.6
1.8
2.5
1.6 3.4
2.8 3.2
3.6 1.9 3.6 3.3 2.3 2.3 2.3 1.3 1.8 4.1
nd 3.6
1.3 3.6
1.3
1.3
1.3
3.5
3.6
3.3
2.6
2.3
1.8
2.3
nd nd 3.7
nd 4.0
nd nd 1.3 1.3 3.6
nd 1.3 nd 3.6
Jan.
March
1.3
nd
nd nd 2.8 2.3 2.3 nd 3.0
nd 3.3 nd nd nd 3.3
1.3
1.8
1.8
1.6
nd nd nd 3.0
nd 2.6 nd 3.5
nd nd nd nd 2.7 2.3 nd nd nd 2.8
nd
nd
nd nd
1.3 2.0
nd 2.8 1.3
1.3 1.6
nd 3.3 nd nd nd 2.3 3.4
1.8
nd 3.3 nd
2.3
1.6
nd 3.0
nd 3.3
44 TABLE 4 Seasonal changes of viable counts (log no./g) of different genera of bacteria decomposing macromolecular substrates in the sediment of a carp culture pond
various
May
July
Sept.
Nov.
Jan.
March
Pseudomonas Enterobacteriaceae Vibrionaceae Coryneforms Bacillus Staphylococcus Total
nd” nd nd nd nd nd nd
nd nd nd nd nd nd nd
nd nd nd nd nd nd nd
4.3 4.3 6.2 nd 4.3 3.3 6.2
nd nd 5.6 5.3 4.3 nd 5.8
nd nd 6.2 nd nd nd 6.2
Casein
Acinetobacter Pseudomonas Enterobacteriaceae Vibrionaceae Flavobacterium Coryneforms Bacillus Streptococcus Staphylococcus Micrococcus Clostridium Bacteroidaceae Anaerobic cocci Total
4.3 5.3 nd 5.5 nd 4.3 4.1 nd 2.6 3.8 nd 4.4 nd 5.8
nd nd nd 5.7 nd 3.3 5.6 nd 5.7 nd nd nd nd 6.1
5.3 6.1 6.6 6.6 4.1 5.0 4.3 4.3 5.8 nd 3.2 5.1 3.5 7.0
nd 4.3 4.3 6.0 nd 4.3 4.3 nd nd nd nd 3.0 nd 6.0
nd 4.3 nd 4.8 nd 4.1 5.6 nd 3.3 nd nd nd nd 5.7
nd 4.3 nd 5.8 nd 4.6 3.3 nd 4.3 4.3 5.1 3.3 nd 5.9
Tributyrin
Acinetobacter Pseudomonas Enterobacteriaceae Vibrionaceae Flavobacterium Coryneforms Bacillus Staphylococcus Micrococcus Clostridium Bacteroidaceae Total
4.3 5.3 nd 5.5 nd nd 4.1 3.9 nd 2.8 3.8 5.8
nd nd nd nd nd nd 5.4 5.7 nd nd nd 5.9
4.3 5.3 5.3 6.6 nd nd nd 4.3 nd nd nd 6.6
5.6 4.3 4.3 6.0 4.3 4.3 4.3 nd 4.5 nd nd 6.2
nd 4.3 nd 4.8 nd 5.3 5.6 3.3 nd nd nd 5.8
nd 3.3 nd 5.7 nd nd nd nd nd nd 3.9 5.7
Starch
Acinetobacter Moraxella Pseudomonas Enterobacteriaceae Vibrionaceae Flavobacterium Coryneforms Bacillus Staphylococcus Micrococcus Total
4.3 5.3 5.3 nd 5.5 4.1 4.3 4.1 3.9 3.8 5.9
nd nd nd 3.6 5.7 nd 3.3 5.8 5.7 3.3 6.2
nd nd nd 6.6 6.6 4.1 5.3 4.8 5.8 nd 6.9
5.4 nd nd 5.1 6.2 nd 4.6 4.3 3.3 nd 6.3
nd 3.3 4.6 nd 5.6 4.3 5.3 5.6 nd nd 6.0
nd nd 4.3 4.3 6.2 4.3 4.6 3.6 nd nd 6.2
Cellulose
Coryneforms Bacillus Bacteroidaceae Total
3.6 3.3 1.3 3.8
nd nd 4.3 4.3
nd nd nd nd
nd nd nd nd
nd nd nd nd
nd nd nd nd
Substrate
Component
Chitin
“nd = Not detected.
45
bacteria were detected at defined sampling times, and that the decomposers of another three substrates were present at all sampling times. These findings suggest that chitin and cellulose are refractory substances while casein, tributyrin and starch are bacteriologically labile in a carp culture pond, as in soil (Alexander, 1977). In fish farming, large amounts of organic matter, originating from excess feed, excreta and dead bodies of aquatic organisms, are always supplied to pond water and finally accumulate on the pond bottom. This organic matter is thought to be a major food source for the heterotrophs in the pond. Fish feed, in particular, contains easily digestible substances including casein and starch (Nose, 1980). These substances enhance the growth of decomposers, resulting in the enrichment of the bacteria. The amount of feed supplied depends upon water temperature since more feed is supplied in summer than in winter. The COD of the water may reflect the amount of feed supplied to the carp pond. In addition, other sources of organic matter such as phytoplankton, especially genus Microcystis, may not be negligible. At present, it remains unresolved which factor is the major source of organic matter in the carp pond. Nevertheless, the results obtained in this study showed that bacteria with an ability to decompose various organic substances were present in a carp pond, especially in the sediment, throughout the year. Moreover, the contribution of anaerobic bacteria to the mineralization of organic matter in the culture pond is to be expected when conditions for growth, including temperature and pH, are satisfied. ACKNOWLEDGEMENTS
We thank Mr. M. Miki and other members of the Tokyo Metropolitan Fisheries Experimental Station for their valuable assistance. This study was partly supported by a Grant-in-Aid from the Ministry of Education, Science and Culture of Japan.
REFERENCES Alexander, M., 1977. Introduction to Soil Microbiology, 2nd edition. John Wiley and Sons, New York, NY, 467 pp. Azuma, R., Ogimoto, K. and Suto, T.,1962. Anaerobic culture method with steel wool. Jpn. J. Bacterial., 17: 802-806. Boffi, VB., 1969. Biochemical patterns of some heterotrophic marine bacteria grown in defined media. J. Gen. Microbial., 55: 227-242. Colwell, R.R. and Morita, R.Y. (Editors), 1974. Effect of the Ocean Environment on Microbial Activities. University Park Press, Baltimore, MD, 587 pp. Kawai, A., Takatori, N. and Sugiyama, M., 1975. Aquaculture environments and microbes. In: The Japanese Society of Scientific Fisheries (Editor), Marine Ecosystem and Microbes. Koseisha-Koseikaku, Tokyo, pp. 112-125.
46 Klug, M.J. and Reddy, C.A. (Editors), 1984. Current Perspectives in Microbial Ecology. American Society for Microbiology, Washington, DC., 710 pp. Nose, T., 1980. Absorption and digestibility of nutrients. In: C. Ogino (Editor), Nutrient and Feed of Fish. Koseisha-Koseikaku, Tokyo, pp. 37-60. Ram, N.M., Zur, 0. and Avnimelech, Y., 1982. Microbial changes occurring at the sediment-water interface in an intensively stocked and fed fish pond. Aquaculture, 27: 63-72. Rodier, J., 1975. Analysis of Water. John Wiley and Sons, New York, NY, pp. 468473. Sakata, T., Sugita, H., Mitsuoka, T., Kakimoto, D. and Kadota, H., 1981. Isolation and distribution of obligate anaerobic bacteria from the intestines of freshwater fish. Bull. Jpn. Sot. Sci. Fish., 46: 1249-1255. Skinner, F.A. and Shewan, J.M. (Editors), 1977. Aquatic Microbiology. Academic Press, London, 369 pp. Sugita, H., Fushino, T., Oshima, K. and Deguchi, Y., 1985a. Microflora in the water and sediment of freshwater culture ponds. Bull. Jpn. Sot. Sci. fish., 51: 91-97. Sugita, H., Ushioka, S., Kihara, D. and Deguchi, Y., 198513.Changes in the bacterial composition of water in a carp rearing tank. Aquaculture, 44: 243-247. ZoBell, C.E., 1946. Marine Microbiology. Chronica Botanica Press, Waltham, MS, 240 pp.
39
-
Printed
in The Netherlands
Substrate Specificity of Heterotrophic Bacteria in the Water and Sediment of a Carp Culture Pond HARUO SUGITA, KEN OSHIMA,
TOSHINORI
Department of Fisheries, College Of&ricdtuFe Shimouma 3, Setagaya, Tokyo 154 (Japan) (Accepted
8 December
FUSHINO
and YOSHIAKI
DEGUCHI
and Veterinary Medicine, Nihon University,
1986)
ABSTRACT Sugita, H., Oshima, K., Fushino, T. and Deguchi, Y., 1987. Substrate specificity of heterotrophic bacteria in the water and sediment of a carp culture pond. Aquacu/ture, 64: 39-46. A total of 489 strains of bacteria were isolated from the water and sediment of a carp culture pond, and their ability to decompose five types of organic matter was examined. Casein, tributyrin and starch were hydrolyzed by 26-65% of the isolated bacteria while chitin and cellulose were refractory and decomposed by only 1.2-8.8% of the isolates. The decomposers of casein, tributyrin and starch were present in the water and sediment throughout the year and Vibrionaceae were the major decomposers. Chitinolytic and cellulolytic bacteria were detected during defined periods.
INTRODUCTION
It is well known that heterotrophic bacteria assimilate materials directly from the abiotic portion of an ecosystem or from materials released by excretion or death of organisms in the aquatic ecosystems. These materials are utilized as energy sources and building blocks for bacterial growth. Since ZoBell (1946) pointed out the importance of heterotrophic bacteria in the marine ecosystem (Boffi, 1969)) many studies concerning the ecology and physiology of heterotrophic bacteria have been performed in natural waters (e.g., Colwell and Morita, 1974; Skinner and Shewan, 1977; Klug and Reddy, 1984). However, there have been few studies on the quantitative and qualitative aspects of microbiology in fish culture ponds. Kawai et al. (1975) and Ram et al. (1982 ) reported the ecology of bacteria which possess a special function in culture environments; however, no information regarding the taxonomic aspect of bacteria was given. Previously we dealt with seasonal changes of microflora of heterotrophs in the water and sediment of culture ponds rearing carp (Cyprinus carpio) and goldfish ( Carassius aurutus) , and reported that aerobic Gramnegative bacteria including Pseudomonas, Vibrio-Aeromonas group, Flavobac-
0044-8486/87/$03.50
0 1987 Elsevier Science Publishers
B.V.
terium, Acinetobacter, Moraxella and Enterobacteriaceae, and anaerobic Bacteroidaceae predominated in the water of culture ponds (Sugita et al., 1985a). In addition to these bacterial groups, Bacillus, Micrococcus and Clostridium were also major components in the sediment of culture ponds. In the present paper, we describe the biochemical properties of bacteria and the seasonal changes of decomposers of various organic substances in the water and sediment of the carp rearing pond. MATERIALS AND METHODS
The carp ( Cyprinus carpio) rearing pond is ca. 2500 m2 and located in Tokyo Metropolitan Fisheries Experimental Station, Mizumoto, Tokyo. About 5600 kg of carp were maintained and fed artificial diets, mainly pellet diets (Nihon Haigo Shiryo) which contained > 39.0% crude protein, < 15.0% ash, < 5% crude fiber, > 4% oil, > 1.4% calcium, and > 1.3% phosphorus. The water and sediment were collected on 20 May, 16 July, 27 September, 11 November 1982, and 19 January and 4 March 1983 (Sugita et al., 1985a). Each diluted sample was plated onto l/20 PYBGF agar, PYBGF agar (Sugita et al., 1985b), PEA blood agar (BBL),MacConkey agar (Eiken) , EG blood agar (Nissui) , NBGT-1/3S blood agar (Sakata et al., 1981) , modified FM agar (Nissui) , Bacteroides agar (Nissui) and FM-CW blood agar (Eiken) . The first four media were aerobically incubated, and the last five agars anaerobically (Sugita et al., 1985a). After incubation, the bacterial colonies were divided into types according to colonial characteristics, and then three representatives of each colony type were picked up and purified. Methods for characterization and identification of aerobic and anaerobic bacteria have been described previously (Sugita et al., 1985a). A total of 489 strains, of which 120 were isolated from the water and 369 from the sediment, were studied. The substrate specificity tests performed in the present study were the hydrolysis of chitin, casein, tributyrin, starch and Avicel-cellulose. These five organic substrates were chosen as representatives of mucopolysaccharide, protein, lipid, and polysaccharides, respectively. The test media used for detecting these properties of isolates were l/20 PYBGF ( Sugita et al., 1985b) and Trypticase soy agar (BBL) for aerobic bacteria, and EG agar (Nissui) supplemented with 5% Fildes solution, a peptic digestion of blood, for anaerobic bacteria; the organic substrates under study were added separately in the concentrations: 5% precipitated chitin, 2% casein ( NBC), 2% tributyrin ( Wako) , 0.5% soluble starch (Wake), and 0.5% Avicel-cellulose (Asahi Ksaei) . The l/20 PYBGF agar contained 0.05% Trypticase peptone (BBL) ,0.025% Phytone peptone (BBL), 0.01% Lab-lemco powder (Oxoid), 0.01% Bacto-yeast extract (Difco), 0.01% glucose and 1% agar (Oxoid, No. 1); pH 7.5. Each isolate was streaked on the test media, and incubated at 20°C for l-3 weeks,
41 TABLE 1 Environmental
parameters
Parameters Temperature PI-I DO (ppm) COD (ppm)
( ‘C )
in the water of a carp culture pond May
July
Sept.
Nov.
Jan.
March
20.6 8.9 9.7 8.0
23.9 9.4 11.3 9.7
23.8 8.5 10.4 9.2
14.8 7.6 7.7 8.2
5.2 7.8 11.2 5.3
9.7 8.8 12.5 5.5
according to the organic substrate, under aerobic or anaerobic conditions. Anaerobiosis was established using the jar technique of Azuma et al. (1962). The hydrolysis of chitin, casein, tributyrin and cellulose was recognized as clear zones surrounding the growth. Starch hydrolysis was determined by a clearing of the medium around the colonies by flooding the plates with Lug01 iodine solution. After determination of the substrate specificity of isolates, the number of each bacterial genus with positive reactions was calculated into a count per 1 g or ml of material. The maximum counts for each genus among the nine agar media were regarded as accurate viable counts of the corresponding genus capable of hydrolyzing given organic substances. Chemical oxygen demand (COD) was determined by the hot method using potassium permanganate ( Rodier, 1975 ) . Other water quality parameters, temperature, pH and dissolved oxygen, were measured as described by Sugita et al. (1985a). RESULTS
AND DISCUSSION
The water in a carp culture pond had the characteristics given in Table 1. The maximum values. of COD and temperature were observed in July, and there was a positive correlation between the two parameters (r=0.947), using linear regression analysis. A total of 360 strains of aerobes, composed of 11 bacterial genera, and a total of 129 strains of anaerobes, which contained four bacterial genera, were isolated from the water and sediment of a carp culture pond, and examined for the ability to hydrolyze five organic substances ( Table 2 ) . The proportions of the bacterial strains possessing the ability to decompose each substrate varied with the organic substance and bacterial genus tested: mucopolysaccharide (chitin), O-32.2%; protein (casein), O-73.3%; lipid (tributyrin) , O-53.3%; polysaccharide (starch), O-88.9%; and polysaccharide (cellulose), O-5.1%. Generally, Vibrionaceae, coryneforms, Bacillus and Staphylococcus had the ability to decompose a wide range of organic substances. Cellulose was decomposed by only 1.2% of the isolates, which con-
42 TABLE 2 Characteristics culture pond Component
of different
(no. strain)
Acinetobacter (22) Moraxella (6) Pseudomonas (52 ) Enterobacteriaceae (28 ) Vibrionaceae (90) Flavobacterium (30) Coryneforms (39) Bacillus (59) Streptococcus (3 ) Staphylococcus (23) Micrococcus (8 ) Clostridium (26) Bacteroidaceae (92 ) Anaerobic cocci (10) Spirochete (1) Total (489)
genera of bacteria
Hydrolysis
isolated
from the water and sediment
of a carp
( %)
Chitin
Casein
Tributyrin
Starch
Cellulose
0.0 16.7 1.9 10.7 32.2 6.7 5.1 5.1
0.0 0.0
21.3 0.0 44.2 42.9 73.3 46.7 61.5 57.6 33.3 65.2 25.0 50.0 41.3 10.0 0.0
40.9 0.0 30.8 25.0 53.3 26.7 7.7 27.1 0.0 26.1 12.5 19.2 a.7 0.0 0.0
18.2 66.7 46.2 60.7 88.9 50.0 79.5 62.7 0.0 78.3 37.5 ntb nt nt nt
0.0 0.0 0.0 0.0 0.0 0.0 5.1 1.7 0.0 0.0 0.0 0.0 3.3 0.0 0.0
8.8
50.9
26.0
64.7
1.2
0.0 4.3
0.0 3.8
0.0
“Positive % . hnt = not tested.
sisted of coryneforms, Bacillus and Bacteroidaceae isolated from the sediment. Spirochetes could not hydrolyze any organic substances tested. Seasonal changes of bacterial groups which had the ability to decompose each organic substance in the water and sediment of a carp culture pond are given in Tables 3 and 4. Chitinolytic bacteria were detected in November, January, and March, with bacterial densities of 103/ml in the water and 105-106/g in the sediment. Vibrionaceae was the major component. Cellulolytic bacteria were detected in the sediment in May and July, with bacterial densities of 103-104/g, composed of coryneforms, Bacillus and Bacteroidaceae. Proteolytic, lipolytic and amylolytic bacteria (13, 11 and 10 genera, respectively) were detected in all the specimens of water and sediment. The maximum viable counts of the decomposer for the three organic substrates were all observed in September. The major decomposer of these substances was Vibrionaceae. This result seems to be reasonable because this family of bacteria, perhaps genus Aeromonas, has the ability to decompose a variety of organic substances (Table 2 ) , and it was the dominant organism in the water and sediment of freshwater culture ponds (Sugita et al., 1985a). The results obtained in this study show that the chitinolytic and cellulolytic
43 TABLE 3 Seasonal changes of viable counts (log no./ml) of different genera of bacteria decomposing macromolecular substrates in the water of a carp culture pond
various
Substrate
Component
May
July
Sept.
Nov.
Chitin
Moraxella Pseudomonas Enterobacteriaceae Vibrionaceae Flavobacterium Bacillus Clostridium Total
nd nd nd nd nd nd nd nd
nd nd nd nd nd nd nd nd
nd nd nd nd nd nd nd nd
nd nd 2.6 3.6 nd nd
Acinetobacter Pseudomonas Enterobacteriaceae Vibrionaceae Flavobacterium Coryneforms Bacillus Staphylococcus Clostridium Bacteroidaceae Total
nd 1.3 1.6 3.3 2.7 nd 1.3 1.6 1.3 2.3 3.5
nd 1.3 1.3 2.8 nd 1.3 2.7 2.8 nd nd 3.3
3.2
nd nd nd 3.6 2.6 nd nd nd 1.6 2.5 3.7
nd nd nd 2.8 2.3
2.3 2.3 1.6 3.3
1.3
1.3
Acinetobacter Morarella Pseudomonas Enterobacteriaceae Vibrionaceae Flavobacterium Bacillus Staphylococcus Clostridium Total
nd nd nd 1.6 3.3 1.6 nd nd nd 3.3
nd nd nd nd 2.4 nd nd nd nd 2.4
3.2
nd nd
Acinetobacter Moraxella Pseudomonas Enterobacteriaceae Vibrionaceae Coryneforms Bacillus Staphylococcus Total
nd
nd nd
3.2 nd 4.0 1.6 3.7 2.3 2.3 2.8 4.2
Casein
Tributyrin
Starch
‘nd= Not detected.
1.3 1.8 2.1
1.3 1.3
3.3
2.8
1.3
1.6
1.8
2.5
1.6 3.4
2.8 3.2
3.6 1.9 3.6 3.3 2.3 2.3 2.3 1.3 1.8 4.1
nd 3.6
1.3 3.6
1.3
1.3
1.3
3.5
3.6
3.3
2.6
2.3
1.8
2.3
nd nd 3.7
nd 4.0
nd nd 1.3 1.3 3.6
nd 1.3 nd 3.6
Jan.
March
1.3
nd
nd nd 2.8 2.3 2.3 nd 3.0
nd 3.3 nd nd nd 3.3
1.3
1.8
1.8
1.6
nd nd nd 3.0
nd 2.6 nd 3.5
nd nd nd nd 2.7 2.3 nd nd nd 2.8
nd
nd
nd nd
1.3 2.0
nd 2.8 1.3
1.3 1.6
nd 3.3 nd nd nd 2.3 3.4
1.8
nd 3.3 nd
2.3
1.6
nd 3.0
nd 3.3
44 TABLE 4 Seasonal changes of viable counts (log no./g) of different genera of bacteria decomposing macromolecular substrates in the sediment of a carp culture pond
various
May
July
Sept.
Nov.
Jan.
March
Pseudomonas Enterobacteriaceae Vibrionaceae Coryneforms Bacillus Staphylococcus Total
nd” nd nd nd nd nd nd
nd nd nd nd nd nd nd
nd nd nd nd nd nd nd
4.3 4.3 6.2 nd 4.3 3.3 6.2
nd nd 5.6 5.3 4.3 nd 5.8
nd nd 6.2 nd nd nd 6.2
Casein
Acinetobacter Pseudomonas Enterobacteriaceae Vibrionaceae Flavobacterium Coryneforms Bacillus Streptococcus Staphylococcus Micrococcus Clostridium Bacteroidaceae Anaerobic cocci Total
4.3 5.3 nd 5.5 nd 4.3 4.1 nd 2.6 3.8 nd 4.4 nd 5.8
nd nd nd 5.7 nd 3.3 5.6 nd 5.7 nd nd nd nd 6.1
5.3 6.1 6.6 6.6 4.1 5.0 4.3 4.3 5.8 nd 3.2 5.1 3.5 7.0
nd 4.3 4.3 6.0 nd 4.3 4.3 nd nd nd nd 3.0 nd 6.0
nd 4.3 nd 4.8 nd 4.1 5.6 nd 3.3 nd nd nd nd 5.7
nd 4.3 nd 5.8 nd 4.6 3.3 nd 4.3 4.3 5.1 3.3 nd 5.9
Tributyrin
Acinetobacter Pseudomonas Enterobacteriaceae Vibrionaceae Flavobacterium Coryneforms Bacillus Staphylococcus Micrococcus Clostridium Bacteroidaceae Total
4.3 5.3 nd 5.5 nd nd 4.1 3.9 nd 2.8 3.8 5.8
nd nd nd nd nd nd 5.4 5.7 nd nd nd 5.9
4.3 5.3 5.3 6.6 nd nd nd 4.3 nd nd nd 6.6
5.6 4.3 4.3 6.0 4.3 4.3 4.3 nd 4.5 nd nd 6.2
nd 4.3 nd 4.8 nd 5.3 5.6 3.3 nd nd nd 5.8
nd 3.3 nd 5.7 nd nd nd nd nd nd 3.9 5.7
Starch
Acinetobacter Moraxella Pseudomonas Enterobacteriaceae Vibrionaceae Flavobacterium Coryneforms Bacillus Staphylococcus Micrococcus Total
4.3 5.3 5.3 nd 5.5 4.1 4.3 4.1 3.9 3.8 5.9
nd nd nd 3.6 5.7 nd 3.3 5.8 5.7 3.3 6.2
nd nd nd 6.6 6.6 4.1 5.3 4.8 5.8 nd 6.9
5.4 nd nd 5.1 6.2 nd 4.6 4.3 3.3 nd 6.3
nd 3.3 4.6 nd 5.6 4.3 5.3 5.6 nd nd 6.0
nd nd 4.3 4.3 6.2 4.3 4.6 3.6 nd nd 6.2
Cellulose
Coryneforms Bacillus Bacteroidaceae Total
3.6 3.3 1.3 3.8
nd nd 4.3 4.3
nd nd nd nd
nd nd nd nd
nd nd nd nd
nd nd nd nd
Substrate
Component
Chitin
“nd = Not detected.
45
bacteria were detected at defined sampling times, and that the decomposers of another three substrates were present at all sampling times. These findings suggest that chitin and cellulose are refractory substances while casein, tributyrin and starch are bacteriologically labile in a carp culture pond, as in soil (Alexander, 1977). In fish farming, large amounts of organic matter, originating from excess feed, excreta and dead bodies of aquatic organisms, are always supplied to pond water and finally accumulate on the pond bottom. This organic matter is thought to be a major food source for the heterotrophs in the pond. Fish feed, in particular, contains easily digestible substances including casein and starch (Nose, 1980). These substances enhance the growth of decomposers, resulting in the enrichment of the bacteria. The amount of feed supplied depends upon water temperature since more feed is supplied in summer than in winter. The COD of the water may reflect the amount of feed supplied to the carp pond. In addition, other sources of organic matter such as phytoplankton, especially genus Microcystis, may not be negligible. At present, it remains unresolved which factor is the major source of organic matter in the carp pond. Nevertheless, the results obtained in this study showed that bacteria with an ability to decompose various organic substances were present in a carp pond, especially in the sediment, throughout the year. Moreover, the contribution of anaerobic bacteria to the mineralization of organic matter in the culture pond is to be expected when conditions for growth, including temperature and pH, are satisfied. ACKNOWLEDGEMENTS
We thank Mr. M. Miki and other members of the Tokyo Metropolitan Fisheries Experimental Station for their valuable assistance. This study was partly supported by a Grant-in-Aid from the Ministry of Education, Science and Culture of Japan.
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