- Email: [email protected]
The vitamin-B12-producing bacteria in the water and sediment of a carp culture pond
Aquaculture, 119 ( 1994) 425-43 1 Elsevier Science B.V., Amsterdam
425
AQUA 40 105
Short Communication
The vitamin-B,,-producing bacteria in the water and sediment of a carp culture pond H. Sugita=, A. Kuruma”, C. Hirato”, T. Ohkoshib, R. Okadab and Y. Deguchi” “Department of Fisheries, Nihon University, Tokyo, Japan ?!?aitama Prefectural Fisheries Experimental Station, Saitama, Japan (Accepted
12 October 1993)
ABSTRACT The heterotrophic bacteria isolated from a carp (Cyprinus carpio) culture pond were examined for vitamin-B,,-producing and -consuming abilities. Total viable counts in the surface water and sediment in the period during May to November ranged from 3.7 x 10’ to 2.2~ lo5 CFU.ml-’ and from 1.3x lo5 to 6.0~ 10’ CFU.g-‘, respectively. Of 746 strains tested, 8% of the bacteria consumed vitamin B,* and the remaining 92% produced it. More than 50% of the obligate anaerobes, especially the genus Clostridium, could produce large amounts of vitamin Biz while 21°h of aerobes/facultative anaerobes and no microaerophiles could produce it at the same level. These results strongly suggest that obligate anaerobes, as well as aerobes/facultative anaerobes, are important in supplying vitamin B,z to phytoplankton in freshwater culture ponds.
INTRODUCTION
In stagnant culture ponds, microalgae are known to play important roles in the freshwater fish cultivation: (a) supplement of oxygen, (b ) adsorption of nutrient salts, (c) rise and maintenance of water temperature, (d) decrease of light intensity, and (e) its nutritional values for fish. In the Japanese culture pond, a blue-green alga, Microcystis aeruginosa, is known to be one of the representative microalgae (Nomura, 1982). Recently, M. aeruginosa was found to require a large amount of vitamin Br2 for their growth (Shibukawa et al., 1986). This fact strongly suggests that the population density of M. aeruginosa, as well as that of other many species of unicellular algae (Provasoli and Carclucci, 1974), is regulated by the amount of vitamin Br2 in the Correspondence to: Dr. H. Sugita, Department tagaya, Tokyo, 154, Japan.
of Fisheries,
Nihon University,
0044-8486/94/$07.00 0 1994 Elsevier Science B.V. All rights reserved. SSDZ0044-8486(93)E0247-7
Shimouma,
Se-
426
H. SUGITA ET AL.
water. Since this substance is produced mainly by bacteria and phytoplankton in the aquatic ecosystem, the vitamin-Br2-producing ability of aerobic/facultatively anaerobic bacteria isolated from various waters was studied by several workers (Kurata and Kimata, 1968; Gillespie and Morita, 1972; Nishijima, 1985). On the other hand, Sugitaet al. (1985,1987,1991b, 1992) found that both obligate anaerobes and microaerophiles also occur as residents in the water and sediment of culture ponds and coastal regions. However, there is little information on the vitamin-BrTproducing ability of these bacteria, except for a report on coastal waters (Sugita et al., 1993). This study was undertaken, therefore, to evaluate the vitamin-Br2-producing ability of heterotrophic bacteria, including aerobes/facultative anaerobes, microaerophiles and obligate anaerobes, isolated from the water and sediment in a freshwater culture pond. MATERIALS AND METHODS
About 90 kg of carp (Cyprinus carpio) were reared in a stagnant water pond of about 1450 m2 in area and 65-78 cm in depth, which is located in Saitama Prefectural Fisheries Experimental Station, Kazo, Saitama. Crumbled or pelleted diets (Nihon Nosan Kogyo, Yokohama) were mainly fed during the period from May to November 199 1. Water samples were collected monthly using a sterile bottle from the surface water. Concomitantly, sediment samples were collected using an Ekman-Berge bottom sampler (Rigosha, Tokyo). All the samples were kept on ice, transported to the laboratory and processed within 4 h after collection, as described below. Each sample was diluted with a known quantity of phosphate buffer solution (pH 7.6) containing 0.05% L-cysteine hydrochloride and 0.1% Bactoagar (Difco, Detroit, MI), and placed onto PYBGF, 1/20PYBGF (Sugita et al., 1983) and FM-CW blood agar (Eiken Chem., Tokyo). The PYBGF agar medium contains 1% Trypticase peptone (BBL, Cockeysville, MD), 0.5% Phytone peptone (BBL),0.24% Lab-lemco powder (Oxoid, Hampshire), 0.2% Bacto-yeast extract (Difco ) , 0.1% glucose, 0.05% L-cysteine hydrochloride (only for anaerobic cultivation) and 1% agar No. 1 (Oxoid). The l/20 PYBGF agar medium contains 0.05% Trypticase peptone, 0.025% Phytone peptone, 0.0 1% Lab-lemco powder, 0.0 1% Bacto-yeast extract, 0.0 1% glucose and 1% agar. The pH value of both media was adjusted to 7.5. The inoculated plates were incubated at 20’ C for 10 days under either aerobic (PYBGF and l/20 PYBGF), microaerophilic (PYBGF and 1/2OPYBGF) or anaerobic (PYBGF and FM-CW) conditions. The microaerophilic condition ( 5% CO21 10% 02/85% N2) was established in a TE-HER CP02 02-CO2 incubator (Hirasawa, Tokyo). The anaerobic condition was established in an ANX-1 anaerobic glove box (Hirasawa), containing 10% H2/ 10% CO2/8O% N2. After incubation, bacterial colonies were counted. About 20 colonies were isolated
VITAMIN-B,,-PRODUCING
BACTERIA IN CARP CULTURE POND
421
at random from each plate and examined for ability to grow under aerobic and anaerobic conditions. All the isolates were divided into three types: (a) aerobes/facultative anaerobes, which grow either aerobically or both aerobically and anaerobically; (b ) microaerophiles, which can grow microaerophilically but do not grow both aerobically and anaerobically; and (c) obligate anaerobes, which grow only anaerobically. The isolates were identified as described previously (Sugita et al., 1988, 1992). On suitably diluted samples, colonies of each bacterial genus were counted and expressed as number (CFU, colony-forming units) per ml or g of material. For each bacterial genus, the highest count obtained on the different agar media was regarded as the estimated viable count for that type. The total viable count was obtained by summation of viable counts of all bacterial genera. The isolates were incubated in 1/2PYBGF broth at 20 oC for 5 days under aerobic, microaerophilic or anaerobic conditions. The 1/2PYBGF broth contains 0.5% Trypticase peptone, 0.25% Phytone peptone, 0.12% Lab-lemco powder, 0.1% Bacto-yeast extract, 0.05% glucose and 0.05% L-cysteine hydrochloride (only for anaerobic cultivation). The culture was then, assayed microbiologically for vitamin B12 (as cyanocobalamin) using L&tobaciZZus Zeichmannii IAM 12066 ( =ATCC7830) by the procedure of Sato ( 1983), and the amount of vitamin B12 produced or consumed was calculated from the difference between the inoculated and uninoculated broths. RESULTS AND DISCUSSION
Water temperatures at the surface and bottom layers of the carp culture pond during May to November ranged from 13.1 to 34.1 “C and from 11.5 to 29.5 ’ C,respectively. A total of 520 bacteria were isolated aerobically from water and sediment samples of the carp culture pond. They were composed ofdcinetobacte? (52), Bacillus ( 1 lo), coryneforms (62 ), Enterobacteriaceae ( 8 ), Flavobacterium (14), MoraxeZZa (34), Pseudomonas (47) and Vibrionaceae (193 strains). The Vibrionaceae strains were further divided into Aeromonas hydrophila ( 17 1 ), other Aeromonas ( 8 ) and Plesiomonas shigelloides ( 14). Of 536 bacteria isolated microaerophilically, 19 strains were found to be microaerophiles. A total of 5 10 bacteria isolated anaerobically were composed of Bacteroides type A ( 16 ), other Bacteroidaceae (4 1) , Clostridium ( 185 ) and facultative anaerobes (268 ) . Population densities of bacterial genera in the surface water and sediment of the carp culture pond in the period from May to November are shown in Tables 1 and 2. Total viable counts in the surface water and sediment ranged from 3.7~10~ to 2.2~ lo5 CFU-ml-’ and from 1.3x lo5 to 6.0~10~ CFU-g-l, respectively. In the water samples, Aeromonas (especially A. hydrophila ) , Bacillus, Bacteroidaceae, Clostridium, coryneforms and Pseudo-
428
H. SUGITA ET AL.
TABLE 1 Microflora of water samples collected from a carp culture pond Genus
May
June
July
Aug.
Sep.
Oct.
Nov.
Aeromonas hydrophila Other Aeromonas Plesiomonas shigelloides Enterobacteriaceae Pseudomonas Acinetobacter Moraxella Flavobacterium Coryneforms Bacillus Microaerophiles Clostridium Bacteroides type A Other Bacteroidaceae
4.73” ndb nd 4.15 4.15 5.04 4.43 4.73 3.15 3.45 nd 3.90 nd 3.46
3.66 nd nd nd 2.82 nd 2.82 nd 2.52 2.52 nd nd 2.43 1.60
2.48 2.92 nd nd 3.26 3.08 3.38 nd 2.48 2.82 nd 2.76 nd nd
3.45 nd 2.92 nd nd nd nd nd 2.15 2.15 nd 1.78 3.48 3.78
3.63 2.43 3.19 nd nd nd nd 2.43 2.73 2.43 nd 1.60 2.46 1.60
4.03 nd nd 2.89 2.89 2.89 nd 3.49 2.89 2.89 nd 2.18 1.88 1.70
3.11 2.32 nd 2.80 2.62 3.02 2.62 2.32 2.04 nd 3.68 2.56 nd 2.56
Total viable counts
5.34
3.82
3.84
3.98
2.57
3.51
2.89
“Viable counts (log CFU- ml- ’ ) . bnd = not detected.
TABLE 2 Microflora of sediment samples collected from a carp culture pond Genus
May
June
JOY
Aug.
Sep.
Oct.
Nov.
Aeromonas hydrophila Other Aeromonas Plesiomonas shigelloides Enterobacteriaceae Pseudomonas Acinetobacter Moraxella Flavobacterium Coryneforms Bacillus Microaerophiles Clostridium Bacteroides type A Other Bacteroidaceae
nd” nd nd nd 6.95 6.78 7.08 5.63 7.38 6.95 nd 5.11 nd 4.18
nd nd nd nd 4.26 nd 4.26 nd 3.78 4.89 nd 4.49 nd nd
5.61b 4.23 nd nd 5.91 6.46 nd nd 5.61 6.57 nd 5.64 nd nd
nd nd nd nd 5.11 5.41 4.78 4.48 5.30 5.86 4.72 5.54 nd 4.36
4.99 4.20 nd nd 5.60 5.77 nd nd 5.30 4.99 4.41 5.15 nd 5.30
5.08 nd 5.00 5.30 5.60 4.18 nd nd 5.70 5.84 nd 5.23 3.75 3.75
5.53 nd nd nd 4.28 nd nd nd 6.16 5.65 nd 5.48 nd nd
TVC
7.78
5.12
6.73
5.76
5.53
5.23
5.50
%d=not detected. bViable counts (log CFU-g-r).
VITAMIN-B12-PRODUCING
BACTERIA IN CARP CULTURE POND
429
m~na.s were predominantly isolated from 5-7 out of 7 samples. In the sediment samples, Pseudomonas, coryneforms, Bacillus and Clostridium were detected in all the samples with high densities ranging from 6.0 x lo3 to 2.4x 10’ CFU.g-l, and Acinetobacter also sometimes became a predominant component with densities up to 6.0x lo6 CFU-g-‘. Microaerophiles were isolated from only 14 and 29% samples of water and sediments, respectively, with low densities, indicating that they were minor components in the carp culture pond. A total of 746 strains of bacteria, including aerobes/facultative anaerobes ( 520 strains), microaerophiles ( 19) and obligate anaerobes (206)) were examined for their ability to produce and consume vitamin B12 and the results are summarized in Fig. 1. The vitamin B12 consumers in the water and sediment samples comprised 4.6 and 9.6% of the aerobic/facultatively anaerobic isolates, 13.3% and 0% of the microaerophilic ones, and 5.4 and 9.3% of the obligately anaerobic ones, respectively. Contrary to this, the vitamin Blz producers with the highest productivity ( 2 1.Ol ngmml-’ ) of the isolates from water and sediments was 26.9 and 15.0% for aerobes/facultative anaerobes, and 5 1.8 and 56.7% for obligate anaerobes, respectively, while no microaerophile had such a high activity. These results indicate that more than half of the obligately anaerobic strains isolated from the carp culture pond could produce markedly large amounts of vitamin B12while low percentages (less than 27%) of aerobes/facultatively anaerobic and microaerophilic ones could produce it. Similar phenomena were observed in coastal waters and fish guts (Sugita et al., 1991a, 1993). Moreover, Clostridium occurred at high densi-
(b)
(a) Aeromonas hydmphila OlherAeromonas Pfesiomonas shige~~oides Enfembacteriaceae Pseudomonas Acfnetobacfer Moraxeffa Flavobacterium Coryneforms Bacilluo Microaerophiles Clostridium Bactemides type A Bacfemidaceae 0
50
100 150 No. Strains
200
50
100 No. Strains
Fig. 1. The productivity of vitamin BIz of each bacterial genus, isolated from the surface water (a) and sediment (b) of a carp culture pond.
430
H. SUGITA ET AL.
ties such as 3.1 x lo4 to 4.4x lo5 CFU*g- ’ in the sediment, which are comparable to the population level of aerobes/ facultative anaerobes, although this micro-organism was detected at low densities ranging from undetectable (<2xlOCFU.m1-‘) to7.9x103CFU~m1-1inthesurfacewater (Table2). These results strongly suggest that obligate anaerobes, as well as aerobes/facultative anaerobes, are important in supplying vitamin Blz to the microalgae in freshwater culture ponds and that many organisms, including carp, benelit finally from them. Several workers suggested that in marine environments, large amounts of vitamin Br2 are produced in the sediment and continuously supplied to seawater (Kurata, 1970; Nishijima, 1985 ). This suggestion may be true for the carp culture pond, because it is known that population densities of microalgae are significantly influenced by the physico-chemical conditions of pond sediments (Nomura, 1982). It is therefore important to analyze the relationship among the amounts of vitamin B12, and population densities of vitamin-B,,-producing bacteria and microalgae for a better understanding of the significance of bacteria in culture ponds. Further studies along these lines should be performed.
REFERENCES Gillespie, P.A. and Morita, R.Y., 1972. Vitamin Brz production and depletion in a naturally occurring eutrophic lake. Appl. Microbial., 23: 341-348. Kurata, A., 1970. Vitamin Br2 transudation from marine bottom muds. J. Oceanogr. Sot. Jpn., 26: 81-86. Kurata, A. and Kimata, M., 1968. On the distribution of marine bacteria producing vitamin Brz and the vitamin production of them. Mem. Res. Inst. Food Sci. Kyoto Univ., 3 1: 26-34. Nishijima, T., 1985. Ecological studies on the outbreak of red tides with reference to B group vitamins in coastal waters. Mem. Fat. Agr. Koch Univ., 43: l-154. Nomura, M., 1982. Rearing management. In: M. Nomura (Editor), Cultivation Techniques of Freshwater Fish. Koseisha-Koseikaku, Tokyo, pp. 122- 150 (in Japanese). Provasoli, L. and Carlucci, A.F., 1974. Vitamins and growth regulators. In: W.D.P. Stewart (Editor), Algal Physiology and Biochemistry. Blackwell, Oxford, pp. 741-787. Sato, K., 1983. Assay methods of vitamin B r2. Vitamins (Japan), 57: 609-616. Shibukawa, K., Takamura, Y., Takao, E., Irie, M. and Yagi, O., 1986. Studies on the growth factors of an axenic clone of the cyanobacterium Microcystis aeruginosa K-3A. Jpn. J. Limnol., 47: 247-255. Sugita, H. and Deguchi, Y., 1983. Media for the isolation of aerobic and facultatively anaerobic bacteria in freshwater environments. Bull. Jpn. Sot. Sci. Fish., 49: 1737-1740. Sugita, H., Oshima, K., Fushino, T. and Deguchi, Y., 1985. Microflora in the water and sediment of freshwater culture ponds. Bull. Jpn. Sot. Sci. Fish., 5 1: 9 l-97. 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. Aquaculture, 64: 39-46. Sugita, H., Tsunohara, M., Ohkoshi, T. and Deguchi, Y., 1988. The establishment of an intestinal microflora in developing goldfish (Carassius auratus) of culture ponds. Microb. Ecol., 15: 333-344.
VITAMIN-B,2-PRODUCING
BACTERIA IN CARP CULTURE POND
431
Sugita, H., Miyajima, C. and Deguchi, Y., 1991a. The vitamin Brz-producing ability of the intestinal microflora of freshwater fish. Aquaculture, 92: 267-276. Sugita, H., Okamoto, N. and Deguchi, Y., 1991b. Distribution of microaerophilic bacteria in the water and sediment of the Edo River mouth. Jpn. J. Limnol., 52: 199-203. Sugita, H., Takayama, M., Ohkoshi, T. and Deguchi, Y., 1992. Occurrence of microaerophilic bacteria in the water and sediment of a grass carp culture pond. Aquaculture, 103: 135-140. Sugita, H., Sasanuma, S., Tanaka, K. and Deguchi, Y., 1993. Vitamin BIz-producing ability of bacteria isolated from coastal waters. Biosc. Biotech. Biochem., 57: 138-139.
425
AQUA 40 105
Short Communication
The vitamin-B,,-producing bacteria in the water and sediment of a carp culture pond H. Sugita=, A. Kuruma”, C. Hirato”, T. Ohkoshib, R. Okadab and Y. Deguchi” “Department of Fisheries, Nihon University, Tokyo, Japan ?!?aitama Prefectural Fisheries Experimental Station, Saitama, Japan (Accepted
12 October 1993)
ABSTRACT The heterotrophic bacteria isolated from a carp (Cyprinus carpio) culture pond were examined for vitamin-B,,-producing and -consuming abilities. Total viable counts in the surface water and sediment in the period during May to November ranged from 3.7 x 10’ to 2.2~ lo5 CFU.ml-’ and from 1.3x lo5 to 6.0~ 10’ CFU.g-‘, respectively. Of 746 strains tested, 8% of the bacteria consumed vitamin B,* and the remaining 92% produced it. More than 50% of the obligate anaerobes, especially the genus Clostridium, could produce large amounts of vitamin Biz while 21°h of aerobes/facultative anaerobes and no microaerophiles could produce it at the same level. These results strongly suggest that obligate anaerobes, as well as aerobes/facultative anaerobes, are important in supplying vitamin B,z to phytoplankton in freshwater culture ponds.
INTRODUCTION
In stagnant culture ponds, microalgae are known to play important roles in the freshwater fish cultivation: (a) supplement of oxygen, (b ) adsorption of nutrient salts, (c) rise and maintenance of water temperature, (d) decrease of light intensity, and (e) its nutritional values for fish. In the Japanese culture pond, a blue-green alga, Microcystis aeruginosa, is known to be one of the representative microalgae (Nomura, 1982). Recently, M. aeruginosa was found to require a large amount of vitamin Br2 for their growth (Shibukawa et al., 1986). This fact strongly suggests that the population density of M. aeruginosa, as well as that of other many species of unicellular algae (Provasoli and Carclucci, 1974), is regulated by the amount of vitamin Br2 in the Correspondence to: Dr. H. Sugita, Department tagaya, Tokyo, 154, Japan.
of Fisheries,
Nihon University,
0044-8486/94/$07.00 0 1994 Elsevier Science B.V. All rights reserved. SSDZ0044-8486(93)E0247-7
Shimouma,
Se-
426
H. SUGITA ET AL.
water. Since this substance is produced mainly by bacteria and phytoplankton in the aquatic ecosystem, the vitamin-Br2-producing ability of aerobic/facultatively anaerobic bacteria isolated from various waters was studied by several workers (Kurata and Kimata, 1968; Gillespie and Morita, 1972; Nishijima, 1985). On the other hand, Sugitaet al. (1985,1987,1991b, 1992) found that both obligate anaerobes and microaerophiles also occur as residents in the water and sediment of culture ponds and coastal regions. However, there is little information on the vitamin-BrTproducing ability of these bacteria, except for a report on coastal waters (Sugita et al., 1993). This study was undertaken, therefore, to evaluate the vitamin-Br2-producing ability of heterotrophic bacteria, including aerobes/facultative anaerobes, microaerophiles and obligate anaerobes, isolated from the water and sediment in a freshwater culture pond. MATERIALS AND METHODS
About 90 kg of carp (Cyprinus carpio) were reared in a stagnant water pond of about 1450 m2 in area and 65-78 cm in depth, which is located in Saitama Prefectural Fisheries Experimental Station, Kazo, Saitama. Crumbled or pelleted diets (Nihon Nosan Kogyo, Yokohama) were mainly fed during the period from May to November 199 1. Water samples were collected monthly using a sterile bottle from the surface water. Concomitantly, sediment samples were collected using an Ekman-Berge bottom sampler (Rigosha, Tokyo). All the samples were kept on ice, transported to the laboratory and processed within 4 h after collection, as described below. Each sample was diluted with a known quantity of phosphate buffer solution (pH 7.6) containing 0.05% L-cysteine hydrochloride and 0.1% Bactoagar (Difco, Detroit, MI), and placed onto PYBGF, 1/20PYBGF (Sugita et al., 1983) and FM-CW blood agar (Eiken Chem., Tokyo). The PYBGF agar medium contains 1% Trypticase peptone (BBL, Cockeysville, MD), 0.5% Phytone peptone (BBL),0.24% Lab-lemco powder (Oxoid, Hampshire), 0.2% Bacto-yeast extract (Difco ) , 0.1% glucose, 0.05% L-cysteine hydrochloride (only for anaerobic cultivation) and 1% agar No. 1 (Oxoid). The l/20 PYBGF agar medium contains 0.05% Trypticase peptone, 0.025% Phytone peptone, 0.0 1% Lab-lemco powder, 0.0 1% Bacto-yeast extract, 0.0 1% glucose and 1% agar. The pH value of both media was adjusted to 7.5. The inoculated plates were incubated at 20’ C for 10 days under either aerobic (PYBGF and l/20 PYBGF), microaerophilic (PYBGF and 1/2OPYBGF) or anaerobic (PYBGF and FM-CW) conditions. The microaerophilic condition ( 5% CO21 10% 02/85% N2) was established in a TE-HER CP02 02-CO2 incubator (Hirasawa, Tokyo). The anaerobic condition was established in an ANX-1 anaerobic glove box (Hirasawa), containing 10% H2/ 10% CO2/8O% N2. After incubation, bacterial colonies were counted. About 20 colonies were isolated
VITAMIN-B,,-PRODUCING
BACTERIA IN CARP CULTURE POND
421
at random from each plate and examined for ability to grow under aerobic and anaerobic conditions. All the isolates were divided into three types: (a) aerobes/facultative anaerobes, which grow either aerobically or both aerobically and anaerobically; (b ) microaerophiles, which can grow microaerophilically but do not grow both aerobically and anaerobically; and (c) obligate anaerobes, which grow only anaerobically. The isolates were identified as described previously (Sugita et al., 1988, 1992). On suitably diluted samples, colonies of each bacterial genus were counted and expressed as number (CFU, colony-forming units) per ml or g of material. For each bacterial genus, the highest count obtained on the different agar media was regarded as the estimated viable count for that type. The total viable count was obtained by summation of viable counts of all bacterial genera. The isolates were incubated in 1/2PYBGF broth at 20 oC for 5 days under aerobic, microaerophilic or anaerobic conditions. The 1/2PYBGF broth contains 0.5% Trypticase peptone, 0.25% Phytone peptone, 0.12% Lab-lemco powder, 0.1% Bacto-yeast extract, 0.05% glucose and 0.05% L-cysteine hydrochloride (only for anaerobic cultivation). The culture was then, assayed microbiologically for vitamin B12 (as cyanocobalamin) using L&tobaciZZus Zeichmannii IAM 12066 ( =ATCC7830) by the procedure of Sato ( 1983), and the amount of vitamin B12 produced or consumed was calculated from the difference between the inoculated and uninoculated broths. RESULTS AND DISCUSSION
Water temperatures at the surface and bottom layers of the carp culture pond during May to November ranged from 13.1 to 34.1 “C and from 11.5 to 29.5 ’ C,respectively. A total of 520 bacteria were isolated aerobically from water and sediment samples of the carp culture pond. They were composed ofdcinetobacte? (52), Bacillus ( 1 lo), coryneforms (62 ), Enterobacteriaceae ( 8 ), Flavobacterium (14), MoraxeZZa (34), Pseudomonas (47) and Vibrionaceae (193 strains). The Vibrionaceae strains were further divided into Aeromonas hydrophila ( 17 1 ), other Aeromonas ( 8 ) and Plesiomonas shigelloides ( 14). Of 536 bacteria isolated microaerophilically, 19 strains were found to be microaerophiles. A total of 5 10 bacteria isolated anaerobically were composed of Bacteroides type A ( 16 ), other Bacteroidaceae (4 1) , Clostridium ( 185 ) and facultative anaerobes (268 ) . Population densities of bacterial genera in the surface water and sediment of the carp culture pond in the period from May to November are shown in Tables 1 and 2. Total viable counts in the surface water and sediment ranged from 3.7~10~ to 2.2~ lo5 CFU-ml-’ and from 1.3x lo5 to 6.0~10~ CFU-g-l, respectively. In the water samples, Aeromonas (especially A. hydrophila ) , Bacillus, Bacteroidaceae, Clostridium, coryneforms and Pseudo-
428
H. SUGITA ET AL.
TABLE 1 Microflora of water samples collected from a carp culture pond Genus
May
June
July
Aug.
Sep.
Oct.
Nov.
Aeromonas hydrophila Other Aeromonas Plesiomonas shigelloides Enterobacteriaceae Pseudomonas Acinetobacter Moraxella Flavobacterium Coryneforms Bacillus Microaerophiles Clostridium Bacteroides type A Other Bacteroidaceae
4.73” ndb nd 4.15 4.15 5.04 4.43 4.73 3.15 3.45 nd 3.90 nd 3.46
3.66 nd nd nd 2.82 nd 2.82 nd 2.52 2.52 nd nd 2.43 1.60
2.48 2.92 nd nd 3.26 3.08 3.38 nd 2.48 2.82 nd 2.76 nd nd
3.45 nd 2.92 nd nd nd nd nd 2.15 2.15 nd 1.78 3.48 3.78
3.63 2.43 3.19 nd nd nd nd 2.43 2.73 2.43 nd 1.60 2.46 1.60
4.03 nd nd 2.89 2.89 2.89 nd 3.49 2.89 2.89 nd 2.18 1.88 1.70
3.11 2.32 nd 2.80 2.62 3.02 2.62 2.32 2.04 nd 3.68 2.56 nd 2.56
Total viable counts
5.34
3.82
3.84
3.98
2.57
3.51
2.89
“Viable counts (log CFU- ml- ’ ) . bnd = not detected.
TABLE 2 Microflora of sediment samples collected from a carp culture pond Genus
May
June
JOY
Aug.
Sep.
Oct.
Nov.
Aeromonas hydrophila Other Aeromonas Plesiomonas shigelloides Enterobacteriaceae Pseudomonas Acinetobacter Moraxella Flavobacterium Coryneforms Bacillus Microaerophiles Clostridium Bacteroides type A Other Bacteroidaceae
nd” nd nd nd 6.95 6.78 7.08 5.63 7.38 6.95 nd 5.11 nd 4.18
nd nd nd nd 4.26 nd 4.26 nd 3.78 4.89 nd 4.49 nd nd
5.61b 4.23 nd nd 5.91 6.46 nd nd 5.61 6.57 nd 5.64 nd nd
nd nd nd nd 5.11 5.41 4.78 4.48 5.30 5.86 4.72 5.54 nd 4.36
4.99 4.20 nd nd 5.60 5.77 nd nd 5.30 4.99 4.41 5.15 nd 5.30
5.08 nd 5.00 5.30 5.60 4.18 nd nd 5.70 5.84 nd 5.23 3.75 3.75
5.53 nd nd nd 4.28 nd nd nd 6.16 5.65 nd 5.48 nd nd
TVC
7.78
5.12
6.73
5.76
5.53
5.23
5.50
%d=not detected. bViable counts (log CFU-g-r).
VITAMIN-B12-PRODUCING
BACTERIA IN CARP CULTURE POND
429
m~na.s were predominantly isolated from 5-7 out of 7 samples. In the sediment samples, Pseudomonas, coryneforms, Bacillus and Clostridium were detected in all the samples with high densities ranging from 6.0 x lo3 to 2.4x 10’ CFU.g-l, and Acinetobacter also sometimes became a predominant component with densities up to 6.0x lo6 CFU-g-‘. Microaerophiles were isolated from only 14 and 29% samples of water and sediments, respectively, with low densities, indicating that they were minor components in the carp culture pond. A total of 746 strains of bacteria, including aerobes/facultative anaerobes ( 520 strains), microaerophiles ( 19) and obligate anaerobes (206)) were examined for their ability to produce and consume vitamin B12 and the results are summarized in Fig. 1. The vitamin B12 consumers in the water and sediment samples comprised 4.6 and 9.6% of the aerobic/facultatively anaerobic isolates, 13.3% and 0% of the microaerophilic ones, and 5.4 and 9.3% of the obligately anaerobic ones, respectively. Contrary to this, the vitamin Blz producers with the highest productivity ( 2 1.Ol ngmml-’ ) of the isolates from water and sediments was 26.9 and 15.0% for aerobes/facultative anaerobes, and 5 1.8 and 56.7% for obligate anaerobes, respectively, while no microaerophile had such a high activity. These results indicate that more than half of the obligately anaerobic strains isolated from the carp culture pond could produce markedly large amounts of vitamin B12while low percentages (less than 27%) of aerobes/facultatively anaerobic and microaerophilic ones could produce it. Similar phenomena were observed in coastal waters and fish guts (Sugita et al., 1991a, 1993). Moreover, Clostridium occurred at high densi-
(b)
(a) Aeromonas hydmphila OlherAeromonas Pfesiomonas shige~~oides Enfembacteriaceae Pseudomonas Acfnetobacfer Moraxeffa Flavobacterium Coryneforms Bacilluo Microaerophiles Clostridium Bactemides type A Bacfemidaceae 0
50
100 150 No. Strains
200
50
100 No. Strains
Fig. 1. The productivity of vitamin BIz of each bacterial genus, isolated from the surface water (a) and sediment (b) of a carp culture pond.
430
H. SUGITA ET AL.
ties such as 3.1 x lo4 to 4.4x lo5 CFU*g- ’ in the sediment, which are comparable to the population level of aerobes/ facultative anaerobes, although this micro-organism was detected at low densities ranging from undetectable (<2xlOCFU.m1-‘) to7.9x103CFU~m1-1inthesurfacewater (Table2). These results strongly suggest that obligate anaerobes, as well as aerobes/facultative anaerobes, are important in supplying vitamin Blz to the microalgae in freshwater culture ponds and that many organisms, including carp, benelit finally from them. Several workers suggested that in marine environments, large amounts of vitamin Br2 are produced in the sediment and continuously supplied to seawater (Kurata, 1970; Nishijima, 1985 ). This suggestion may be true for the carp culture pond, because it is known that population densities of microalgae are significantly influenced by the physico-chemical conditions of pond sediments (Nomura, 1982). It is therefore important to analyze the relationship among the amounts of vitamin B12, and population densities of vitamin-B,,-producing bacteria and microalgae for a better understanding of the significance of bacteria in culture ponds. Further studies along these lines should be performed.
REFERENCES Gillespie, P.A. and Morita, R.Y., 1972. Vitamin Brz production and depletion in a naturally occurring eutrophic lake. Appl. Microbial., 23: 341-348. Kurata, A., 1970. Vitamin Br2 transudation from marine bottom muds. J. Oceanogr. Sot. Jpn., 26: 81-86. Kurata, A. and Kimata, M., 1968. On the distribution of marine bacteria producing vitamin Brz and the vitamin production of them. Mem. Res. Inst. Food Sci. Kyoto Univ., 3 1: 26-34. Nishijima, T., 1985. Ecological studies on the outbreak of red tides with reference to B group vitamins in coastal waters. Mem. Fat. Agr. Koch Univ., 43: l-154. Nomura, M., 1982. Rearing management. In: M. Nomura (Editor), Cultivation Techniques of Freshwater Fish. Koseisha-Koseikaku, Tokyo, pp. 122- 150 (in Japanese). Provasoli, L. and Carlucci, A.F., 1974. Vitamins and growth regulators. In: W.D.P. Stewart (Editor), Algal Physiology and Biochemistry. Blackwell, Oxford, pp. 741-787. Sato, K., 1983. Assay methods of vitamin B r2. Vitamins (Japan), 57: 609-616. Shibukawa, K., Takamura, Y., Takao, E., Irie, M. and Yagi, O., 1986. Studies on the growth factors of an axenic clone of the cyanobacterium Microcystis aeruginosa K-3A. Jpn. J. Limnol., 47: 247-255. Sugita, H. and Deguchi, Y., 1983. Media for the isolation of aerobic and facultatively anaerobic bacteria in freshwater environments. Bull. Jpn. Sot. Sci. Fish., 49: 1737-1740. Sugita, H., Oshima, K., Fushino, T. and Deguchi, Y., 1985. Microflora in the water and sediment of freshwater culture ponds. Bull. Jpn. Sot. Sci. Fish., 5 1: 9 l-97. 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. Aquaculture, 64: 39-46. Sugita, H., Tsunohara, M., Ohkoshi, T. and Deguchi, Y., 1988. The establishment of an intestinal microflora in developing goldfish (Carassius auratus) of culture ponds. Microb. Ecol., 15: 333-344.
VITAMIN-B,2-PRODUCING
BACTERIA IN CARP CULTURE POND
431
Sugita, H., Miyajima, C. and Deguchi, Y., 1991a. The vitamin Brz-producing ability of the intestinal microflora of freshwater fish. Aquaculture, 92: 267-276. Sugita, H., Okamoto, N. and Deguchi, Y., 1991b. Distribution of microaerophilic bacteria in the water and sediment of the Edo River mouth. Jpn. J. Limnol., 52: 199-203. Sugita, H., Takayama, M., Ohkoshi, T. and Deguchi, Y., 1992. Occurrence of microaerophilic bacteria in the water and sediment of a grass carp culture pond. Aquaculture, 103: 135-140. Sugita, H., Sasanuma, S., Tanaka, K. and Deguchi, Y., 1993. Vitamin BIz-producing ability of bacteria isolated from coastal waters. Biosc. Biotech. Biochem., 57: 138-139.