Abstract: The bacterial population of water from the Marine Research Aquarium was examined qualitatively and quantitatively. Lower concentrations (1.3x102) were found in the initial phase of the aquarium tank and increased moderately day by day. A total of 1109 strains were isolated from 30 water samples. The isolates comprised of 16 species 10 genera; 86.65% of these isolates were gram-negative rods. The Vibrio isolates comprised 48.9% of the total population. The generic compositions of the bacterial isolates were Flavobacterium, Pseudomonas, Salmonella, Bacillus, Staphylococcus, Alcaligenes, Aeromonas, Enterobacter and Cytoplaga. The results show that the survival rate of the fish species Pomacanthus imperator, P. semicirculatus, Chaetodon collare, C. vagabundus, C. plebius are poor probably due to abundance of the Vibrio population. The higher concentration of other genera may also increases the mortality rate among these ornamental fishes. Majority of the bacterial strains isolated are also considered as human pathogens.
Introduction
Generally, organic matter in recirculating aquariums is derived from uneaten feed or diets, dead bodies, wounded fishes and excreta of fish. These inorganic matters can directly affect total bacterial populations of the aquaria. The water quality of recirculating system is mainly affected by the bacterial flora in the water column and filter materials as well as the different elements of this closed system. Lesser survival can be observed in fishes that are maintained in water with high bacterial pathogens. In the present study, the recycling of water in the aquarium was done mainly through three different facilities i.e., 1) a mechanical filter in the bottom of the aquarium tanks to remove suspended materials 2) a canister filter in individual tanks that contains charcoal and bioballs to transform the toxic soluble matter into non-toxic compounds. 3) A UV sterilizer to control bacterial populations. All these materials provide a substratum for bacterial growth in a closed system. The nitrifying bacteria from the biological filter medium control the stability of water in addition to their nitrifying action.
The low efficiency of biological filter media causes the bloom of opportunistic pathogenic bacteria in the rearing tanks (Blancheton and Canaguier, 1995). The exhibition aquaria and closed aquaculture systems reproduce aquatic environments with high densities of fish and the fishes kept in such intensive systems are very sensitive to the microbial flora of the water (Blanch et al., 1999). Moreover, bacterial populations in aquaculture facilities and in exhibition aquaria may differ from those of the natural environment and may affect the nutrition and health of the fish. The influence of associated bacteria in water, food and initial stages of fish larvae, on the survival of fish in captivity has been demonstrated by Nicolas et al. (1989) and Westerrdahl et al. (1994). In addition, aquaria containing ornamental fish are often found in school classrooms, medical and dental offices, eating establishments, department stores, nursing homes and even in hospital wards. It is apparent that the presence of potentially pathogenic microorganisms and opportunistic pathogenic bacteria in these aquaria would present a risk to the public health. The knowledge about these aquarium pathogenic organisms is important to hobbyist. Thus, the present study was initiated to qualitatively and quantitatively determine the bacterial population in the water supplied with ornamental fish.
Materials and Methods
The ornamental fishes were purchased from Gulf of Mannar southeast coast of India and transported more than 350 km in polyethylene package filled by one third of pure oxygen. These fishes were quarantined in UV sterilized water for three days; only after this initial quarantine treatment, the fishes were introduced into the newly established experimental cum public aquaria. The schematic diagram of the newly established recirculating Marine Research Aquarium system used in this study and the sampling locations are shown in Fig. 1. In the present experimental aquaria, 20% of the water was recycled through the various filters every day. The survival of the fishes were regularly monitored throughout the study period and the total heterotrophic bacterial populations also examined. Total number of fish species introduced and their sizes are shown in Table 1.
Sampling for Bacteriological Examinations
The water samples were taken during three different phase i.e., Phase I
(1st day), Phase II (15th day) and Phase III (30th day). Totally 30 samples
were taken during the present experiment.
| Fig. 1: | Technical view of recirculating system in Marine Research Aquarium |
| Table 1: | The fish species characteristics of the sampling aquaria |
Quantitative Bacteriological Examination
Enumeration of Total Heterotrophic Bacteria (THB) was made using the pour
plate method on sterile Zobell marine agar medium (Hi-media chemicals Pvt. Ltd,
Bombay, India) with the composition of Peptone-5.0 g, Yeast extract 1.0 g, K2HPO4-0.5
g, FeSo4-Trace, Agar-15 g, Distilled water-1000 mL and pH-7.2. For
plating 1 mL of the serially diluted sample was pipetted out into sterile petri
plates. Sterile media was then poured into petri plates aseptically and swivel
led for through mixing. After solidification, the plates were incubated in an
inverted position for 72 h at 28±1°C. Bacterial colonies developed
on Zobell marine agar after the incubation period were counted and their density
expressed as Colony Forming Units (CFU) per milliliter.
Qualitative Bacteriological Examination
The different morphological colonies were picked from the Zobell marine
agar plates and restreaked in appropriate nutrient agar plates. The tests used
to characterize the isolates are described by Edwards and Eving (1972), Skerman
(1967) and Smith et al. (1972). Identification is facilitated by examination
of colonial morphology and pigmentation as well as examination of the shape,
arrangement, staining characteristics, flagellar arrangement and motility of
the cells. In addition, the ability of the isolates to produce oxidase and catalase,
to ferment lactose and to metabolize glucose fermentativety or oxidatively was
tested. Gram-negative species were further separated on the basis of carbohydrate
utilization, growth in the presence of bile salts, production of urease and
the indole, methyl red, Voges-Proskauer and citrate utilization reactions. Other
tests used were the orthonitrophenyl, β-D-galactopyranosidase (ONPG) test,
the lysine and ornithine decarboxylase tests and the arginine decarboxylase
and dihydrolase tests. Vibrio species were confirmed by testing the sensitivity
of the culture to 2,4 diamino-6,7-diisopropylpteridine. Final identification
of the isolates to species level were done with the schemes of Buchanan and
Gibbons (1975), Krieg (1984) and Sneath (1986).
Results
Quantitative Bacterial Examination
The results in Table 2 show that the samples from the
initial phase contained low concentration (5x102-2.3 x103
CFU mL-1) of total heterotrophic bacteria per mL but it increased
in the following phases (1.7x103-1.60x105 CFU mL-1).
Qualitative Bacteriological Examinations
A total of 1109 isolates were partially characterized from the 30 water
samples. The isolates comprised of 16 species from 10 genera of bacteria; 86.65%
of these isolates were gram-negative rods (Table 3). The most
common species of isolates belonged to the genus Vibrio. These Vibrio
isolates comprised 48.9% of the total isolates and were demonstrated to be present
in 27 of the water samples in all the three phases. Of the Vibrio isolates
170 were identified as V. parahaemolyticus, 155 as V. valnificus,
111 as V. angnillarium and 106 as V. cholerae. Species of Pseudomonas
comprised 16.22% of the total isolates and were isolated from 22 water samples.
This genera is present all the phases except phase III of Arius maculates,
P. imperator and P. semicirculatus tanks. In the majority of these
Pseudomonas isolates were non-pigmented species and were able to grow
at 42°C on Pseudomonas agar. Typical pyocyanin producing Pseudomonas
aeruginosa was also isolated in Pomocentrus coelores fish tank. In
addition, the species Pseudomonas fluerescens produce, fluorescein and
were isolated from the 6 tanks comprised of 65 isolates. The Salmonella
is rich (48 isolates) in P. imperator, P. semicirculatus tanks
followed by A. sebae (32 isolates), C. collare (15 isolates)
C. vagabundus, C. plebius (7 isolates) and Thallosoma lunare
(5 isolates).
| Table 2: | Bacterial concentration in different aquaria samples |
| Table 3: | Frequency of isolation of bacterial species from the samples |
Among the other isolates Bacillus megaterium, B. cereus comprised 7.84% , Staphylococcus sp. 3.25%, Alcaligenes fecalis 2.25%, Aeromonas hydrophilla 2.8%, Enterobacter aerogenes 2.61%, Cytoplaga sp. 3.52 % and Flavobacterium sp. 2.97% and they are isolated from very few of the samples.
Discussion
The present findings show that marine aquarium water in the recycling system contains significant number of a wide variety of bacteria. Position of various filtration system in the present aquarium clearly shows that the experimental tank contains nearly pure water without any bacterial cells. The bacteria were introduced into the water from uneaten feed or diets, dead bodies, wounded fishes and excreta of fish and these paths differed based on the species. For example the fish species P. imperator and C. collare were underfeeding in the initial phases and results in increase of uneaten feed within the tank. It directly influenced the bacterial population which in turn may affect the health of the fish. The food ingested microbial composition has been reported through the estimation of intestinal micro-flora by various authors (Tanasomwang and Muroga, 1988; Blanch et al., 1999). The presence of high concentration of Vibrio in the tanks 3, 4, 5 also resulted in lesser survival rate of the fishes maintained in these tanks. The influence of the associated bacteria, V. anguillarum in water, food and initial stages of fish on the survival of fish in captivity has been demonstrated by Westerdahl et al. (1994). Blanch et al. (2001) also monitored the bacteria of the aquarium water, particularly the vibrio populations and determined a higher composition of Vibrio populations from several exhibition aquaria with a shared water supply. P. aeruginosa, V. cholerae, V. parahaemolyticus isolated in the present samples are able to survive and multiply in the gut, mucus and tissues of fish and they are also potential pathogens of man (Janssen, 1970; Trust and Money, 1972; Weistreich and Lechtman, 1973). In recirculating aquaria microbial populations multiplies day by day. This is directly affect the fish health that are maintained in high concentrations of harmful microorganisms. Since bacteria present in an aquarium and they could also readily be transferred to humans, besides affecting the fishes the presence of potential pathogens in the aquarium could become a hazard to human health.
Acknowledgments
We thank the authorities of Annamalai University for allowing use of Marine Research Aquarium in the present study.