4. Discussion
The number of bacteria fixed to the surfaces of the tank and the PVC pipes was approximately constant, with an average of 10
7
viable heterotrophic bacteria CFU per cm
2
of biofilm. Percival et al. 1998 found 3.6 × 10
2
CFU cm
− 2
on a stainless steel surface in a drinking water environment. Fera and Prieur 1986 found a
biofilm of 10
7
total cells per cm
2
in sea water for three types of surfaces: aluminum, stainless steel and polycarbonate filters. This value, expressed as the number of
viable bacteria, is equivalent to approximately 10
4
CFU cm
− 2
Kogure et al., 1979. The higher concentration of bacteria on the biofilm in our rearing system
could be explained either by the slower linear speed of about 1 m s
− 1
, compared with 0.01 m s
− 1
in our study, or by the higher nutrient concentration in the rearing media. Higher numbers of bacteria up to 10
10
cm
− 2
have been observed on a biofilm by Ganderson et al. 1992 and Personne´ et al. 1995.
The number of bacteria remained stable at a fixed ingested feedreplacement water ratio, although the number of bacteria was different in each component of
the rearing system Fig. 3. In accordance with our observations, Sich and Van Rijn 1992 found that the free bacterial populations were not homogeneously dis-
tributed throughout their recirculating water system. Their tilapia 10 kg m
− 3
facilities had a denitrifying unit, and they used a settler instead of a mechanical filter. They also had a nitrifying biofilter, but no UV disinfection unit. In their
system, they found a total of 10
8
bacteria ml
− 1
approximately 10
5
CFU ml
− 1
in the tank and the biological filter, which rose to a total of 10
10
cells ml
− 1
in the denitrifying unit outlet. Korzeniewski and Korzeniewska 1982 found 10
5
CFU ml
− 1
in trout cages. Our values of between 10
4
and 10
5
CFU ml
− 1
are similar to those found in trout cages and are lower than those observed by Sich and Van Rijn
1992 in their recirculating water system. Different enumeration techniques and
Fig. 8. Vibrionaceae and Bergey’s group IV distribution in June.
enrichment media were used by each of these authors, which makes comparison difficult and approximate. A generation time of 2.7 h was found for the free viable
heterotrophic populations. This value is within the normal range for microbial communities observed in biological wastewater treatment systems Pollard and
Grennfield, 1997. This should be compared to 26 h for nitrite bacteria and 60 h for nitrate bacteria Shilo and Rimon, 1982; Belser, 1984.
The time was too short for free bacteria to grow significantly in number between two consecutive passages through the UV disinfection unit. These results led us to
propose that the biofilm at the surface of the biological filter was the main source of bacteria, as already described by Blancheton and Canaguier 1995. This was
supported by the identification of the bacteria, which showed that all of the biotypes present in the fixed populations were also represented in the free bacteria
in our system. This hypothesis can easily be tested in the tank, where the sites colonized by the bacteria, the number of bacteria per cm
2
and the growth rate were 22 m
2
, 1.5 × 10
7
CFU cm
− 2
and 0.25 h
− 1
, respectively. Taking into account the dilution rate 1.4 h
− 1
and the average growth rate 0.25 h
− 1
, the increase in the number of bacteria 1.3 × 10
4
CFU ml
− 1
cannot be explained without taking into consideration the fixed populations. If we consider that the fixed and free bacteria
have a similar growth rate, a fixed population of only 7.5 × 10
5
CFU cm
− 2
, which corresponds to only 5 of the biofilm in the tank 1.5 × 10
7
CFU cm
− 2
, would be sufficient to explain the increase in the number of free bacteria observed between
the inlet and the outlet of the tank. This percentage of dividing bacteria can be seen in biofilms or flocs Fontana et al., 1992. Hence the biological filter with its large
surface area is the main producer of bacteria of the rearing system. The same type of calculation can be made for each component of the system, but the percentage
of dividing bacteria varies with the hydraulic characteristics and the colonized surface. Further investigations will be devoted to this phenomenon in order to
model each compartment of bacterial growth biofilms and free populations as a function of the ingested feedreplacement water ratio, and the hydraulic and other
physical characteristics of the system. Fish biomass had no influence on the concentration of the principal genera of bacteria Fig. 4 when the ratio of ingested
feed to inflowing replacement water was kept constant: the number of viable free bacteria in our system was stable. When this ratio was increased, the number of free
and fixed bacteria also increased Fig. 6 due to the fact that more nutrients had been provided for the biofilm, increasing its growth rate Pelmont, 1993 and the
number of fixed and free bacteria.
Very little has been published about the kind of bacteria to be found in a recirculating water system; a few studies have looked mainly at the nitrifying or the
denitrifying populations. Bergey’s group IV Bergey’s Manual of Determinative Bacteriology, 1994 is very heterogeneous and contains bacteria commonly found in
aquatic environments; they can be both strict aerobic bacteria or microaerophilic bacteria such as Pseudomonas. In contrast, the Vibrionaceae family contains
populations that can grow under anoxic and anaerobic conditions. Vibrio have been isolated in freshwater, estuarine and seawater environments, although most of them
are probably saprophytic West and Colwell, 1984. Some Vibrio species are
pathogenic for man, others are pathogenic for marine vertebrates and invertebrates. This is why conditions, which favor Vibrionaceae growth must be avoided in order
to prevent any increase in the risk to the health of the fish in the system. Our results confirmed those of Paat et al. 1989 who found bacteria belonging to genera that
live in aquatic environments Pseudomonas, Fla6obacterium and Vibrio in an experimental carp aquaculture with a recirculating water system. In both of these
aquaculture systems, the rearing conditions did not select for a specific bacterial flora. Only a few Vibrio were ever observed among the fixed bacteria in the pipes,
the tank and the biological filter. The free Vibrio populations increased in number when the biological filter was clogged, a situation which gives rise to anoxic areas
where anaerobic flora such as Vibrionaceae rather than the Bergey’s group IV families are able to grow. The difference in respiratory metabolism explains why
Vibrionaceae could become the dominant bacteria when there is a lack of oxygen. When properly managed, the biological filter only released bacteria belonging to
Bergey’s group IV. With its large surface area, a large number of free bacteria were able to grow on it Fig. 5; thus, the accumulation of organic matter and the
resulting lack of oxygen or even the formation of anaerobic patches should be avoided.
5. Conclusions
Kategori