Aquacultural Engineering 22 2000 109 – 120
Populations of heterotrophic bacteria in an experimental recirculating aquaculture system
N. Leonard
a
, J.P. Blancheton
b,
, J.P. Guiraud
a
a
USTL, Laboratoire GBSA, Place E. Bataillon,
34000
Montpellier, France
b
IFREMER, Chemin de Maguelone,
34250
Pala6as-Les-Flots, France
Abstract
The aim of this work was to identify the main viable heterotrophic bacteria in a marine fish farm with a recirculating water system and to study their growth dynamics. The
experiments were performed with sea bass Dicentrarchus labrax in an experimental recircu- lating water system. The bacteria identified were typical of the marine environment:
Pseudomonas, Oceanospirillum, Marinobacter, Paracoccus and Erythrobacter genus from Bergey’s group IV, two genus of Vibrionaceae, one strain of Vibrio and one strain of
Aeromonas. These populations were stable when the ingested feedreplacement water ratio was kept constant. Fixed bacteria formed large biofilms, which released about 10
4
CFU ml
− 1
into the tank. The biological filter, with its large surface area, was the largest source of bacteria in the farm, but the UV disinfection unit kept the number of free bacteria stable.
When properly managed, the majority of bacteria that grew on the biological filter were from Bergey’s group IV; no Vibrio were ever detected on it. © 2000 Elsevier Science B.V. All
rights reserved.
Keywords
:
Heterotrophic bacteria; Recirculating aquaculture system; Fish www.elsevier.nllocateaqua-online
1. Introduction
A fish farm using a recirculating water system contains both fish and microbial organisms. The bacterial population comprises both autotrophic and heterotrophic
flora Sich and Van Rijn, 1992; Sugita et al., 1992. The autotrophic population has been well documented but represents only 20 of the total eubacterial rRNA on a
Corresponding author. Tel.: + 33-4-67504100; fax: + 33-4-67682885. E-mail address
:
jpblanchifremer.fr J.P. Blancheton 0144-860900 - see front matter © 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 1 4 4 - 8 6 0 9 0 0 0 0 0 3 5 - 2
biological filter Hovanec and Delong, 1996. Very few studies have been conducted on the non-pathogenic heterotrophic populations; those studies that have been
published are limited in scope and do not apply to our production systems. However, it would appear that the biological filter does determine bacterial growth
Blancheton and Canaguier, 1995. The aim of the present study was to identify the main viable bacterial groups both the fixed and the free populations and to study
the stability over time of the number and kind of organisms present at different points in the experimental aquaculture system, with a view to determining the
parameters that control bacterial growth. Bacteria were counted using standard cell culture techniques. To increase the number of bacteria which could be grown, a
revivification of the bacteria was performed such that only growing and substrate- responsive bacteria were taken into consideration Peele and Colwell, 1981. The
experiments were performed with sea bass Dicentrarchus labrax in an experimen- tal recirculating water system at the IFREMER station at Palavas.
2. Materials and methods
2
.
1
. Experimental aquaculture system A general diagram of the recirculating water system and the sampling sites used
in this study are shown in Fig. 1. This system was described by Blancheton and Cove`s 1993 and its operational characteristics are indicated in Table 1. Total
system volume was approximately 30 m
3
, and the fish biomass D. labrax was maintained at between 700 and 900 kg. The replacement water flow rate was
initially set at 1 m
3
h
− 1
and then varied between 2 and 0.3 m
3
h
− 1
.
2
.
2
. Microbiological analyses
2
.
2
.
1
. Sampling Four 500 ml water samples were taken every month once disinfection the
sampling valves at the inlet and outlet of each component of the system had been
Fig. 1. Fish rearing system. System components are described in Table 1.
Table 1 Characteristics of the different components of the recirculation system
Components Numbers on the
Functions Characteristics
Fig. 1 1
10 m
3
, 22 m
2
walls each Fish tank
2 Rearing tanks tank
2 Particle separa-
Feces and uneaten feed collection fecal trap AquaOptima
tor Removal of small particles
Mechanical mesh filter
3 filter 60 mm
4 CO
2
stripping counter current airwater
Packed column packed column
Pumping tank 1.5 m
3
pH regulation at 7.6 — replace- 5
ment water adding Pump
30 m
3
h
− 1
6 Bacteria control
75 W 7
UV disinfection unit
Nitrification Biological filter
3 m
3
microporous packing: 8
Biogrog Thermo-regulation: 22 9 1°C
Heat exchanger 9
Bubbling of pure oxygen ]
6 mg l
− 1
10 Oxygenation
disinfected and rinsed Fig. 1. Every month three biofilm samples were taken from the wall of the rearing tank, the pipes and the biological filter. The level of water
in the tank was lowered to allow a 100-cm
2
sample of biofilm to be scraped with a sterile blade from the wall of the tank 20 cm below the water surface. In addition,
100 cm
2
of biofilm was scraped from a PVC pipe with a sterile blade and another sample of biofilm was taken from the top of the biological filter packing. The
biological filter was systematically back-washed twice a week; sampling was always carried out 1 day after the back-wash.
2
.
2
.
2
. Re6i6ification and ultrasonic treatment A 45-min revivification was performed in yeast extract medium 0.3 g l
− 1
NaCl 34 g l
− 1
; Kogure et al., 1979; Peele and Colwell, 1981; Singh et al., 1990. The pH was adjusted to 7.6. In order to remove the biofilm from the filter media and to
separate the bacteria aggregates, the sample was given 10-min ultrasonic treatment 20 kHz, 50 W at the beginning of the revivification. For free bacteria, 10 ml of the
sample were mixed with 90 ml of the revivification media; the solid sample was mixed with 200 ml of revivification media.
2
.
2
.
3
. Enumeration media Counts of viable heterotrophic bacteria CFU were made in Marine Agar Difco
2216 after 10 days of culture at 25°C. Dilutions were prepared in 34 g l
− 1
sterile
sodium chloride solution. The revivification step corresponded to the first dilution. Plates were set up in duplicate for each plated dilution.
2
.
2
.
4
. Growth rate The average growth rate of the free viable heterotrophic bacteria sampled in the
rearing tank was determined in a sea water medium at 25°C. The CFU were evaluated every 2 h. This experiment was performed in duplicate and the results
were expressed as mean 9 standard deviation.
2
.
2
.
5
. Identification Twenty percent of colonies were randomly selected for isolation in a pure culture
from plates with between 30 and 300 colonies. The bacteria isolated in pure culture 19 – 24 h were identified using colony morphology, color, gram stain, bacterial
morphology, catalase activity, oxidase activity, respiratory metabolism and vibrio- static O.129 2,4 Diaamino 6,7 diisopropyl pteridine resistance. In addition, API 20
NE strips Biome´rieux, France were used to identify gram negative bacteria using a medium consisting of yeast extract 0.3 g l
− 1
and NaCl 30 g l
− 1
. Bacteria were identified according to Bergey’s Manual Bergey’s Manual of Determinative Bacte-
riology, 1994.
2
.
3
. Accuracy of the results For liquid samples, the data were expressed as means with the 95 confidence
limits: mean 9 1.96 s
2
n; s
2
n is considered as the variance, 1.96 corresponds to the Student-t-value for a sampling greater than 30 and a 95 confidence limit, n is
the number of plate counts Guiraud, 1998. For the solid samples, the analysis was performed in duplicate and the data expressed as mean 9 standard deviation. The
bacteria concentration in the biological filter was expressed as CFU per g of wet packing because the surface area of the packing material is not precisely
known.
2
.
3
.
1
. Statistics Because the variances were not homogeneous andor the residual departed from
normality, all the data were log transformed prior to statistical analysis. Data were analyzed with one way analyses of variance ANOVA, with the treatment or the
sampling point as the factor. To study the relative importance of each system component in terms of production of bacteria, a Newman – Keuls test was carried
out after the ANOVA to group the different sampling points, at an ingested feedreplacement water ratio of 1 kg m
− 3
. To analyze the influence of fish biomass on bacterial growth in the rearing tank, a Pearsons correlation was performed
between fish biomass and the number of bacteria in the rearing tank expressed as the difference between the number of bacteria at the inlet and the outlet of this
tank.
3. Results
