1. HRTand nutrients a¡ect bacterial communities grown on
recirculation aquaculture system e¥uents
Oliver Schneider1, Mariana Chabrillon-Popelka2, Hauke Smidt2, Olga Haenen3, Vasiliki Sereti1,
Ep H. Eding1 & Johan A. J. Verreth1
1
Aquaculture and Fisheries Group, Wageningen University, Wageningen, The Netherlands; 2Laboratory of Microbiology, Wageningen University,
Wageningen, The Netherlands; and 3CIDC-Lelystad, NRL for Fish and Shellfish Diseases, Wageningen University, Wageningen, The Netherlands
Correspondence: Oliver Schneider, Abstract
Aquaculture and Fisheries Group,
Wageningen University, PO Box 338, 6700 AH
In a recirculation aquaculture system the drumfilter effluent can be used as
Wageningen, substrate for heterotrophic bacterial production, which can be recycled as feed.
The Netherlands. Tel.: 100 31 317 485147; Because the bacteria might contain pathogens, which could reduce its suitability as
fax: 100 31 317 483937; feed, it is important to characterize these communities. Bacteria were produced in
e-mail: oliver.schneider@wur.nl growth reactors under different conditions: 7 h hydraulic retention time (HRT) vs.
2 h, sodium acetate vs. molasses, and ammonia vs. nitrate. The community of the
Received 26 January 2006; revised 26 drumfilter effluent was different from those found in the reactors. However, all
November 2006; accepted 2 December 2006.
major community components were present in the effluent and reactor broths.
First published online 16 March 2007.
HRT influenced the bacteria community, resulting in a DGGE profile dominated
DOI:10.1111/j.1574-6941.2007.00282.x
by a band corresponding to an Acinetobacter sp.-related population at 2 h HRT
compared to 7 h HRT, where bands indicative of a-proteobacterial populations
Editor: Michael Wagner most closely related to Rhizobium and Shinella spp. were most abundant. Molasses
influenced the bacterial community. It was dominated by an Aquaspirillum
Keywords serpens-related population. Providing total ammonia nitrogen (TAN) in addition
bacteria; community; aquaculture; waste to nitrate led to the occurrence of bacteria close to Sphaerotilus spp., Flavobacter-
conversion; 16S rRNA gene. ium mizutaii and Jonesia spp. It was concluded from these results that a 6–7 h HRT
is recommended, and that the type of substrate is less important, and results in
communities with a comparably low pathogenic risk.
already applied in integrated and activated ponds. In such
Introduction ponds, waste conversion does not only improve water
In recirculation aquaculture systems (RAS), feed is con- quality but also feed conversion ratios, because the pro-
verted into fish and faecal and nonfaecal loss. These two duced bacteria biomass may be consumed by fish or shrimp
waste sources are composed mainly of solid waste, and (Avnimelech et al., 1989; Edwards, 1993; Burford et al., 2003;
dissolved waste: ammonia and phosphate. The waste is Hari et al., 2004).
treated by mechanical filtration to remove the solids from To produce bacterial biomass utilizing the effluent stream
the system water and by biofiltration to nitrify ammonia to of the drum filter, a bacterial reactor has to be integrated
less hazardous nitrate. The effluent from the mechanical into the system (Fig. 1). The nutrient ratios in the slurry
filter is the major discharge of such systems. It comprises coming from the filter are normally not ideal for bacteria
solid (faecal loss) and dissolved waste (nonfaecal loss). This production. Optimal C : N ratios for heterotrophic bacteria
slurry contains dissolved waste, because the solid particles production are about 12–15 g : 1 g (Lechevallier et al., 1991;
are backwashed from the filter screen using system water, Henze et al., 1996; Avnimelech, 1999). Fish receiving high
which contains dissolved waste. The effluent of the RAS is protein diets produce carbon deficient waste. This is due to
either directly discharged to the environment, or digested in the amount of nitrogen, which accumulates in the RAS
lagoons or septic tanks, or thickened and/or applied as system water. The C : N resulting ratio in the effluent is
fertilizer for land based agriculture (Chen et al., 1997; 2–3 g : 1 g (Table 1).
Losordo et al., 2003). A possible alternative approach is to Therefore, the slurry requires organic carbon supplemen-
convert the waste into heterotrophic bacterial biomass. This tation. Sources and levels of carbon supplementation, sludge
biomass can be reutilized as aquatic feed. Such processes are composition [total ammonia nitrogen (TAN) or nitrate]
FEMS Microbiol Ecol 60 (2007) 207–219
c2007 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved

2. 208 O. Schneider et al.
Screen filter (60 µm)
Flow
equalizer
Trickling filter
Org. C
Source
pH control
Pure oxygen
Biofilter sump
Bacteria reactor
Pump sump
Bacteria collection
Fig. 1. Simplified scheme of a conventional recirculation aquaculture system for African catfish extended by the bacteria growth reactor and the flow
equalizer.
Table 1. Waste composition measured in the influent of the bioreactors and the influence of different carbon and nitrogen sources
(mg LÀ1) were evaluated. The second objective was to assess if the
Waste concentration produced bacteria biomass contains potential pathogens,
TAN 1.3 Æ 0.8 (0.3–4.8) which could reduce its suitability as feed. To address these
NO2-N 3.3 Æ 1.3 (0.7–12.4) objectives, we used complementary cultivation-dependent
NO3-N 182 Æ 58 (76–419) and 16S rRNA gene-targeted biomolecular approaches,
Kj-N 59 Æ 43 (13–260) aiming at the identification of bacterial populations present
TOC 422 Æ 159 (64–884) under the various process conditions,
Ortho-P 15.1 Æ 7.7 (6.2–40.1)
Ash 1776 Æ 717 (857–4957)
TS 3530 Æ 1033 (1936–7300) Materials and methods
TSS 1472 Æ 1041 (200–5770)
VSS 707 Æ 460 (40–2226) System setup
Conductivity 2000 –3000 mS
Two bacteria growth reactors were connected in parallel to a
Concentrations as averages Æ SD (minimum and maximum).
flow equalizer which received the effluent of a screen filter
TAN, total ammonia nitrogen; NO2-N, nitrite-N; NO3-N, nitrate-N; Kj-N,
(60-mm mesh size, Fig. 1). The screen filter was part of a
Kjeldahl nitrogen corrected for TAN concentrations; TOC, total organic
carbon; ortho-P, ortho-phosphate phosphorus; TS, total solids; TSS, total
recirculation aquaculture system (RAS), which was com-
suspended solids; VSS, volatile suspended solids. posed of four culture tanks, a biofilter and two sumps. In the
equalizer the slurry was aerated and agitated. The equalizer
was integrated into the system to allow for constant waste
flows towards the bacteria reactor, because the screenfilter
and sludge and hydraulic retention time (SRT, HRT) are all backwashes in pulses, depending on its automated flushing
factors influencing the bacteria community forming the cycle. The hydraulic retention time (HRT) of the drumfilter
produced biomass. Furthermore, the community composi- effluent in the equalizer was 4 h and the drumfilter backwash
tion depends also on the natural autochthonous microbiota volume about 120–140 L kgÀ1 feed.
from the sludge and system water. If the produced biomass is
reused as aquatic feed, it is important to evaluate the
Fish husbandry
biomass for potential bacteria pathogens.
The first study objective was to characterize the bacterial Fish were obtained from a commercial African catfish
community in the system water, in the slurry coming from hatchery (Fleuren and Nooijen, The Netherlands). Fish were
the flow equalizer, and of the produced bacterial biomass in stocked initially in four different cohorts of 140 fish each
the reactor. The effect of different hydraulic retention times (70, 170, 320 and 560 g individual average weight) into the
c 2007 Federation of European Microbiological Societies FEMS Microbiol Ecol 60 (2007) 207–219
Published by Blackwell Publishing Ltd. All rights reserved

3. Bacteria communities produced on RAS effluents 209
four tanks. Every 28 days the oldest cohort was harvested. conditions in the reactor (4 2 mg LÀ1). Oxygen was mon-
The emptied tank was restocked with 140 fish of about itored online using pH/Oxi 304i meters (WTW, Germany)
70 g. The final fish weight ranged between 823 and connected to a PC. This PC controlled the oxygenation of
1038 g. Therefore a complete production cycle from 70 to the broth. The oxygen control program reacted on a set-
about 1000 g lasted 112 days. Fish were fed with commercial point concentration of 3 mg LÀ1 oxygen inside the broth. pH
diet (Biomeerval, Skretting, France), containing 7% levels were maintained between 7.0 and 7.2 by addition of
moisture, 49% crude protein, 11% crude fat, 22% carbohy- acid or base (HCl, NaOH, 0.5–1N) stirred by a pH controller
drates, of which 2% crude fiber, 11% crude ash and (Liquisys M, Endress-Hauser, Germany). The reactor tem-
1.7% phosphorous (based on manufacturer information). perature was 28 1C, fixed by a water bath. The reactor was
The realized feeding level was between 16 and 19 g kgÀ1 continuously agitated by a rotor (RZR 2102, Heidolph,
metabolic body weight (W0.8) day-1. Diurnal waste fluctua- Germany) and the agitation speed was fixed to 350 r.p.m.
tions were minimized by applying a 24 h feeding regime. The
monthly harvesting/restocking scheme minimized changes Experimental designs and sampling
in both biomass within the system and in feed load. This
In this study, the bacterial communities corresponding to
stocking and feeding strategy assured minimal fluctuations
the content of bioreactors which operated under four
of waste production during a production cycle.
different conditions were analyzed (Table 2). In addition,
the communities of the system water and flow equalizer
Bacteria reactors were characterized. To achieve the different culture condi-
The reactors were made of glass in the workshop of tions, two flows were combined in the reactor influent: the
Wageningen University. The reactors had a working volume waste flow containing the fish waste from the flow equalizer
of 3.5 L and were equipped with baffles to improve the and the supplement flow containing the three organic
hydrodynamics (Fig. 2). From the flow equalizer the slurry carbon supplements. In the fourth operation condition,
was continuously pumped into the bacterial culture reactor TAN was added to the supplement flow. The supplements
by a peristaltic pump (Masterflex L/S, Masterflex). The SRT were mixed with distilled water and pumped by a peristaltic
was equal to the HRT as no sludge was returned. Pure pump (PD5001, Heidolph, Germany) into the reactors at a
oxygen was diffused by air-stones to maintain aerobic flow rate which was about 5% of the total flow rate. These
experimental conditions allowed comparing the effects of
Degassing pipe different HRTs, different carbon sources, and different
Inlets for
Oxygen inlet nitrogen sources. Because bacteria prefer TAN over nitrate
acetate
acid
base Waste inlet as a nitrogen source, the effect of those two nitrogen sources
could be investigated. Nitrate was available from the RAS
effluent stream, but it was decreasingly taken up by the
bacteria in the presence of increasing TAN concentrations. A
more detailed description of the experiments is provided in
Schneider (2007) and further in Schneider (2006), Schnei-
der et al. (2006a, b). From the three sampling points (system
water at the fish tanks influent, flow equalizer and bacteria
reactor), samples were siphoned and either analyzed as
Stirrer aqueous samples (50 mL), or sample material was collected
with 2 blades
pH electrode
over time (10.5 L) and centrifuged at 12g for 20 min (Table
Oxygen electrode
2). The supernatant was discarded, and the solid fraction
was collected and freeze dried.
Reactor outlet
Isolation, biochemical and 16S rRNA gene
ribotyping of cultured bacteria
Baffles Aqueous samples (1– 4) were homogenized, and each
homogenate was inoculated on to brain heart infusion
(BHI) agar with 5% sheep blood (home made at CIDC-
Lelystad, The Netherlands), and in parallel on to Cytophaga
agar (Oxoid), and incubated at 22 1C for 5 –7 days. After
Air stone
bacterial growth occurred, morphologically different colo-
Fig. 2. Schematic drawing of the bacteria growth reactor. nies were randomly selected for further typing in a pure
FEMS Microbiol Ecol 60 (2007) 207–219
c 2007 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved

4. 210 O. Schneider et al.
Table 2. Sample scheme for the four experimental conditions
Biochemical analysis and DNA isolation and
Sample HRT (h) Sample-ID 16S rRNA gene ribotyping PCR amplification
System water 1 Aqueous sample (50 mL) –
Equalizer 2 Aqueous sample (50 mL) Lyophilized (10.5 L)
Reactor
1.7 g C LÀ1 sodium acetate 7 3 Aqueous sample (50 mL) Lyophilized (10.5 L)
1.7 g C LÀ1 sodium acetate 2 4 Aqueous sample (50 mL) Lyophilized (10.5 L)
2.5 g C LÀ1 molasses 6 5 – Aqueous sample (50 mL)
1.7 g LÀ1 sodium acetate plus 250 mg LÀ1 TAN 6 6 – Aqueous sample (50 mL)
Volumes represent the original sample volume.
plate culture. These were cultured to a monoculture, using denaturation 95 1C for 30 s, annealing at 56 1C for 40 s, and
BHI with 5% sheep blood and identified according to extension at 72 1C for 1 min; and a final extension at 72 1C
standard biochemical tests (Bergey, 1984; Austin Austin, for 5 min. PCR products were verified by electrophoresis on
1987; Barrow Feltham, 1993). If identification was not a 1% (w/v) agarose gel containing ethidium bromide.
possible by these conventional methods, further typing was
carried out using molecular methods, using the Microseq DGGE analysis
500, 16S rRNA gene bacterial identification kits (Applied
Amplicons were separated by DGGE based on the protocol
Biosystems, Foster City, CA), according to the method
of Muyzer Smalla (1998) using the Decode system (Bio-
provided by the manufacturer.
Rad Laboratories, Hercules) with the following modifica-
tions. The polyacrylamide gels consisted of 8% (v/v) poly-
DNA isolation and PCR amplification for acrylamide (ratio of acrylamide to bisacrylamide: 37.5 :1)
molecular characterization of bacterial and 0.5 Â Tris-acetate-EDTA buffer (pH 8.0). Denaturing
communities acrylamide of 100% was defined as 7 M urea and 40%
formamide. The polyacrylamide gels were prepared with
For molecular analyses, DNA was isolated from freeze-dried
denaturing gradients ranging from 30% to 55% to separate
samples with the Fast DNASPIN kit (for soil; QBIOgene,
the generated amplicons of the total bacterial communities.
Cambridge, United Kingdom). Briefly, 0.1 g of freeze-dried
The gels were poured from the top using a gradient maker
material from each sample were placed in Lysing Matrix E
and a pump (Econopump; Bio-Rad) set at a rate of
Tubes with 122 mL of MT buffer and 978 mL of PBS and
4.5 mL minÀ1. Prior to the polymerization of the denaturing
processed three times for 30 s at speed 5.5 m sÀ1. The rest of
gel (gradient volume, 28 mL), a 7.5 mL stacking gel without
the protocol was carried out according to the manufacturer’s
denaturing chemicals was added. Electrophoresis was per-
instructions.
formed first for 5 min at 200 V and then for 16 h at 85 V in
PCR was performed with Taq polymerase kit (Invitrogen,
0.5 Â Tris-acetate-EDTA buffer (pH 8.0) at a constant
Carlsbad, CA) with the universal primer set 0968-a-S-GC-f
temperature of 60 1C. The gels were stained with AgNO3
(5 0 -AACGCGAAGAACCTTA-3 0 ) and S-D-Bact-L1401-a-A-
according to the method of Sanguinetti et al. (1994) and
17 r (5-CGGTGTGTACAAGACCC-3 0 ; N¨ bel et al., 1996),
u
dried overnight at 60 1C. Gels were scanned at 400DPI, and
which amplify the V6 to V8 regions of the bacterial 16S
analyzed with gel analysis software (BIONUMERICS 4.0; Applied
rRNA gene. The first primer has a 40 nucleotide GC rich
Maths BVBA, Sint-Martens-Latem, Belgium).
sequence at the 5 0 end (CGC CGG GGG CGC GCC CCG
GGC GGG GCG GGG GCA CGG GGG G), which allows the
Cloning of the PCR-amplified products
detection of sequence variations of amplified DNA frag-
ments by subsequent denaturing gradient gel electrophoresis 16S rRNA gene-targeted PCR amplicons (1500 bp) were
(DGGE; Muyzer et al., 1993). Each PCR reaction mixture generated with the set of primers 27-f (5-
contained (final volume, 50 mL) 20 mM Tris-HCl (pH 8.4), GTTTGATCCTGGCTCAG-3) and S-D-Bact-1492-a-A-19 r
3 mM MgCl2, each deoxynucleoside triphosphate at a con- (5-CGGCTACCTTGTTACGAC-3; Lane, 1991) and were
centration of 0.2 mM, each primer at a concentration of purified with NucloeSpin Extract II (Macherey-Nagel, The
0.2 mM, 1.25 U of Taq polymerase, and 1 mL of template Netherlands) according to the manufacturer’s instructions.
DNA. Samples were amplified in a Whatman Biometra PCR products were cloned into Escherichia coli XL1-Blue
Thermocycler (G¨ ttingen, Germany) using the following
o competent cells (Stratagene) using the Promega pGEM-T
program: predenaturation at 95 1C for 2 min; 35 cycles of easy vector system (Promega, Madison, WI). Ligation and
c 2007 Federation of European Microbiological Societies FEMS Microbiol Ecol 60 (2007) 207–219
Published by Blackwell Publishing Ltd. All rights reserved

5. Bacteria communities produced on RAS effluents 211
transformation reactions were performed according to the construction of a 16S rRNA gene based phylogenetic tree,
protocol described by the manufacturer. PCR was per- using the neighbour joining method (Saitou Nei, 1987).
formed on cell lysates of ampicillin-resistant transformants Phylogenetic placement was performed in comparison with
using vector specific primers T7 (TAATACGACTCACTA- reference sequences with Jukes–Cantor correction and
TAGG) and Sp6 (GATTTAGGTGACACTATAG) to confirm application of a phylum-level filter as implemented in ARB
the size of the inserts. A total of 96 amplicons of the correct (release February 2005). Chimeric sequences were identified
size (per sample) were subjected to amplified ribosomal by comparison of phylogenetic affiliation of the two respec-
DNA restriction analysis (ARDRA) using the restriction tive 5 0 - and 3- partial sequences. For the tree shown in Fig. 4
enzymes MspI, CfoI, and AluI. From each sample, clones and 5 0 -partial sequences obtained from clones and
corresponding to a unique RFLP pattern were used to sequences determined by ribotyping of cultured isolates
amplify V6–V8 regions of 16S rRNA genes with the primers were used from E. coli position 118 to 412.
968f-GC-f and 1401r as described previously, and they were
selected for subsequent sequence analysis according to their Nucleotide sequence accession numbers
migration position in the DGGE gel compared to the
Partial 16S rRNA gene sequences of the 16S rRNA gene
amplicons of the original DGGE profile of the sample.
clones have been deposited in the GenBank database under
accession numbers DQ788530–DQ788539.
Sequence analysis
PCR amplicons (1.4 kb) of transformants selected by the Results
above-described ARDRA/DGGE screening procedure were
purified with NucloeSpin Extract II (Macherey-Nagel, The Isolation, and biochemical and 16S rRNA gene
Netherlands) according to the manufacturer’s instructions. ribotyping of cultured bacteria
The samples were subjected to DNA sequence analysis
The results from the biochemical and 16S rRNA gene
(BaseClear Lab services, The Netherlands) with the primers
ribotyping for the system water, the equalizer and different
SP6 and T7, yielding two partial sequences (5 0 and 3 0 ) per
reactor broths are given in Table 3. While the system water
clone of c. 500 nucleotides.
and the flow equalizer contained five and seven different
Sequences were analyzed for similarity with sequences
bacteria, only four and three different bacteria were detected
deposited in public databases using the BLAST tool (McGinnis
in the reactor samples.
Madden, 2004) at the National Center for Biotechnology
Information database (http://www.ncbi.nlm.nih.gov/BLAST).
Molecular analysis of bacterial community
Alignment and further phylogenetic analysis of the
structure
sequences were performed using the ARB software package
(Ludwig et al., 2004). The resulting alignments were manu- The phylogenetic affiliations of the clones corresponding to
ally checked and corrected when necessary, and unambigu- prevalent bands in the DGGE sample profile were deter-
ously aligned nucleotide positions were used for mined by sequence analysis (Fig. 3, Table 4, Fig. 4). In the
Table 3. Results from the biochemical and 16S rRNA gene typing for the system water, the equalizer and different reactor broths
System water Equalizer 1.7 g C LÀ1, HRT 7 h 1.7 g C LÀ1, HRT 2 h
% of matching (homology)
Sample ID 1 2 3 4 by ribotyping Method
Bacillus sp. 1 – 1
Edwardsiella sp. 1 99 2
Proteus vulgaris 1 – 1
Aeromonas hydrophilia 1 1 1 – 1
Aeromonas sobria 1 1 – 1
Acinetobacter Iwoffi 1 – 1
Pseudomonas sp. 1 1 – 2
Comamonas sp. 1 99 2
Arcobacter butzlerii sp. 1 1 99 2
Chryseobacterium sp. 1 100 2
Flavobacterium sp. – 1
Myroides sp. 1 1 1 98 and 93 1,2
Sphingobacterium sp. 1 99 2
C, carbon; HRT, hydraulic retention time; method 1, biochemical procedure; method 2, 16S rRNA gene ribotyping.
FEMS Microbiol Ecol 60 (2007) 207–219
c2007 Federation of European Microbiological Societies
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6. 212 O. Schneider et al.
1.7g/L–1 sodium acetate, 2.5gC/1 molasses. 1.7gC L–1sodium 1.7gC L–1 sodium
250mg L–1 TAN, 6 h 6h acetate, 7 h acetate, 2 h Equalizer
1
2
3
4
5
6
7
12 8
11
9
10
Fig. 3. 16S rRNA gene-targeted PCR-DGGE analysis of bacterial communities in samples 2–6. Identification of bands was performed by DGGE analysis
of clones. Clones corresponding to bands 1 and 3 were all chimeric, and sequences are therefore not considered.
flow equalizer (sample 2), the predominant bands corre- phylogenetic relations between the detected phylotype and
sponded to sequences most closely related to Sarcina sp., related sequences are displayed in Fig. 4.
Flavobacterium columnare and Catellibacterium terrae
(bands 2, 4, 5). Clones corresponding to bands 1 and 3 were
found to be chimeric, unfortunately prohibiting unambig-
Discussion
uous identification. In sample 3 (1.7 g C LÀ1 sodium acetate, The integrated application of complementary cultivation-
7 h HRT) and in sample 4 (1.7 g C LÀ1 sodium acetate, 2 h dependent and biomolecular approaches allowed for the
HRT), similar profiles were found. For sample 3, DGGE qualitative and semi-quantitative comparison of the bacteria
fingerprinting suggested that the microbial community was communities present in the system water and the flow
dominated by a-proteobacterial populations most closely equalizer, and those that developed in bioreactors operated
related to Rhizobium spp. and Shinella zoogloeoides. In at four different conditions.
sample 4, the most predominant/abundant population was In general, only a limited number of bacterial populations
related to Acinetobacter lwoffi, while this phylotype was less were identified that were common to both the system
abundant in sample 3. Alpha-proteobacterial populations water and the flow equalizer. Examples were Aeromonas sp.
were only detected as minor community components. In and Myroides sp. RAS configuration might have caused
sample 5 (2.5 g C LÀ1 molasses, 6 h HRT), the most abun- such differences in the two bacteria communities. The
dant phylotype was most closely related to Aquaspirillum drumfilter effluent originates from water with a higher
serpens. Comparison of DGGE fingerprints suggested that organic waste load than the tank influent water, which
also a-proteobacterial populations corresponding to bands was treated by the drumfilter. This treatment can reduce
7 and 8 were present, while other minor bands could not be the chemical oxygen demand (COD) load in the system
identified. In sample 6 (1.7 g C LÀ1 sodium acetate, 250 mg water by 50% (own unpublished data). This reduction
TAN/l and 6 h HRT), the main identified components of the affects bacterial numbers, namely by removal of those
microbial community were populations related to Jonesia populations which grow in flocks and on solid particular
spp., Sphaerotilus spp. and Flavobacterium mizutaii. The waste, and of substrates, which are no longer available
c 2007 Federation of European Microbiological Societies FEMS Microbiol Ecol 60 (2007) 207–219
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7. Bacteria communities produced on RAS effluents 213
Fig. 4. Phylogenetic tree of bacterial 16S rRNA gene sequences retrieved from the different samples and cultured isolates (^, 16S rRNA gene
ribotyping; , biochemical procedures; V, PCR-DGGE). The tree was constructed from sequences obtained in this study and reference sequences by
neighbor joining procedures, using a bacterial filter, as implemented in ARB (Ludwig et al., 2004). Accession numbers of reference sequences and 16S
rRNA gene clones are provided in the figure. The reference bar indicates 10% sequence divergence.
for bacteria growth. The bacterial strains, found in the all experiments fish were healthy and the system performed
system water and the flow equalizer, included suspected well. The detected bacteria had, therefore, no visible nega-
pathogens at different levels (Tables 3–5). Despite the fact, tive impact on fish health. In general, the bacterial phylo-
however, that potential pathogens were detected, during types found in the system water and flow equalizer are
FEMS Microbiol Ecol 60 (2007) 207–219
c 2007 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved

8. 214 O. Schneider et al.
Table 4. Results from the DNA isolation and PCR amplification for the equalizer and different reactor broths
1.7 g C acetate 1.7 g C acetate 2.5 g C LÀ1 molasses, 1.7g C LÀ1, 250 mgTAN LÀ1,
Equalizer LÀ1 HRT 7 h LÀ1 HRT 2 h HRT 6 h HRT 6 h
Sample ID 2 3 4 5 6 Band-ID
Sarcina ventriculi (95) 1 (1) (1) (1) (1) 2
Flavobacterium collumnare (94) 1 (1) (1) (1) (1) 4
Catellibacterium terrae (95) 1 (1) (1) (1) (1) 5
Rhodobacter sphaeroides (93)
Gammaproteobacterium Bioluz (98) (1) 1 (1) (1) 6
Acinetobacter johnsonii (98)
Rhizobium sp. OK-55(97) 1 (1) (1) (1) 7
Rhizobium sp. OK-55(97) 1 (1) 8
Aquaspirillum serpens (98) 1 9
Jonesia denitrificans (91) 1 10
Sphaerotilus sp. IF5 (98) 1 11
Sphingobacterium multivorum (90) 1 12
Named bacteria are the closest match to the analysed sequences; sequence similarity is given in parantheses. Where most closely related sequences
correspond to phylotypes that are not or poorly characterized, the closest cultured reference strain is also provided.
1, identified as present in the sample; (1), presence predicted from identical band migration; C, carbon; HRT, hydraulic retention time.
typical for aquatic, fish farm and wastewater environments was not reported. To grow at a HRT of 2 h, a growth rate of
(Table 5). at least 0.5 hÀ1 is required, which is out of range for
The communities obtained from the reactors operated at Rhizobium spp. at high conductivities. Shorter HRT (e.g.
four different operation conditions were different from the 2 h compared to 7 h) might therefore bear the risk to culture
community of the flow equalizer. Only Arcobacter sp. and mainly potentially pathogenic bacteria.
Myroides sp. were found in both the flow equalizer and in A community similar to that obtained with sodium
one reactor broth samples (sample 3), when communities acetate (sample 3) was found for the reactor using molasses
were analyzed by biochemical and 16S rRNA gene ribotyp- as substrate (sample 5). The major difference was a commu-
ing of culture isolates. Based on comparison of 16S rRNA nity shift from strains represented by bands 7 and 8 to a
gene-targeted PCR-DGGE community fingerprints, all bac- population close to Aquaspirillum serpens, which was not
teria present in the equalizer were also present in the reactor detected as major component in sample 3. Such changes can
broth (Table 4). However, the major community compo- occur if both bacteria utilize similar substrates and can grow
nents in the reactor were composed of other populations, under similar conditions (Table 5). Furthermore, the
which were not found in the equalizer. HRT seemed to have molasses was not sterile. Bacteria other than those existing
a minor effect on the bacterial community as is shown by the in the system might have been introduced through the
results of samples 3 and 4, which differed only in their HRT substrate. Whether the bacteria, close to Aquaspirillum
(7 vs. 2 h). However, in sample 3, a-proteobacterial popula- spp., were more capable to utilize molasses than the Rhizo-
tions close to Rhizobium spp. and Shinella zoogloeoides were bium spp./Shinella zoogloeoides related populations has
the major community components, whereas in sample 4 (2 h nevertheless not been reported elsewhere.
HRT), a population most closely related to Acinetobacter When TAN was applied in addition to sodium acetate, the
spp. was the major component. This suggests that the bacteria community changed significantly (samples 3 and
previously dominating a-proteobacterial populations were 6). Nearly all bacteria, which were detected in sample 3 were
out-competed at this low HRT. This corroborates data from also present in sample 6, but another three were also found
Singleton et al. (1982), who reported growth rates for in sample 6. These bacteria were close to Sphaerotilus spp.,
Rhizobium spp. as 0.7–0.2 and 0.4–0.2 hÀ1 for water Sphingobacterium/Flavobacterium spp. and Jonesia spp. (Fig.
conductivities of 1200 and 6000 mS cmÀ1, respectively. 4). Pathogenicity has not been reported for any of these
The experimental conditions were in this range strains (Table 5). Sphingobacterium spp. grow well on swine
(2000–3000 mS cmÀ1). In contrast, Acinetobacter spp. grown manure, where TAN is a major nitrogen source (Leung
on sodium acetate have higher growth rates of 0.2–0.8 hÀ1 at Topp, 2001). Furthermore, Sphaerotilus- and Jonesia spp.-
25 1C compared to the high conductivity conditions related populations have been found in wastewater and mud
(Oerther et al., 2002). Unfortunately water conductivity (Table 5). All three might be then superior to other,
c 2007 Federation of European Microbiological Societies FEMS Microbiol Ecol 60 (2007) 207–219
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9. Bacteria communities produced on RAS effluents 215
Table 5. Habitat and growth conditions and pathogenicity for bacterial phylotypes or their closest related strains found in the different samples
Habitat and growth Pathogenicity focussing
Bacteria conditions on animals and fish Reference
Bacillus sp. Saprophytic Some strains, f.i. B. cereus (in carp and Weber (1997); Austin Austin
Waste water, paper mill slime striped bass), B. mycoides (in channel (1999); Oppong et al. (2003);
catfish), and B. subtilis (in carp) Tchobanoglous et al. (2003)
Edwardsiella sp. 23–28 1C Some fish pathogenic enterobacteria: Austin Austin (1987); Abbott
Aquatic habitats and especially fish, E.tarda (eel), E.ictaluri (channel Janda (2001)
amphibians, reptiles, and birds catfish), different effects on various
species, reaching from fatal to none
Proteus vulgaris Saprophytic Only few indication Austin Austin (1987); Weber
Soil, water, integral part of gut flora (1997); Manos Belas (2001);
Tanaka R et al. (2004)
Aeromonas hydrophilia Facultative anaerobic, 4–37 1C Facultative opportunistic found as Kinne (1984); Rice et al. (1984);
Different salinities well on healthy fish Austin Austin (1987); Meyer-Reil
Aquatic habitats, waste water Koester (1993); Weber (1997);
found frequently at fish farms Leonard et al. (2000)
Aeromonas sobria Facultative anaerobic 4–37 1C Facultative opportunistic or not Kinne (1984); Austin Austin (1987);
Different salinities necessarily attributed as pathogenic Meyer-Reil Koester (1993); Weber
Aquatic habitats, waste water found as well on healthy fish (1997)
frequently on fish farms
Acinetobacter iwoffi Aerobic 20–30 1C Facultative opportunistic, few Rice et al. (1984); Austin Austin
Different salinities indications (1987); Meyer-Reil Koester (1993);
Soil, aquatic habitats, waste water Fang et al. (2002); Wagner Loy
frequently on fish farms (2002)
Pseudomonas sp. Mesophilic temperatures Some facultative opportunistic, or Adamse (1968a); Austin Austin
Different salinities pathogenic strains reported: f.i. P. (1999); Palleroni et al. (1999)
Soils, water, sewage, animals, anguilliseptica (in eel, sea bream
plants and sea bass)
Sphaerotilus natans Aerobic/anaerobic Not reported Adamse (1968b); Pasveer (1968);
Freshwater Spring (2002); Schonborn (2003)
Sludges, waste water
Comamonas sp. Aerobic Rare opportunistic pathogens, no Etchebehere et al. (2001); Gumaelius
20–37 1C evidence of pathogenic effect on et al. (2001); Willems de Vos (2002)
Waste water, activated sludge, healthy people
animals’ blood
Aquaspirillum serpens Aerobic Not reported Payne (1981); Pot et al. (1999); Tal
Different salinities et al. (2003); Thomsen et al. (2004)
Denitrifcication reactors as well in
marine recirculation systems
Rhizobium/ Facultative aerobic Not reported Payne (1981); O’Hara Daniel (1985);
Mesorhizobium Soil, denitrification reactors, Batut Boistard (1994); Encarnacion
culturable on wastewater sludge, et al. (1995); Sadowsky Graham
aquatic (2000); Rebah et al. (2001);
systems, denitrification reactors Etchebehere et al. (2002); Liu et al.
(2005)
Shinella zoogloeoides Aerobic Not reported Kargi Karapinar (1995); Dugan et al.
Aquatic systems, domestic sewage (1999)
and aerobic sewage-treatment
systems
Castellibacterium sp./ Fresh to salt water Not reported Kersters et al. (2003); Tanaka Y et al.
Rhodobacter sp. Activated sludge, marine sludge (2004); Cytryn et al. (2005a, b)
Arcobacter butzlerii/sp. Aerobic Possibly involved Moreno et al. (2003); Tanaka R et al.
15–37 1C (2004); Lehner et al. (2005)
Gut microbiota, surface and
ground waters
Seawage and activated sludge
FEMS Microbiol Ecol 60 (2007) 207–219
c2007 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved

10. 216 O. Schneider et al.
Table 5. Continued.
Habitat and growth Pathogenicity focussing
Bacteria conditions on animals and fish Reference
Chryseobacterium sp. Aerobic Pathogenic, f.i. C. scophthalmum (in Urdaci et al. (1998); Austin Austin
Different salinities turbot), C. balustinum (in marine fish) (1999); Jooste Hugo (1999);
Soil, plants, aquatic habitat, Bernardet Nakagawa (2000);
activated sludge Mustafa et al. (2002);
Bernardet (2005)
Flavobacterium sp. Aerobic Facultative, mostly found externally, Kinne (1984); Austin Austin (1987);
5–42 1C may induce skin necrosis after stress Murray et al. (1990); Meyer-Reil
Salinity below 1% found as well on healthy fish, some Koester (1993); Bernardet
Soil, aquatic habitat frequently species are very pathogenic Nakagawa (2000); Bernardet et al.
at fish farms (2005)
Myroides sp. Aerobic Opportunistic Gonzalez et al. (2000); Hugo et al.
25–30 1C (2000)
Human instestine, soil, water
Sphingobacterium sp. Aerobic Not reported Holmes (1999); Leung Topp (2001);
Soil, activated sludge, gut fauna, Tanaka R et al. (2004)
liquid swine manure
Sarcina ventriculi Obligate anaerobic, but not Not reported Goodwin Zeikus (1987); Jung et al.
oxygen sensitive (1993); Snell-Castro et al. (2005)
30–37 1C
Gut fauna
Jonesia quinghaiensis Aerobic Not reported Schumann et al. (2004)
20–30 1C
Different salinities
Mud
outcompeted populations in the utilization of TAN, result- ing community were not identified. Those bacteria have
ing in higher growth rates. been found in other studies, focusing on the system as a
Given the pathogenic risk associated with short HRTs, it whole by investigating its components (Tal et al., 2003;
is advisable to choose for HRTs of 6–7 h. The choice of Cytryn et al., 2005). Investigations of heterotrophic bacteria
organic C donor seems of less importance, as the obtained communities yielded some similar results, such as the
communities in the presence of sodium acetate or molasses, detection of Pseudomonas spp., Aeromonas spp., Aquaspir-
respectively, did not change in their pathogenicity. More- illum spp. and others (Leonard et al., 2000; Tal et al., 2003).
over, the addition of TAN did not increase the risk of Anyway, it is unlikely to find completely identical bacterial
potentially pathogenic populations, as revealed by the communities in RAS, because of differences in their envir-
comparison of samples 3 and 6. Two considerations have to onmental conditions (marine vs. freshwater), configurations
be made. The ‘native’ nitrogen source supplied in the RAS (e.g. presence of UV, foam fractionators), and in the
effluent stream is nitrate. To utilize this nitrogen species, the cultured animals.
system design did not change and the reactor can easily be
installed after the drumfilter. If TAN should be used, the
system would have to be modified to eliminate nitrification.
Conclusion
The only advantage to using TAN might then be the The bacteria community found in the system water and in
potentially higher nutritional value of the obtained bacteria the flow equalizer contained some possible opportunistic
biomass. This advantage would have to be confirmed by pathogens, but did not result in severe disease symptoms or
additional experiments. Generally, the pathogenic risk and production losses during the fish culture operation. The
nutritional value of all obtained bacterial material has to be bacteria community of the flow equalizer was semi-quantita-
further investigated in feeding trials, if the bacteria biomass tively different from the communities found in the bacteria
should be used as aquatic feed. reactors. However, all major community components were
To compare the occurrence of bacteria found in the present in both equalizer slurry and reactor broths. Hydrau-
system water, the flow equalizer and in the bacteria reactor lic retention times (7 h vs. 2 h) influenced the bacteria
with bacteria found in RAS in general is difficult, because community composition, resulting in a more abundant
literature data is scarce. Because no biofilter material was fraction of potentially pathogenic populations related to
investigated in this study, bacteria belonging to the nitrify- Acinetobacter at 2 h HRT compared to 7 h HRT. At 7 h
c 2007 Federation of European Microbiological Societies FEMS Microbiol Ecol 60 (2007) 207–219
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11. Bacteria communities produced on RAS effluents 217
bacteria close to Rhizobium spp. and Shinella zoogloeoides Bernardet JF, Vancanneyt M, Matte-Tailliez O, Grisez L, Tailliez P,
formed the major components of the community. The use of Bizet C, Nowakowski M, Kerouault B Swings J (2005)
molasses instead of sodium acetate caused a shift in compo- Polyphasic study of Chryseobacterium strains isolated from
sition to a bacterial community dominated by a population diseased aquatic animals. Syst Appl Microbiol 28: 640–660.
similar to Aquaspirillum serpens. Providing TAN in addition Burford MA, Thompson PJ, McIntosh RP, Bauman RH
to nitrate as nitrogenous substrate led to the occurrence of Pearson DC (2003) Nutrient and microbial dynamics in high-
bacteria close to Sphaerotilus spp., Flavobacterium mizutaii intensity, zero-exchange shrimp ponds in Belize. Aquaculture
and Jonesia spp. It was concluded from those results that a 219: 393–411.
reactor operation regime of 6–7 h HRT is recommended, and Chen SL, Coffin DE Malone RF (1997) Sludge production and
that the type of substrate (sodium acetate or molasses, TAN management for recirculating aquacultural systems. J World
or nitrate) is less important. Considering conventional RAS Aquac Soc 28: 303–315.
configurations, nitrate might be preferred over TAN. How- Cytryn E, Minz D, Gelfand I, Neori A, Gieseke A, De Beer D
ever, for all the obtained bacteria communities, additional Van Rijn J (2005a) Sulfide-oxidizing activity and bacterial
tests are required to investigate their pathogenic risk and community structure in a fluidized bed reactor from a zero-
nutritional values as aquatic feed in more detail. discharge mariculture system. Environ Sci Technol 39:
1802–1810.
Cytryn E, van Rijn J, Schramm A, Gieseke A, de Beer D Minz D
Acknowledgements (2005b) Identification of bacteria potentially responsible for
oxic and anoxic sulfide oxidation in biofilters of a recirculating
The authors would like to thank the hatchery and the
mariculture system. Appl Environ Microbiol 71: 6134–6141.
laboratory staff of the AFI group for their support during Dugan PR, Stoner DL Pickrum HM (1999) The genus Zooglea.
the study. This study was financed by the European Union in The Prokaryotes: An Evolving Electronic Resource for the
the frame of the ZAFIRA project (ICA4-CT-2001-10025). Microbiological Community (Dworkin M, Falkow S, Rosenberg
E, Schleifer K-H Stackebrandt E, eds). Springer, Berlin.
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