Short-term changes in microbial communities in the water column around 
the fish farm in the Bay of Piran
Kratkotrajne spremembe mikrobne združbe v vodnem stolpcu okoli ribogojnice  
v Piranskem zalivu
Valentina Turk*, Tinkara Tinta
Marine Biology Station, National Institute of biology, Fornače 41, 6330 Piran, Slovenia
*Correspondence: valentina.turk@nib.si
Abstract: A multidisciplinary approach was used to study the impact of fish far-
ming on coastal bacterial communities in the inner part of the Bay of Piran (northern 
Adriatic). Differences in bacterial abundance, production and the occurrence of selected 
bacterial groups were studied in the water column around the cage and at different 
distances from the centre of the fish cage towards the open water, i.e., reference ma-
rine station. We also examined the effect of fish feeding on the surrounding system 
in a short-term in situ experiment based on the simultaneous collection of seawater 
samples from different locations around the fish cage before and after feeding of 
fish. Our study suggests that fish feeding has a moderate short-term effect on water 
column parameters, including bacterial abundance and production, only at a limited 
distance from the fish cages. The nitrifying, ammonia-oxidizing bacterial groups, as 
determined by the fluorescent in situ hybridization method, were represented at a 
higher percentage in the seawater samples in the middle and around the fish cages. 
β- Proteobacteria, γ-Proteobacteria and the Cytophaga-Flavobacterium group were 
represented to a higher percentage at sampling sites in the middle of the Bay of Piran 
and at the reference marine station. The Vibrio group was detected at all sampling 
sites. The accumulation of organically enriched fish food and waste products released 
into the seawater during the short-term experiment resulted in a significant increase 
in particulate matter, orthophosphate and ammonium. In response to the increase in 
inorganic nutrients, we observed a significant increase in bacterial production, while 
no significant differences were observed in bacterial abundance in such short time.
Keywords: aquaculture, bacterial abundance, bacterial community composition, 
bacterial production, fluorescent in situ hybridisation, pollution
Izvleček: Vpliv ribogojstva na bakterijske združbe smo proučevali z multidisci-
plinarnim pristopom v notranjem delu Piranskega zaliva (severni Jadran). Razlike v 
številčnosti bakterij, bakterijski produkciji in pojavljanju izbranih bakterijskih skupin 
smo preučevali v vodnem stolpcu okoli ribje kletke in na izbranih lokacijah od središča 
ribje kletke do odprtih vod. Spremembe pred in po hranjenju rib smo preučili tudi v 
kratkoročnem poskusu in situ, ki je temeljil na istočasnem vzorčenju morske vode na 
različnih lokacijah okoli ribje kletke. Naša študija kaže, da hranjenje rib kratkoročno 
zmerno vpliva na parametre vodnega stolpca, vključno s številčnostjo bakterij in pro-
 ACTA BIOLOGICA SLOVENICA 
 LJUBLJANA 2021 Vol. 64, Št. 2: 9–23
10Acta Biologica Slovenica, 2021, 64 (2), 9–23
dukcijo, in le na omejeni razdalji od ribjih kletk. Nitrifikacijske bakterije, ki oksidirajo 
amonij, določene s fluorescentno in situ hibridizacijsko metodo, so bile zastopane 
v višjem deležu v vzorcih morske vode v sredini in neposredni okolici ribje kletke. 
β-Proteobakterije, γ-Proteobakterije in skupina Cytophaga-Flavobacterium so bile 
zastopane v višjem odstotku na bolj oddaljenih vzorčnih mestih. Potencialno patogena 
vrsta Vibrio je bila prisotna na vseh mestih vzorčenja. Kopičenje organsko obogatene 
ribje hrane in odpadnih produktov, ki se sproščajo v morsko vodo v kratkem času po 
hranjenju, je povzročilo znatno povečanje koncentracij partikulatne organske snovi, 
ortofosfata in amonija. Kot odgovor na povečanje anorganskih hranil smo izmerili 
bakterijsko aktivnost, medtem ko razlik v biomasi v vodnem stolpcu ni bilo opaziti.
Ključne besede: akvakultura, bakterijska abundance, bakterijska produkcija, 
bakterijska vrstna sestava, fluorescentna in situ hibridizacija,  onesnaženje 
Introduction
In the last 20 years, aquaculture has expanded 
rapidly both globally and in the Mediterranean, 
with a focus on the production of sea bass Di-
centrachus labrax and sea bream Sparus aurata 
(FAO  2018). The rapid expansion of aquaculture 
has raised increasing concerns about negative 
environmental impacts, given numerous well-
documented cases (Bouwman et al. 2013). These 
are primarily the impacts of waste products from 
farming – organic matter, nutrients, chemicals 
and pharmaceuticals – and the transfer of genes, 
parasites and diseases between wild and farmed 
species (Hargrave et al. 1993, McGhie et al. 2000, 
Karakassis 2000, Kim et al. 2004, Armstrong et al. 
2005, Bouwman et al. 2013, Cabello et al. 2013, 
Wang et al. 2020). The numerous documented 
negative environmental impacts of aquaculture 
are mainly related to the accumulation of uneaten 
feed and faeces in seawater and on the seabed 
beneath fish cages (Hargrave et al. 1997, Karakas-
sis 2000, Pitta et al. 2006, Vezzulli et al. 2008, 
Reimers et al. 2013). Fish food consists largely of 
proteins and the main components of this organic 
enrichment affect biogeochemical processes and 
often lead to eutrophication, oxygen deficiency 
and hypoxia on the seabed (Hargrave et al. 1993, 
Karakassis 2000). The effects of high organic 
matter loading on sediment below aquaculture 
farms have been extensively studied, showing 
changes in the structure of benthic communities 
that affect the entire food web and lead to an 
overall decline in species diversity (Hargrave et 
al. 1997, Karakassis et al. 1998, Christensen et al. 
2000, Grego et al. 2009, Mirto et al. 2012, Luna 
et al. 2013, Reimers et al. 2013). 
Marine microbes drive biogeochemical cycles 
in coastal areas (Kirchman 1994). They regulate 
carbon and energy transfer in the food web, the 
maintenance of water quality, and the health of 
marine ecosystems (Azam and Malfatti 2007). 
Marine microbes respond rapidly to environmental 
perturbations (Galand et al. 2016, Ape et al. 2019) 
and play an important role in the degradation and 
remineralization of organic matter. Several studies 
have shown that organic wastes from fish farms 
stimulate microbiological metabolism, most of 
which can be attributed to changes in enzymatic 
activity (Caruso et al. 2003, Vezzulli et al. 2004), 
nitrification, denitrification or sulphate reduction 
(McCaig et al. 1999, Christensen et al. 2000, Asami 
et al. 2005, Kondo et al. 2012, Dowle et al. 2015)
ammonium concentrations, nitrification rates, and 
ammonia oxidizer most-probable-number counts 
were determined in samples of sediment collected 
from beneath a fish cage and on a transect at 20 and 
40 m from the cage. The data suggest that nitrogen 
cycling was significantly disrupted directly beneath 
the fish cage, with inhibition of nitrification and 
denitrification. Although visual examination indi-
cated some slight changes in sediment appearance 
at 20 m, all other measurements were similar to 
those obtained at 40 m, where the sediment was 
considered pristine. The community structures of 
proteobacterial β-subgroup ammonia-oxidizing 
bacteria at the sampling sites were compared by 
PCR amplification of 16S ribosomal DNA (rDNA. 
Increases in bacterial abundance (Mirto et al. 2000, 
Vezzulli et al. 2002, Bissett et al. 2007), virus-like 
11Turk and Tinta: Impact of aquaculture on bacterioplankton 
particles (Garren et al. 2008, Luna et al. 2013), 
and bacterial production (Navarro et al. 2008, 
Garren et al. 2008), as well as changes in bacte-
rial community composition (Bissett et al. 2006, 
Garren et al. 2008, Castine et al. 2009, Kawahara 
et al. 2009, Quero et al. 2015, Galand et al. 2016, 
Martins et al. 2018, Kolda et al. 2020) have been 
detected in the sediments bellow fish cages. 
Increased deposition of organic matter on 
the seabed below fish cages (Kovac et al. 2003, 
Lojen et al. 2005) has been confirmed for our 
study area and negatively affects the sediment 
chemical properties and the abundance and 
structure of meiofauna (Grego et al. 2009, 2020). 
The impact of aquaculture on microbes have 
been studied mainly in sediments, much less in 
the water column. In our study, we focused on 
the short-term effects of aquaculture activities 
on the bacterial community in the upper layer of 
the water column around the cage and at different 
locations from the centre of the fish cage to the 
open water. We mainly investigated the changes 
in nutrient concentrations, abundance and bacte-
rial production before and after feeding the fish 
in a short-term in situ experiment based on the 
simultaneous collection of seawater samples from 
different sites around the fish cage.
Materials and methods 
Study site and experimental setup 
The study was conducted in October 2005 
in the marine fish farm in the inner part of the 
shallow semi – enclosed Bay of Piran (Gulf of 
Trieste, northern Adriatic Sea) (Fig. 1). The bay 
is about 7 km long and 5 km wide and is charac-
terised by limited hydrodynamics. The fish farm 
consisted of 20 floating cages with a total area of 
2019 m2, where sea bass (Dicentrarchus labrax) 
and gilthead sea bream (Sparus auratus) were 
kept. The depth below the fish cages was about 
13 m. The total annual production in 2005 was 
100 tonnes. The fish were fed three times a day. 
The daily dietary intake was 12 kg, and consisted 
Figure 1:  Sampling sites in the middle and around the fish cage at 6 and 20 m intervals, in the inner part of the 
Bay of Piran, and the reference station in the Gulf of Trieste (ZBOB).
Slika 1:   Mesta vzorčenja morske vode v središču in okolici ribje kletke na razdalji 6 m in 20 m, v notranjem 
delu Piranskega zaliva, ter zunanji referenčni postaji v Tržaškem zalivu (ZBOB).
12Acta Biologica Slovenica, 2021, 64 (2), 9–23
mainly of proteins (44 %) and lipids (22 %) and 
a smaller proportion of oligosaccharides (16 %), 
fibrins (2 %), ash (8 %) and vitamins, according 
to manufacturer. 
To evaluate the impact of aquaculture on the 
pelagic bacterial community, two different studies 
were conducted. In the first study, seawater sam-
ples were collected at stations along the transect 
located 6 m, 20 m, 1500 m, and 5400 m from the 
centre of the fish cage (Fig. 1), at 5 m depth with 
Niskin samplers. At the same time, basic physical 
(temperature, salinity) and chemical parameters 
(total particulate matter, orthophosphate, total 
phosphorous and nitrogen, nitrate, ammonium) 
were analysed to determine the level of organic 
pollution. 
For the second study, a system of anchored 
sampling bottles was constructed at different 
locations: in the centre of the cage (00CC), 
sampling points in a circle around the fish cage 
at a distance of 6 m (00N1, 0EN1, 0ES1, 00S1, 
0WS1, 0WN1), and 20 m (00N2, 0EN2, 0ES2, 
00S2, 0WS2, 0WN2) (Fig. 1). Each bottle (5 L) 
was attached to an underwater metal frame at 
5 m depth. At the time of sampling, all bottles 
were opened simultaneously (by small boats 
and by divers). Seawater samples were collected 
at each sampling site before (bf) and 3 h after 
(af) fish feeding and the results for each param-
eter were expressed as the ratio K= Cbf/Caf in 
percentage. 
Oceanographic parameters and nutrient 
analyses
Basic oceanographic parameters (temperature, 
salinity) were measured using a CTD fine –scale 
probe (Microstructure Profiler MSS90, Sea & 
Sun Technology GmbH), and 24-hour current 
measurements were made (not presented in 
this study). Total particulate matter (TPM) was 
measured gravimetrically, and inorganic nutrients 
(nitrate, ammonium, phosphate) were measured 
colorimetrically (Grasshoff et al. 1983) using a 
UV/VIS spectrometer, Perkin Elmer, Lambda 14. 
Bacterial abundance and production
Seawater samples for total bacterial counts 
were fixed with formaldehyde (2% final concen-
tration) and analyzed by the method of Porter and 
Feig (1980). Samples were stained with the fluo-
rochrome 4’,6’-diamino-2-phenylindole (DAPI, 
1 µg mL-1 final concentration) and counted under 
an epifluorescence microscope (Olympus BX51).
Bacterial carbon production (BCP) was 
measured by incorporation of 3H-leucine into 
newly synthesized proteins in bacterial cells 
(Simon and Azam 1989) using the centrifuga-
tion protocol of Smith and Azam (1992). Ultima 
Gold (PerkinElmer) was added to each sample 
and radioactivity was measured using a liquid 
scintillation counter (Canberra Packard TriCarb 
Liquid Scintillation Analyzer, model 2500 TR). 
Table 1:   List of oligonucleotide probes with sequence and target bacterial group. 
Tabela 1:  Seznam oligonukleotidnih sond s sekvenco in tarčno skupino bakterij.
Probe Sequence Target organisms
Nso 1225 5’- CGC CAT TGT ATT ACG TGT GA – 3’ nitrifying ammonia-oxidizing bacteria
NEU 5’- CCC CTC TGC TGC ACT CTA – 3’ nitrifying ammonia-oxidizing bacteria
EUBI,II,III 5’- GCT GCC TCC CGT AGG AGT – 3’ all bacteria 
BET 42a 5’ – GCC TTC CCA CTT CGT TT – 3’ β-proteobacteria 
GAM 42a 5’ – GCC TTC CCA CAT CGT TT – 3’ γ-proteobacteria 
CF 319a 5’ – TGG TCC GTG TCT CAG TAC – 3’ Cytophaga-Flavobacterium group of CFB phylum 
GV 5’ – AGG CCA CAA CCT CCA AGT AG – 3’ Vibrio group 
13Turk and Tinta: Impact of aquaculture on bacterioplankton 
Fluorescent in situ hybridization (FISH) 
The abundance of selected phylogenetic 
groups was determined by the fluorescence in 
situ hybridization method using Cy3-labelled 
oligonucleotide probes (Thermo Electron Cor-
poration). The sequences and target organisms 
of all 16S rRNA-targeted oligonucleotide probes 
are listed in Tab. 1. 
Group specific probes were EUBI, II, III for all 
bacteria, BET 42a for β-Proteobacteria, GAM42 
for γ-Proteobacteria, CF 319a Cytophaga-Flavo-
bacterium cluster of Cytophaga-Flavobacteria-
Bacteroides CFB phylum and GV for Vibrio group. 
The nitrifying, and ammonia-oxidizing bacteria 
groups were determined using the Nso 1225 probe 
specific for the ammonia-oxidizers and the NEU 
probe complementary to a signature region of most 
halophilic and halotolerant ammonia-oxidizers. 
From each sampling station, 10 ml of sample 
was fixed overnight with 37% formaldehyde. 5 ml 
subsamples were filtered onto 0.2 μm polycarbon-
ate filters (47 mm, Poretics) and rinsed with 3 x 
PBS, 1x PBS solution, and distilled water and 
stored at -20 ºC. For each probe, a filter piece was 
placed on a glass slide covered with Parafilm and 
overlaid with 20 μl hybridization probe prepared 
by mixing 2 μl probe and 18 μl hybridization 
solution. The hybridization solution contained 
360 μl NaCl (5 M), 40 μl Tris-HCl (1M, pH 7.2-
8), 2 μl 10% SDS, the optimal concentration of 
formamide, 400 μl blocking solution (Roche), 
and 500 μl distilled water. The filters were incu-
bated in the hybridization oven at 42 ºC for 3h. 
After hybridization, filters were washed in wash 
solution (1000 μl Tris/HCl (1M), 700 μl NaCl (5 
M), 500 μl EDTA, 25 μl 20% SDS and 47.8 ml 
distilled water) for 20 min at 48 ºC. Washed and 
dried filter pieces were stained with 1 μg mL-1 
4’,6’- diamino-2-phenylindole (DAPI). Cells were 
counted by epifluorescence microscopy (Olympus 
BX51). The abundance of each bacterial group 
detected was expressed as the percentage of each 
group in the total bacterial count.
Statistical analysis
A three-way ANOVA was conducted to test 
the main interactive effects of bacteriological 
and chemical parameters (orthophosphate, total 
phosphate, ammonium, bacterial abundance and 
carbon production), feeding activity (before and 
after feeding), and sampling sites (distance – 0 m, 
6 m, 20 m; and directions – south, north, east, west). 
Table 2:   The results (means and standard deviations) of total particulate matter (TPM), nitrate, ammonium, 
orthophosphate, total phosphorus (Tot P) and nitrogen (Tot N), bacterial biomass and bacterial carbon 
production rate (BCP) in seawater samples from different sites as a function of distance from fish cages.
Tabela 2:  Rezultati (srednja vrednost in standardni odmik) celokupne partikulatne snovi (TPM), nitrata, amonija, 
ortofosfata, celokupnega fosforja (Tot P) in nitrogen (Tot N), bakterijske biomase in bakterijske produkcije 
(BCP) v vzorcih morske vode na različnih merilnih mestih glede na oddaljenost od ribiških kletk.
Distance from the fish cage (m)
Parameter Cage 6 20 200 1500 5400
TPM (mg L-1) 2.40 2.12 ± 0.56 2.55 ± 1.79 2.09 ± 0.35 1.67 1.04
Nitrat (μM L-1) 0.95 0.33 ± 0.07 0.34 ± 0.12 0.38 ± 0.18 0.51 0.20
Ammonium (μM L-1) 0.68 0.59 ± 0.12 0.57 ± 0.04 0.82 ± 0.28 0.76 0.52
Tot N (μM L-1) 31.13 33.80 ± 4.86 31.72 ± 3.92 42.46 ± 9.20 45.68 37.01
Orthophosphate (μM L-1) 0.12 0.10 ± 0.01 0.11 ± 0.04 0.11 ± 0.01 0.15 0.11
Tot P (μM L-1) 0.32 0.29 ± 0.02 0.30 ± 0.04 0.29 ± 0.06 0.33 0.31
Bacterial biomass (μg C L-1) 43.95 53.21 ± 12.3 44.15 ± 5.62 37.38 ± 10.3 27.28 21.37
BCP (μg C L-1day-1) 10.31 9.27 ± 2.72 10.89 ± 2.26 1.65 ± 1.08 1.34 3.9
14Acta Biologica Slovenica, 2021, 64 (2), 9–23
Results 
Effects of fish farm activity on nutrient and 
microbial variables along the pollution 
gradient
Results of chemical and microbiological 
analyses of the upper layer of the seawater column 
did not differ significantly among sampling sites, 
although, higher concentrations of total particulate 
matter, nitrate, and bacterial carbon production 
were measured near the fish cage (Tab. 2).
Total particulate matter values ranged from 
2.12 to 2.55 mg L-1 at sites near the fish cage and 
were twice the values measured at the site 5400 
m from the fish farm. The highest nitrate concen-
tration was at the cage site and was three times 
lower at all other sites (Tab. 2). Concentrations 
of ammonium, orthophosphate, total phosphorus 
(Tot P), and nitrogen (Tot N) in the upper layer of 
the water column did not differ among stations, or 
concentrations were slightly higher at the station 
in the middle of the Bay of Piran. 
Bacterial carbon production differed signifi-
cantly (F=7.932, p < 0.001) between sites and was 
on average up to 5-fold higher near the fish cage 
compared to values measured at the sampling 
site 1500 m away, and 3-fold higher compared 
to bacterial production measured at the reference 
site (Tab. 2, Fig. 2).
Similar trends were observed for bacterial 
abundance, but the difference was not statistically 
significant. The average bacterial abundance in the 
vicinity of the fish cage (0, 6, and 20 m) ranged 
from 2.4 to 3.1 x 109 cells L-1 and decreased 
with increasing distance from the cage. Bacterial 
abundance was more than twice as high at the fish 
farm as at the sampling sites at 1500 m and 5400 
m (Tab. 2, Fig. 2). 
Molecular analyses of targeted ribosomal RNA 
using the FISH method and various oligonucleotide 
probes revealed differences in the composition of 
bacterial groups in seawater along the transect 
(Fig. 3). Based on the literature data, we selected 
specific probes for the bacterial groups expected 
in an environment polluted by aquacultures.
0
2
4
6
8
10
12
0
20
40
60
80
0 6 20 1500 5400
Ba
ct
er
ia
l a
bu
nd
an
ce
 (
µg
 C
 L
-1
)
Distance from the fish cage (m)
Ba
ct
er
ia
l p
ro
du
ct
io
n 
(µ
g 
C
 L
-1
d 
-1
)
Figure 2:  Bacterial biomass (■) and bacterial production (●) at sampling sites at different distances from the fish 
cages in the Bay of Piran. 
Slika 2:   Bakterijska biomasa (■) in bakterijska produkcija (●) na merilnih mestih različnih razdalj od ribjih 
kletk v Piranskem zalivu.
15Turk and Tinta: Impact of aquaculture on bacterioplankton 
The various groups were represented differ-
ently. In the middle and immediate vicinity of the 
fish cage, all identified bacterial groups accounted 
for 30-40% of the total number of bacterial cells 
in the seawater samples, and the composition of 
the remaining portion was not determined. Within 
the identified groups, the nitrifying and ammonia-
oxidizing bacterial groups Nso 1225 and NEU 
were represented with a higher percentage, 10 
and 6 %, respectively. At other sampling sites, 
the β-Proteobacteria, γ-Proteobacteria and the 
Cytophaga-Flavobacterium group of the CFB 
strain (Cytophaga-Flavobacteria-Bacteroides) 
predominated (Fig. 3). At the sampling site 1500 m 
from the fish cage, the identified bacterial groups 
of all selected oligonucleotide probes accounted for 
85 % of the total bacterial count. Compared to the 
other stations, there was a higher percentage of all 
bacterial groups, especially the β-Proteobacteria, 
and Cytophaga-Flavobacterium groups, except for 
the ammonia-oxidizers of the β-Proteobacteria 
subgroup (the Nso 1225 probe), which was repre-
sented with the highest percentage at station 00CC. 
At the reference marine site, all bacterial groups 
were evenly distributed, however accounted for 
only 30 % of the total bacterial count and most 
of the community composition was unknown, 
possibly due to untargeted bacterial groups of the 
α-Proteobacteria that may predominate in coastal, 
more oligotrophic waters. The Vibrio group (up 
to 6% of the total bacteria count) was detected at 
all sampling sites.
Effects of fish feeding on nutrient levels, 
bacterial abundance and production in the 
water column in a short period of time
The results of chemical and bacteriological 
analyses of seawater, sampled simultaneously at 
all sampling sites before and 3 hours after feeding, 
showed changes in the concentrations of several 
parameters. The results of the relative difference 
(%) of total particulate matter, orthophosphate and 
ammonium concentrations after fish feeding in 
relation to before fish feeding at the sites around 
the fish cage (in the middle, 6 m and 20 m away) 
are shown in Fig. 4. 
With few exceptions, all values increased 3 
hours after feeding compared with results before 
fish feeding. Overall, concentrations of total 
particulate matter, increased from 2.12 ± 0.5 
Figure 3:  Percentage of bacterial groups determined by the fluorescent in situ hybridization method (FISH) at 
sampling sites at different distances from fish cages in the Bay of Piran.
Slika 3:   Procent posamezne skupine bakterij določene z metodo Fluorescentne in situ hibridizacije (FISH) na 
merilnih mestih, ki so različno oddaljeni od ribjih kletk v Piranskem zalivu.
16Acta Biologica Slovenica, 2021, 64 (2), 9–23
mg L-1 to 5.65 ± 1.1 mg L-1. A similar trend was 
observed in the concentrations of orthophosphate, 
which doubled from 0.07 ± 0.01 μmol L-1 to 0.14 
± 0.04 μmol L-1, ammonium increased from 0.59 
± 0.1 μmol L-1 to 0.77 ± 0.1 μmol L-1, and nitrate 
from 0.33 ± 0.07 μmol L-1 to 0.77 ± 0.2 μmol L-1. 
A three-way ANOVA showed a significant increase 
in concentrations of orthophosphate (F=25.09, p 
< 0.0001), total phosphorus (F=37.08, p < 0.001), 
ammonium (F=8.61, p < 0.009) after the feeding. 
But we found no significant difference between 
distance and direction of sampling sites.
During the experiment, bacterial carbon pro-
duction increased at almost all stations around the 
cage at radius of 6 m and 20 m. The average rate 
of bacterial carbon production was significantly 
higher 3 hours after feeding (F=18.81, p < 0.001) 
within the 20 m radius and an overall increase 
from 9.66 ± 2.8 µg C L-1 d-1 to 14.54 ± 2.9 µg 
C L-1 d-1 was recorded. The highest increase was 
observed towards the south and southwest, up 
to 163% at the station 00S2 and 145% at station 
0WS1 and in the middle of the cage up to 125% 
(at the station 00CC) (Fig. 5 A). 
Figure 4:  Results of relative difference (%) of total particulate matter (TPM) (green), orthophosphate (blue) and 
ammonium (red) concentrations after fish feeding in relation to before fish feeding at the sites around 
the fish cage (in the middle, 6 m and 20 m away). 
Slika 4:   Rezultati relativne razlike (%) v koncentracijah totalne partikulatne snovi (TPM), ortofosfata in amonija 
po hranjenju rib glede na vrednosti pred hranjenjem v okolici ribje kletke (v centru, 6 m in 20 m stran).
17Turk and Tinta: Impact of aquaculture on bacterioplankton 
Figure 5:  Relative difference (%) in bacterial carbon production (A) and bacterial biomass (B) measured before 
and after fish feeding at the sites around the fish cage (in the middle, 6 m and 20 m away). The size of 
the circle shows the increase (red color) or decrease (grey color).
Slika 5:   Relativna razlika (%) v izmerjeni bakterijski produkciji ogljika (A) in bakterijski biomasi (B) pred in 
tri ure po hranjenju rib v okolici ribje kletke (v centru, 6 m in 20 m stran). Velikost kroga ponazarja % 
povečanja (rdeč krog) ali znižanja (siv krog).
18Acta Biologica Slovenica, 2021, 64 (2), 9–23
Bacterial biomass, recalculated from bacterial 
counts using the 19.8 fg C cell-1 conversion fac-
tor (Lee and Fuhrman 1987), changed between 
sites, but no significant differences in concentra-
tions were observed after feeding. Mean values 
decreased from 48.8 µg C L-1 ± 9.8 to 46.2 ± 
19.7 µg C L-1, showing an increase after feeding 
only at some sites in the southwest direction. No 
changes or even a slight decrease was observed at 
all other sites (Fig. 5 B). The production/biomass 
(P/B) ratio changed from 0.20 ± 0.06 to 0.38 ± 0.2 
after feeding and was on average up to 4.5 times 
higher near the fish cages than at other sampling 
sites, reflecting higher daily biomass production.
Discussion 
Fish farming changes the load of organic 
and inorganic nutrients according to the increase 
of organic wastes (uneaten food, faeces, soluble 
excreta), which potentially negatively affects 
the marine environment, especially shallow 
areas, gulfs and semi-enclosed bays. Our study 
suggests that fish farming has moderate impact 
on water column parameters, including bacterial 
abundance and production, but only at a limited 
distance from fish cages (< 20 m), while at other 
distances no effects were detectable using our 
approach. Direct effects of nutrient pollution 
from fish farms on water quality and secondary 
effects on production and bacterial communities 
have also been reported in previous studies in the 
region (Turk and Malej 2003) and in other coastal 
areas (Price et al. 2015, Šestanović et al. 2016). 
In this study, we only analysed the effects on the 
pelagic microbial community, while the effects of 
increased organic matter on the benthic microbial 
community were not analysed in detail. Uneaten 
food and faeces lead to the accumulation of a large 
amount of organic matter on the seabed below the 
cages in our study area (Lojen et al. 2005) since 
the sedimentation rates in the vicinity of the fish 
cages were significantly higher (eight times higher) 
than background values for Piran Bay (Kovac et 
al. 2003). Accumulation of organic and inorganic 
nutrients showed significant changes in redox 
potential and concentrations of chlorophyll a and 
phaeopigments in the sediment (Štrukelj 2008), 
as well as structure and abundance of meiofauna 
(Grego et al. 2009, 2020). The negative influence 
of fish farm on the phytoplankton and bacterio-
plankton in immediate proximity was confirmed 
with in situ experiment with incubation in dialysis 
bags (Štrukelj 2008).
In addition, we investigated the effects of 
feeding of the sea bass and sea bream on bacterial 
abundance and production in a short-term in situ 
experiment based on the simultaneous sampling 
of seawater from different sites around the fish 
cage before and after fish feeding (Fig. 5). Feeding 
fish with organically enriched fish food resulted 
in significant increases in total particulate matter, 
dissolved organic and inorganic nutrients in the sea-
water of the cage and surrounding area. Increased 
concentrations of ammonium and orthophosphate 
could have triggered intense bacterial metabolism, 
which might be reflected in the recorded increase 
of the bacterial production. The observed higher 
P/B ratio reflects higher daily biomass produc-
tion near the fish cages as compared to other 
sampling sites, although no significant differences 
in bacterial abundance were observed over the 
3 h period. Previous results from the P-limited 
northern Adriatic showed that dissolved phospho-
rus concentrations in fish farms were elevated in 
the water column compared to reference stations 
(Matijević et al. 2009), resulting in changes in 
abundance and production of phytoplankton and 
bacterioplankton (Šestanović et al. 2016). In our 
study area, correlations between the bacterial 
community and orthophosphate concentration 
have been documented (Sjöstedt et al. 2013, 
Malfatti et al. 2014, Tinta et al. 2015), as well as 
rapid turnover and uptake of orthophosphate (Turk 
et al. 1992). Similar effects of organic material 
input from fish farms on bacterial activity, espe-
cially enzymatic activity, have been described in 
different aquaculture areas (Vezzulli et al. 2002, 
Caruso et al. 2003). 
The effects of dissolved wastes (inorganic and 
organic nutrients) depend on the rate at which these 
nutrients are diluted before being assimilated by 
the pelagic ecosystem. It is assumed that in cases 
where the flushing time (i.e., the time required to 
exchange the local water volume with new coastal 
water) is shorter than the typical microbial genera-
tion time and the risks of an increase in nutrient 
concentrations caused by the fish farming do not 
result in a measurable increase in local microbial 
19Turk and Tinta: Impact of aquaculture on bacterioplankton 
biomass (Black 2001). Smaller pelagic organisms 
such as bacteria near fish farms have access to 
highly available organic nutrients and have short 
generation times (Kirchman 2016). Because the 
generation time of heterotrophic bacteria in the 
Bay of Piran ranges from 5 to 82 hours (Turk et 
al. 2020), we did not detect differences in bacterial 
abundance in our 3-hour experiment. Changes in 
the concentrations of inorganic and organic nutri-
ents mainly affect bacterial production. During the 
field experiment currents near the fish cage showed 
north-westerly to south-westerly direction with 
a velocity of 7 cm s-1 (Malačič and Forte 2003), 
which could influence the higher concentrations 
of inorganic nutrients and bacterial production at 
sampling sites located in this direction. 
The method of fluorescent in situ hybridiza-
tion with rRNA-targeted oligonucleotide probes 
is widely used for cultivation-independent iden-
tification, quantification, and visualization of 
microbes in different environments (Amann et al. 
1990). Nitrifying bacteria are particularly difficult 
to culture, because they have a long generation 
time and poor counting efficiency. Analysis with 
oligonucleotide hybridization probes specific for 
different ammonia-oxidizing bacteria (probes Nso 
1225 and NEU) showed that at least two differ-
ent species of ammonia-oxidizing bacteria were 
present with a higher percentage in the seawater 
samples in the middle and around the fish cage. The 
importance of nitrifying bacteria in the context of 
fish culture has been studied previously (McCaig 
et al. 1999, Bissett et al. 2006). McCaig et al. 
(1999) demonstrated the importance of sediment 
contamination in fish farms on nitrification rates 
and the composition of ammonium-oxidizing 
bacteria. Using 16S rDNA clone library analysis, 
Bissett et al. (2006) demonstrated the presence of 
ammonium-oxidation Nitrospira associated clones 
and differences in the microbial community as a 
function of sediment pollution level. The groups 
of β-Proteobacteria, γ-Proteobacteria and Cy-
tophaga – Flavobacterium -Bacteriodes reached a 
higher percentage at more distant sampling sites. 
The importance and seasonal changes in these 
bacterial communities has been demonstrated in 
recent studies but only on benthic communities 
using next -generation sequencing methods (Gar-
ren et al. 2008, Fodelianakis et al. 2014, Quero et 
al. 2015, Martins et al. 2018, Duarte et al. 2019, 
Ape et al. 2019, Kolda et al. 2020, Roquigny et 
al. 2021). Species belonging to the genus Vibrio 
are heterotrophic bacteria that increases rapidly 
when nutrients are available (Thompson et al. 
2006). Some Vibrio species are known pathogens 
of fish (Austin and Austin 2016), and the presence 
of Vibrio near the fish cages could be an important 
reservoir for fish pathogens that could spread over 
large geographic areas.
Conclusions 
Given the rapid development of aquaculture, 
concerns remain about the environmental impact 
of intensive aquaculture on water and sediment 
quality, particularly in shallow and semi-enclosed 
bays. Our study suggests that aquaculture has 
moderate effects on water column parameters, 
and can cause changes in bacterial abundance, 
production and community composition, but only 
at a limited distance from fish cages. However, 
we did not follow change in the sediment where 
in fact these effects might be more pronounced. 
We also confirmed that concentrations of organic 
and inorganic nutrients change in a relatively short 
time after fish feeding, which affect the bacterial 
production. Changes in bacterial activity and 
community composition can lead to changes in 
biogeochemical cycling and ecological status of 
the environment. Sedimentation of organic matter 
has a strong impact on sediment quality and benthic 
community, but changes in the concentration of 
organic and inorganic nutrients in the water col-
umn, as well as microbial abundance, production, 
and community composition in a relatively short 
time are not negligible. Preliminary fluorescence 
in situ hybridization results indicated changes in 
abundance of certain bacterial groups in response 
to pollution, as well as the presence of bacteria 
pathogenic to humans and fish. In the future, 
seasonal changes and long-term consequences 
should be investigated in the water column as 
well as sediment, to better understand changes 
in microbial community structure, and biogeo-
chemical processes. 
20Acta Biologica Slovenica, 2021, 64 (2), 9–23
Povzetek
Dokumentirani številni negativni vplivi 
marikulture na okolje so povezani predvsem s 
kopičenjem ne-zaužite hrane in ekskrecijskih 
produktov v morskih vodi in na/v sedimentu. 
Rezultati naših meritev prostorskih razlik 
števila in hitrosti rasti populacije heterotrofnih 
planktonskih bakterij, kot tudi analize vsebnosti 
hranilnih snovi, so pokazali povišane vrednosti 
merjenih parametrov v bližini ribogojnice, ki pa se 
z oddaljenostjo od vira onesnaženja zmanjšujejo. 
Rezultati analize sestave bakterijskih združb 
z metodo fluorescentne in situ hibridizacije in 
izbranih bakterijskih oligonukleotidnih sond so 
pokazali prisotnost amonij-oksidirajočih bakterij, 
β-Proteobacteria, γ-Proteobacteria and Cy-
tophaga – Flavobacterium kot tudi potencionalno 
patogenega seva Vibrio. Kopičenje organsko 
obogatene ribje hrane in odpadnih produktov, ki 
se sproščajo v morsko vodo znotraj ribje kletke 
in okolici v relativno kratkem času po hranjenju, 
je povzročilo znatno povečanje koncentracij par-
tikulatne organske snovi, ortofosfata in amonija. 
Kot odgovor na povečanje anorganskih hranil smo 
izmerili znatno povečano bakterijsko produkcijo, 
medtem ko razlik v številu bakterij ni bilo opaziti.
Acknowledgements
This research was funded by the European 
6th Framework Programme (ECASA; Ecosystem 
Approach for Sustainable Aquaculture. Project 
No. 006540). The authors acknowledge financial 
support from the Slovenian Research Agency 
(ARRS) (research core funding “Coastal Sea 
Research” (No. P1-0237), and the project “Driv-
ers that structure coastal marine microbiome with 
emphasis on pathogens – an integrated approach” 
(No. J1-9157). We thank Alenka Malej for lead-
ing the project, providing guidance and valuable 
comments during the study and support during 
the manuscript writing. We thank the staff of the 
Marine Biological Station (National Institute of 
Biology) for help in conducting the experiment 
and Milijan Šiško for statistical analysis. 
References
Amann, R.I., Krumholz, L., Stahl, D.A. 1990. Fluorescent-oligonucleotide probing of whole cells for 
determinative, phylogenetic, and environmental studies in microbiology. Journal of Bacteriology, 
172(2), 762-770.
Ape, F., Manini, E., Quero, G.M., Luna, G.M., Sarà, G., Vecchio, P., Brignoli, P., Ansferri, S., Mirto, S. 
2019. Biostimulation of in situ microbial degradation processes in organically-enriched sediments 
mitigates the impact of aquaculture. Chemosphere, 226, 715-725.
Armstrong, S.M., Hargrave, B.T., Haya, K. 2005. Antibiotic use in finfish aquaculture: modes of action, 
environmental fate, and microbial resistance. In Environmental effects of marine finfish aquaculture. 
Edited by B.T. Hargrave. Springer Berlin Heidelberg, Berlin, Heidelberg. pp. 341-357.
Asami, H., Aida, M., Watanabe, K. 2005. Accelerated sulfur cycle in coastal marine sediment beneath 
areas of intensive shellfish aquaculture. Applied and Environmental Microbiology, 71(6), 2925-2933.
Austin, B., and Austin, D.A. 2016. Bacterial fish pathogens: disease of farmed and wild fish. In 6th ed. 
2016. Springer International Publishing : Imprint: Springer, Cham.
Azam, F., and Malfatti, F. 2007. Microbial structuring of marine ecosystems. Nature Reviews Micro-
biology, 5(10), 782-791.
Bissett, A., Bowman, J., Burke, C. 2006. Bacterial diversity in organically-enriched fish farm sediments: 
Bacterial diversity in organically-enriched fish farm sediments. FEMS Microbiology Ecology, 
55(1), 48-56. 
Bissett, A., Burke, C., Cook, P.L.M., Bowman, J.P. 2007. Bacterial community shifts in organically 
perturbed sediments. Environmental Microbiology, 9(1), 46-60. 
Black, K.D. (Editor). 2001. Environmental impacts of aquaculture. In 1. publ. Sheffield Academic Pr. 
[u.a.], Sheffield.
21Turk and Tinta: Impact of aquaculture on bacterioplankton 
Bouwman, A.F., Beusen, A.H.W., Overbeek, C.C., Bureau, D.P., Pawlowski, M., Glibert, P.M. 2013. 
Hindcasts and future projections of global inland and coastal nitrogen and phosphorus loads due 
to finfish aquaculture. Reviews in Fisheries Science, 21(2), 112-156. 
Cabello, F.C., Godfrey, H.P., Tomova, A., Ivanova, L., Dölz, H., Millanao, A., Buschmann, A.H. 2013. 
Antimicrobial use in aquaculture re-examined: its relevance to antimicrobial resistance and to animal 
and human health: Aquacultural antimicrobial use and antimicrobial resistance. Environmental 
Microbiology, 15(7), 1917-1942. 
Caruso, G., Genovese, L., Mancuso, M., Modica, A. 2003. Effects of fish farming on microbial enzyme 
activities and densities: comparison between three Mediterranean sites. Letters in Applied Micro-
biology, 37(4), 324-328.
Castine, S.A., Bourne, D.G., Trott, L.A., McKinnon, D.A. 2009. Sediment microbial community analysis: 
Establishing impacts of aquaculture on a tropical mangrove ecosystem. Aquaculture, 297(1-4), 91-98. 
Christensen, P., Rysgaard, S., Sloth, N., Dalsgaard, T., Schwærter, S. 2000. Sediment mineralization, 
nutrient fluxes, denitrification and dissimilatory nitrate reduction to ammonium in an estuarine 
fjord with sea cage trout farms. Aquatic Microbial Ecology, 21, 73-84.
Dowle, E., Pochon, X., Keeley, N., Wood, S.A. 2015. Assessing the effects of salmon farming seabed 
enrichment using bacterial community diversity and high-throughput sequencing. FEMS Micro-
biology Ecology, 91(8), fiv089.
Duarte, L.N., Coelho, F.J.R.C., Cleary, D.F.R., Bonifácio, D., Martins, P., Gomes, N.C.M. 2019. Bacte-
rial and microeukaryotic plankton communities in a semi-intensive aquaculture system of sea bass 
(Dicentrarchus labrax): A seasonal survey. Aquaculture, 503, 59-69. 
FAO (Food and Agriculture Organization of the United Nations). 2018. The State of World Fisheries 
and Aquaculture. FAO, Rome. 2018.
Fodelianakis, S., Papageorgiou, N., Pitta, P., Kasapidis, P., Karakassis, I., Ladoukakis, E.D. 2014. The 
pattern of change in the abundances of specific bacterioplankton groups is consistent across different 
nutrient-enriched habitats in Crete. Applied and Environmental Microbiology, 80(13), 3784-3792. 
Galand, P.E., Lucas, S., Fagervold, S.K., Peru, E., Pruski, A.M., Vétion, G., Dupuy, C., Guizien, K. 
2016. Disturbance increases microbial community diversity and production in marine sediments. 
Frontiers in Microbiology, 7.
Garren, M., Smriga, S., Azam, F. 2008. Gradients of coastal fish farm effluents and their effect on coral 
reef microbes. Environmental Microbiology, 10(9), 2299-2312. 
Grasshoff, K., Ehrhardt, M., Kremling, K., Almgren, T. (Editors). 1983. Methods of seawater analysis. 
In 2nd rev. and extended ed. Verlag Chemie, Weinheim.
Grego, M., De Troch, M., Forte, J., Malej, A. 2009. Main meiofauna taxa as an indicator for assessing 
the spatial and seasonal impact of fish farming. Marine Pollution Bulletin, 58(8), 1178-1186.
Grego, M., Malej, A., De Troch, M. 2020. The depleted carbon isotopic signature of nematodes and 
harpacticoids and their place in carbon processing in fish farm sediments. Frontiers in Marine 
Science, 7, 572.
Hargrave, B., Duplisea, D., Pfeiffer, E., Wildish, D. 1993. Seasonal changes in benthic fluxes of dis-
solved oxygen and ammonium associated with marine cultured Atlantic salmon. Marine Ecology 
Progress Series, 96, 249-257.
Hargrave, B.T., Phillips, G.A., Doucette, L.I., White, M.J., Milligan, T.G., Wildish, D.J., Cranston, 
R.E. 1997. Assessing benthic impacts of organic enrichment from marine aquaculture. Water, Air, 
& Soil Pollution, 99(1-4), 641-650.
Karakassis, I., Tsapakis, M., Hatziyanni, E. 1998. Seasonal variability in sediment profiles beneath fish 
farm cages in the Mediterranean. Marine Ecology Progress Series, 162, 243-252.
Karakassis, I. 2000. Impact of cage farming of fish on the seabed in three Mediterranean coastal areas. 
ICES Journal of Marine Science, 57(5), 1462-1471.
Kawahara, N., Shigematsu, K., Miyadai, T., Kondo, R. 2009. Comparison of bacterial communities 
in fish farm sediments along an organic enrichment gradient. Aquaculture, 287(1-2), 107-113. 
22Acta Biologica Slovenica, 2021, 64 (2), 9–23
Kim, S.-R., Nonaka, L., Suzuki, S. 2004. Occurrence of tetracycline resistance genes tet (M) and tet (S) 
in bacteria from marine aquaculture sites. FEMS Microbiology Letters, 237(1), 147-156.
Kirchman, D.L. 1994. The uptake of inorganic nutrients by heterotrophic bacteria. Microbial Ecology, 
28(2), 255-271.
Kirchman, D.L. 2016. Growth rates of microbes in the oceans. Annual Review of Marine Science, 
8(1), 285-309.
Kolda, A., Gavrilović, A., Jug-Dujaković, J., Ljubešić, Z., El-Matbouli, M., Lillehaug, A., Lončarević, 
S., Perić, L., Knežević, D., Vukić Lušić, D., Kapetanović, D. 2020. Profiling of bacterial assembla-
ges in the marine cage farm environment, with implications on fish, human and ecosystem health. 
Ecological Indicators, 118, 106785. 
Kondo, R., Shigematsu, K., Kawahara, N., Okamura, T., Yoon, Y.H., Sakami, T., Yokoyama, H., Koizumi, 
Y. 2012. Abundance of sulphate-reducing bacteria in fish farm sediments along the coast of Japan 
and South Korea. Fisheries Science, 78(1), 123-131. 
Kovac, N., Čermelj, B., Vrišer, B., Lojen, S. 2003. Influence of fish farming on coastal marine sediment 
in Slovenia (Piran Bay, northern Adriatic):Final report. National Institute of Biology.
Lee, S., and Fuhrman, J.A. 1987. Relationships between biovolume and biomass of naturally derived 
marine bacterioplankton. Applied and Environmental Microbiology, 53(6), 1298-1303.
Lojen, S., Spanier, E., Tsemel, A., Katz, T., Eden, N., Angel, D.L. 2005. δ15N as a natural tracer of 
particulate nitrogen effluents released from marine aquaculture. Marine Biology, 148(1), 87-96.
Luna, G.M., Corinaldesi, C., Dell’Anno, A., Pusceddu, A., Danovaro, R. 2013. Impact of aquaculture on 
benthic virus–prokaryote interactions in the Mediterranean Sea. Water Research, 47(3), 1156-1168. 
Malačič, V., and Forte, J. 2003. Distribution of the food surplus and faecal particles on the seabed bellow 
a fish farm in the Bay of Piran. Annales, Series Historia Naturalis, (13), 3-8.
Malfatti, F., Turk, V., Tinta, T., Mozetič, P., Manganelli, M., Samo, T.J., Ugalde, J.A., Kovač, N., 
Stefanelli, M., Antonioli, M., Fonda-Umani, S., Del Negro, P., Cataletto, B., Hozić, A., Ivošević 
DeNardis, N., Žutić, V., Svetličić, V., Mišić Radić, T., Radić, T., Fuks, D., Azam, F. 2014. Microbial 
mechanisms coupling carbon and phosphorus cycles in phosphorus-limited northern Adriatic Sea. 
Science of the Total Environment, 470-471, 1173-1183.
Martins, P., Coelho, F.J.R.C., Cleary, D.F.R., Pires, A.C.C., Marques, B., Rodrigues, A.M., Quintino, 
V., Gomes, N.C.M. 2018. Seasonal patterns of bacterioplankton composition in a semi-intensive 
European seabass (Dicentrarchus labrax) aquaculture system. Aquaculture, 490, 240-250. 
Matijević, S., Kušpilić, G., Morović, M., Grbec, B., Bogner, B., Skejić, S., Veža, J. 2009. Physical and 
chemical properties of the water column and sediments at sea bass/sea bream farm in the middle 
Adriatic (Maslinova Bay). Acta Adriatica, (50 (1)), 59-76.
McCaig, A.E., Phillips, C.J., Stephen, J.R., Kowalchuk, G.A., Harvey, S.M., Herbert, R.A., Embley, 
T.M., Prosser, J.I. 1999. Nitrogen cycling and community structure of proteobacterial β-subgroup 
ammonia-oxidizing bacteria within polluted marine fish farm sediments. Applied and Environmental 
Microbiology, 65(1), 213-220. 
McGhie, T.K., Crawford, C.M., Mitchell, I.M., O’Brien, D. 2000. The degradation of fish-cage waste 
in sediments during fallowing. Aquaculture, 187(3-4), 351-366. 
Mirto, S., La rosa, T., Danovaro, R., Mazzola, A. 2000. Microbial and meiofaunal response to intensive 
mussel-farm biodeposition in coastal sediments of the western Mediterranean. Marine Pollution 
Bulletin, 40(3), 244-252. 
Mirto, S., Gristina, M., Sinopoli, M., Maricchiolo, G., Genovese, L., Vizzini, S., Mazzola, A. 2012. 
Meiofauna as an indicator for assessing the impact of fish farming at an exposed marine site. 
Ecological Indicators, 18, 468-476. 
Navarro, N., Leakey, R., Black, K. 2008. Effect of salmon cage aquaculture on the pelagic environment 
of temperate coastal waters: seasonal changes in nutrients and microbial community. Marine Eco-
logy Progress Series, 361, 47-58. 
23Turk and Tinta: Impact of aquaculture on bacterioplankton 
Pitta, P., Apostolaki, E.T., Tsagaraki, T., Tsapakis, M., Karakassis, I. 2006. Fish farming effects on 
chemical and microbial variables of the water column: a spatio-temporal study along the Mediter-
ranean sea. Hydrobiologia, 563(1), 99-108. 
Price, C., Black, K., Hargrave, B., Morris, J. 2015. Marine cage culture and the environment: effects 
on water quality and primary production. Aquaculture Environment Interactions, 6(2), 151-174.
Quero, G.M., Cassin, D., Botter, M., Perini, L., Luna, G.M. 2015. Patterns of benthic bacterial diver-
sity in coastal areas contaminated by heavy metals, polycyclic aromatic hydrocarbons (PAHs) and 
polychlorinated biphenyls (PCBs). Frontiers in Microbiology, 6.
Reimers, C.E., Alleau, Y., Bauer, J.E., Delaney, J., Girguis, P.R., Schrader, P.S., Stecher, H.A. 2013. 
Redox effects on the microbial degradation of refractory organic matter in marine sediments. Ge-
ochimica et Cosmochimica Acta, 121, 582-598. 
Roquigny, R., Mougin, J., Le Bris, C., Bonnin-Jusserand, M., Doyen, P., Grard, T. 2021. Characteri-
zation of the marine aquaculture microbiome: A seasonal survey in a seabass farm. Aquaculture, 
531, 735987.
Šestanović, S., Peković, J., Matijević, S., Ninčević Gladan, Ž. 2016. Effects of fish farming on micro-
bial planktonic communities in the middle Adriatic sea. Aquaculture Research, 47(4), 1040-1054. 
Simon, M., and Azam, F. 1989. Protein content and protein synthesis rates of planktonic marine bacteria. 
Marine Ecology Progress Series, 51, 201-213. 
Sjöstedt, J., Pontarp, M., Tinta, T., Alfredsson, H., Turk, V., Lundberg, P., Hagström, Å., Riemann, 
L. 2013. Reduced diversity and changed bacterioplankton community composition do not affect 
utilization of dissolved organic matter in the Adriatic Sea. Aquatic Microbial Ecology, 71(1), 15-24. 
Smith, D.M., and Azam, F. 1992. A simple, economical method for measuring bacterial protein synthesis 
rates in seawater using 3H-leucine. Marine Microbial Food Webs, 6, 107-114.
Štrukelj, M. 2008. Vpliv marikulture na okolje: diplomsko delo. Univerza Nova Gorica, Nova Gorica.
Thompson, F.L., Austin, B.,Swings, J. (Editors). 2006. The Biology of Vibrios. ASM Press, Washing-
ton, DC, USA.
Tinta, T., Vojvoda, J., Mozetič, P., Talaber, I., Vodopivec, M., Malfatti, F., Turk, V. 2015. Bacterial 
community shift is induced by dynamic environmental parameters in a changing coastal ecosystem 
(northern Adriatic, northeastern Mediterranean Sea) - a 2-year time-series study: Bacterial com-
munity shift in a dynamic coastal ecosystem. Environmental Microbiology, 17(10), 3581-3596.
Turk, V., Rehnstam, A.-S., Lundberg, E., Hagström, Å. 1992. Release of Bacterial DNA by Marine 
Nanoflagellates, an Intermediate Step in Phosphorus Regeneration. Applied and Environmental 
Microbiology, 58(11), 3744-3750. 
Turk, V., and Malej, A. 2003. The influence of fish cage aquaculture on acterioplankton in the Bay of 
piran (Gulf of Trieste, Adriatic Sea. Annales, Series Historia Naturalis 13(1), 37-42.
Turk, V., Malkin, S., Celussi, M., Tinta, T., Cram, J., Malfatti, F., Chen, F. 2020. Ecological role of 
microbes: current knowledge and future prospects. In Geophysical Monograph Series, 1st edition. 
Edited by T.C. Malone, A. Malej, and J. Faganeli. Wiley. pp. 129-145.
Vezzulli, L., Chelossi, E., Riccardi, G., Fabiano, M. 2002. Bacterial community structure and activity 
in fish farm sediments of the Ligurian sea (Western Mediterranean). Aquaculture International, 
10(2), 123-141.
Vezzulli, L., Moreno, M., Marin, V., Pezzati, E., Bartoli, M., Fabiano, M. 2008. Organic waste impact 
of capture-based Atlantic bluefin tuna aquaculture at an exposed site in the Mediterranean Sea. 
Estuarine, Coastal and Shelf Science, 78(2), 369-384. 
Wang, X., Cuthbertson, A., Gualtieri, C., Shao, D. 2020. A Review on mariculture effluent: characte-
rization and management tools. Water, 12(11), 2991.