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Effects of live rock on the reef-building coral Acropora digitifera cultured with high
levels of nitrogenous compounds
Yeong Shyan Yuen
a
, Seitaro S. Yamazaki
a
, Takashi Nakamura
a,b
, Gaku Tokuda
c
, Hideo Yamasaki
a,
*
a
Faculty of Science, University of the Ryukyus, Okinawa, 903-0213, Japan
b
Amakusa Marine Biological Laboratory, Kyushu University, Kumamoto, 863-2507, Japan
c
Tropical Biosphere Research Center, COMB, University of the Ryukyus, Okinawa, 903-0213, Japan
1. Introduction
Aquaculture systems for marine ornamental species including
reef-building corals, in contrast to food species, have yet to be
established. Aquaculture of corals often faces difficulties in
maintenance of water quality that lead to failures during the
propagation processes. High mortality of cultured and captive
corals in aquaria can be partly ascribed to inappropriate culture
conditions and also to mishandling of corals during transportation
(Calfo, 2001; Wabnitz et al., 2003). In closed seawater facilities
(such as aquaria and mariculture systems), limited water exchange
raises critical problems. Increases in inorganic nitrogenous (N)
compounds that are mainly produced by inhabitants such as fishes
is a major cause of water quality degradation and often shows both
direct and indirect harmful effects on corals.
Among various types of N compounds, ammonium (NH
4+
) and
nitrate (NO
3
) ions are well known to cause direct toxicity to
marine invertebrates (Camargo and Alonso, 2006). Accumulation
of NO
3
degrades water quality through the changes in alkalinity,
pH, redox potential and dissolved oxygen level, all of which
indirectly affects the health and growth of marine invertebrates
(Delbeek and Sprung, 1994; Grguric et al., 2005). Since those
inorganic N compounds are nutrients for photosynthetic organ-
isms, an increase in their concentrations potentially induces algal
overgrowth. Thus, for aquaculture of reef-building corals the
control of N compound level is particularly important.
A wide variety of filtrationsystems have been applied to solve the
problem of excessive N. Biological filtration systems have attracted
much attention from aquarists (Delbeek and Sprung, 1994). The
principle of the biological systems is attributed to nitrification and
denitrification activities of bacteria (Hovanec and DeLong, 1996;
Nagadomi et al., 1999). To breakdown N compounds, several
technologies have been established such as in the Monaco System
and the Berlin System (Delbeekand Sprung, 1994). In these systems,
‘‘live rock’’ is an inevitable component. In fact, many aquarists have
emphasized the importance of ‘‘live rock’’ in coral aquaria. In spite of
a long application of ‘‘live rock’’ for coral aquaria, however, there
have been few papers available for scientific confirmation of the
effects of ‘‘live rock’’ on reef-building corals.
‘‘Live rock’’ is not a scientific terminology but is a term widely
used in the aquarium trade to describe a dead coral skeleton that is
Aquacultural Engineering 41 (2009) 35–43
ARTICLE INFO
Article history:
Received 19 December 2008
Accepted 7 June 2009
Keywords:
Ammonia oxidizing bacteria
Crustose coralline algae
Denitrifying bacteria
Live rock
Reef-building coral
Water quality
ABSTRACT
Reef-building corals are sensitive to excessive nitrogenous (N) compounds. To maintain levels of
inorganic nitrogenous compounds low in coral aquaria, various technologies, mechanical, chemical and
biological, have been applied. As one of the biological techniques, ‘‘live rock,’’ which can be defined as a
dead coral skeleton covered with crustose coralline algae (CCA), has long been applied for coral aquaria.
Until recently, however, there has been little evidence for the effectiveness of live rock in removal of N
compounds from coral aquaria. Demonstrating comparative experiments with live rocks, here we report
that the live rock is capable of removing N compounds and reduces the mortality of reef-building coral.
We cultured the reef-building coral Acropora digitifera with the sea cucumber Holoth uria atra as a natural
nitrogen producer. H. atra increased the concentration of the inorganic N compounds (NH
4+
,NO
3
and
NO
2
) that resulted in high coral mortality. The presence of the live rock remarkably reduced the
concentrations and sustained a high coral photosynthetic activity. We detected the functional genes
amoA and nirS within the live rock, suggesting the occurrence of both nitrifying and denitrifying bacteria.
These results support the idea that ‘‘live rock’’ is an effective biofilter that can maintain water quality
suitable for reef-building corals.
ß2009 Elsevier B.V. All rights reserved.
* Corresponding author. Tel.: +81 98 895 8550; fax: +81 98 895 8576.
E-mail addresses: yeongshyan@yahoo.com (Y.S. Yuen),
k078557@eve.u-ryukyu.ac.jp (S.S. Yamazaki), takasuken@yahoo.co.jp
(T. Nakamura), tokuda@comb.u-ryukyu.ac.jp (G. Tokuda),
yamasaki@sci.u-ryukyu.ac.jp (H. Yamasaki).
Contents lists available at ScienceDirect
Aquacultural Engineering
journal homepage: www.elsevier.com/locate/aqua-online
0144-8609/$ – see front matter ß2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquaeng.2009.06.004
Author's personal copy
covered by encrusting algae along with a variety of invertebrate
animals (Delbeek and Sprung, 1994). ‘‘Live rock’’ is now a highly
priced commodity in the aquarium trade (Green and Shirley,
1999). In spite of a long application of ‘‘live rock’’ for coral aquaria,
however, there have been few papers available for scientific
confirmation of the effects of ‘‘live rock’’ on reef-building corals.
One of the prominent features of ‘‘live rock’’ is the presence of
encrusting algae covering its surface. In many public aquaria,
‘‘live rock’’ can be found in coral reef display tanks. Most of
these encrusting algae belong to crustose coralline algae (CCA).
In the fields, CCA plays fundamental role in the formation of
coral reef (Littler and Doty, 1975). Moreover, recent studies have
revealed that CCA facilitates larval settlement and metamor-
phosis of a wide variety of marine invertebrates including reef-
building corals (Morse and Morse, 1991). Thus, it becomes
obvious that CCA plays important roles in natural as well as
artificial system.
The objective of this study was to investigate effects of the live
rock on reef-building corals under conditions of high N com-
pounds. The results presented in this paper support the hypothesis
that the live rock is capable of maintaining a good water quality for
reef-building corals. The association of bacteria colonized within
the carbonate substrate of the live rock is discussed in N
compounds removal activity.
2. Materials and methods
2.1. Samples preparation
In this study, we define ‘‘live rock’’ as dead coral skeleton
encrusted by crustose coralline algae. On the coral reefs of
Okinawa, Japan, a large carbonate structure encrusted by CCA can
be found near reef-building corals as shown in Fig. 1A. In addition,
we often find smaller fragments of dead coral skeleton that are
covered by CCA (Fig. 1B). The live rocks consist of CCA that belongs
to Hydrolithon samoense,Pneophyllum sp. and Spongites sp. were
collected from Ginowan Marina Reef, Okinawa, Japan (26816
0
N,
127843
0
E) in April 2007 (Fig. 1B). Samples were transported to the
laboratory and acclimated in an aquarium kept at 26 8C. Light
intensity of 150
m
mol photon m
2
s
1
was provided with a metal
halide lamp (12:12 h light:dark cycle). Only live rocks with 100%
coverage of CCA were used in this study. Two species of sea
cucumber Holothuria leucospilota and Holothuria atra were also
collected from the same site. They were kept in aerated aquaria
under fluorescent light (50
m
mol photon m
2
s
1
, 12:12 h light:-
dark cycle).
The reef-building coral Acropora digitifera were collected from
Bisezaki Reef, Okinawa, Japan (26871
0
N, 127888
0
E). They were
acclimated for more than a year in an outdoor flow-through
aquarium at the Sesoko Station of Tropical Biosphere Research
Center (University of the Ryukyus, Japan). The coral samples were
cut into nubbins of similar size (approximately 3 cm in length) and
immediately attached to acrylic screws using superglue (Loctite,
Henkel, Ireland) as described by Nakamura and Yamasaki (2005).
The coral nubbins were then acclimated in an aquarium with
recirculated water flow before being used for experiment
(Nakamura et al., 2005). Artificial seawater (New-Ocean Artificial
Seawater, Japan Biochemical) with the salinity adjusted to 35 ppt
was used in all experiments.
2.2. Live rock experiment
Ammonium removal capacity was compared among live rocks
of different weights and surface areas. Single live rocks with wet
weights of 20 g, 65 g and 95 g, respectively were selected and
each size of the live rock was placed into 1-L aquaria. To examine
the effects of the surface area, multiple samples with the total
wet weights of 20 g, 65 g and 95 g, respectively were placed into
1-L aquaria. Surface areas of the live rocks were estimated by
using the aluminum foil method described by Marsh (1970).The
wet weights and surface areas of the live rock used are shown in
Table 1. Aquaria without the live rocks were used as control.
Ammonium chloride (NH
4
Cl) was added into the seawater as the
final concentration of 100
m
M in the treatments. The concentra-
tion of NH
4+
in seawater was analyzed after 24 h from the
treatments.
Fig. 1. Live rocks encrusted by crustose coralline algae (CCA). (A) A large carbonate structure encrusted by CCA in the field; (B) small pieces of the live rocks covered by CCA
were used in this study; (C) top view of an aquarium set-up containing sea cucumbers, corals and the live rocks; (D) a schematic diagram of the set-up for coral experiment.
From left to right, aquaria containing: sea cucumbers only (control); sea cucumbers and corals; sea cucumbers, corals and live rocks. Plus sign (+) indicates the presence of the
organism whereas negative sign () indicates the absence of the organism.
Y.S. Yuen et al. / Aquacultural Engineering 41 (2009) 35–43
36
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2.3. Sea cucumber experiment
H. leucospilota and H. atra (wet weight approximately 200 g)
were placed into two independent 3-L aquaria. Small pieces of the
live rock (approximately 200 g) were added into these aquaria.
Aquaria kept at 26 8C were illuminated by fluorescent light
(50
m
mol photon m
2
s
1
) and aerated using air pumps during
the experiment. Water samples were collected every 24 h intervals
for nutrient analysis.
2.4. Coral experiment
Five to six individuals of H. atra with the total wet weight of
approximately 200 g were placed into a 3-L aquarium. They were
confined to the bottom of the aquarium using a polyethylene mesh
fixed at 7 cm above the bottom of the aquarium to prevent a direct
contact with the coral nubbins and the live rocks (Fig. 1C and D).
Twelve A. digitifera nubbins were attached to the side walls of the
aquaria so that the lateral side of coral nubbins was exposed to
light. The live rocks (approximately 230 g) were placed onto the
mesh. An identical set of treatments but without the live rocks was
set up to observe short-term effects of eutrophication on A.
digitifera. In addition, an aquarium containing only H. atra was used
to monitor the increase in nutrient level in seawater. All aquaria
were aerated using air pumps and illuminated using a metal halide
lamp of 65
m
mol photon m
2
s
1
with 12:12 photoperiod. Sea-
water temperature was kept at 25
0.5 8C. The experimental set-up
is represented in Fig. 1D.
2.5. Water quality measurements
Seawater pH was measured with a portable pH meter
(HORIBA, Japan) every 4 h except during the night. Water
samples were collected from all the aquaria at the same interval
as pH measurements. The water samples were frozen at 60 8C
until used for nutrient analysis. NH
4+
,NO
3
and NO
2
in
seawater were quantified with an automated wet-chemistry
analyzer (Bran+Luebbe QuAAtro, Germany).
2.6. Assessment of coral photosynthetic activity
To assess photosynthetic activity of the symbiotic algae in
hospite, we used a Diving-PAM fluorometer (Walz, Effeltrich,
Germany) to measure the chlorophyll afluorescence emitted from
the algae. Maximum potential yield of photosystem II (PSII) or
dark-adapted F
v
/F
m
is a reliable measure of the photochemical
efficiency of PSII (Demmig and Bjorkman, 1987). The decrease in F
v
/
F
m
value has been applied as a good indicator to monitor
photodamage (or photoinhibition) of photosynthesis (Franklin
et al., 1992). In this study we used F
v
/F
m
to assess the photodamage
of in hospite algal photosynthesis (Takahashi et al., 2004). The
measurement was carried out daily. Photosynthetic performance
was also assessed with rapid light curves (RLC), an indication of
the potential ability of coral to respond to rapid light fluctuation
(Ralph and Gademann, 2005). The samples were exposed to
eight incremental steps of irradiance ranging from 0 to
1270
m
mol photons m
2
s
1
using a program installed in the
Diving-PAM.
2.7. pH experiment
To assess whether the increase in seawater pH is due to the
dissolution of calcium carbonate from the live rock, a control
experiment was carried out using small pieces of skeleton of
massive coral Porites sp. (approximately 25 g). Changes in seawater
pH were measured with a portable pH meter (HORIBA, Japan).
2.8. Statistical analysis
Effects of eutrophic water on the photosynthetic performance
of A. digitifera was assessed with an analysis of variance (ANOVA).
Means were compared with post hoc test. Statistical analyses were
performed using StatView 5.01 (SAS Institute Inc., USA).
2.9. Nucleic acid extraction, PCR and RT-PCR
One set of the live rock was incubated in 100
m
MofNH
4+
under light conditions (65
m
mol photon m
2
s
1
) with aeration
while another set of the live rock was incubated in 50
m
Mof
NO
3
under dark condition without aeration. After 3 days
incubation period, DNA was extracted from the seawater, CCA
thallus and carbonate substrate underneath the CCA thallus
whereas RNA was extracted from the carbonate substrate only.
Cross-section of the live rock reveals different layers including
the CCA thallus, the green layer underneath the CCA thallus and
the whitish carbonate substrate (see, Fig. 7A). Surface tissue of
CCA thallus was scraped using a file, the fine powder was
collected for DNA extraction. Using a hammer and a chisel, CCA
thallus was separated from the live rock and the whitish part of
the carbonate substrate was then mechanically broken with
mortar and pestle. DNA was extracted from the broken substrate.
Seawater (around 300 ml) was filtered through a 0.45
m
mfilter
paper to remove any sediment or suspension. The filtrate was
then filtered through a 0.22
m
m filter paper. The filter paper was
used for DNA extraction. DNAs were extracted using the
UltraClean
TM
Soil DNA Isolation Kit (MO BIO Lab. Inc., USA)
according to the manufacturer’s instructions. For RNA extraction,
fine powder of the whitish part of the carbonate substrate was
incubated in 50
m
l of 5% lysozyme in TE buffer for 10 min at room
temperature and then total RNA was extracted using RNA/DNA
kit (QIAGEN, Hilden, Germany) according to the manufacturer’s
instructions. Isopropanol-precipitated RNAs were dissolved in
10
m
l of RNase-free water provided in the kit.
The gene for 16S rRNA as well as the genes involved in
nitrification process (amoA) and denitrification (nirS) were
amplified by PCR from DNAs isolated from the live rock and
seawater. Amplifications were carried out in a total volume of
25
m
l using a PCR thermal cycler (ASTEC, model PC707, Japan). The
reaction mixture contained 2.5
m
lof10Ex Taq buffer, 1.5
m
lof
dNTP mixture, 1.0
m
l of forward primer, 1.0
m
l of reverse primer,
2.0
m
l of DNA template, 0.25
m
l of Taq DNA polymerase and
17.0
m
l of distilled water (TaKaRa Ex Taq
TM
Hot Start Version kit,
TaKaRa Bio Inc., Japan). A 16S rRNA gene fragment was amplified
with the universal bacterial primers, F341 (5
0
-CCTACGGGAGG-
CAGCAG-3
0
) and 907R (5
0
-CCGTCAATTCCTTTRAGTTT-3
0
).
The thermal cycling program was as follow: 5 min of initial
denaturation at 94 8C, followed by 65 8C for 1 min and 72 8C for
1 min, this was followed by 19 cycles of denaturation at 94 8C for
1 min, a touchdown primer annealing from 64 8Cto558C for 1 min
(decreased 0.5 8C for each second cycle), primer extension for
1 min at 72 8C, followed by an additional 9 cycles of denaturation at
94 8C, a constant annealing temperature of 55 8C for 1 min, a
Table 1
Surface area and weight of the live rocks used in this study.
Wet weight (g) Surface area (cm
2
)
20 65 95
Single – light 39 76 96
Multiple – light 48 132 224
Single – dark 40 74 92
Multiple – dark 47 151 220
Y.S. Yuen et al. / Aquacultural Engineering 41 (2009) 35–43
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primer extension at 72 8C for 1 min, followed by a final extension at
72 8C for 5 min.
Functional genes of amoA and nirS were amplified with the
primer set amoA-1F (5
0
-GGG GTT TCT ACT GGT GGT-3
0
)-amoA-2R
(5
0
-CCC CTC KGS AAA GCC TTC TTC-3
0
)(Rotthauwe et al., 1997) and
cd3aF (5
0
-GTS AAC GTS AAG GAR ACS GG-3
0
)-R3cd (5
0
-GAS TTC
GGR TGS GTC TTG A-3
0
), respectively (Throba
¨ck et al., 2004).
The thermal cycling program for amoA was as follow: 4 min of
initial denaturation at 94 8C, followed by eight cycles of
denaturation at 94 8C for 1 min, a touchdown primer annealing
from 62 8Cto568C for 1 min (decreased 0.8 8C for each second
cycle), primer extension for 1 min at 72 8C, followed by an
additional 26 cycles with a constant annealing temperature of
56 8C for 1 min, a primer extension at 72 8C for 1 min, followed by a
final extension at 72 8C for 3 min. The program for nirS included
5 min of initial denaturation at 94 8C, annealing at 60 8C for 1 min,
extension at 72 8C for 1 min, followed by 17 cycles of denaturation
at 94 8C for 1 min, a touchdown primer annealing from 59 8Cto
51 8C for 1 min (decreased 0.5 8C for each second cycle), primer
extension for 1 min at 72 8C, followed by an additional 17 cycles
with a constant annealing temperature of 51 8C for 30 s, a primer
extension at 72 8C for 1 min, followed by a final extension at 72 8C
for 3 min. PCR products were confirmed by 2% agarose gel
electrophoresis.
Reverse-transcription PCR (RT-PCR) was carried out using a
OneStep RT-PCR kit (QIAGEN, Hilden, Germany) with the primers
for amoA and nirS genes. The reaction mixture contained 5
m
lof5
QIAGEN OneStep RT-PCR buffer, 1.0
m
l of dNTP mixture, 1.0
m
lof
QIAGEN OneStep RT-PCR enzyme mix, 1.5
m
l of forward primer,
1.5
m
l of reverse primer, 1.0
m
l of RNA template and 14
m
lof
RNase-free water. To ascertain the absence of the bacterial DNA
contamination, RNA samples were also assigned to normal PCR
without reverse transcription under the same conditions as
mentioned for the DNA amplifications. PCR products were
confirmed by 2% agarose gel electrophoresis.
3. Results
3.1. Size effects
We first examined size-dependence of the biofiltration
efficiency of the live rock. As a model N compound, NH
4+
was
tested using different sized live rocks. Fig. 2 shows percentage of
NH
4+
removed from seawater in 24 h. The experiment was
conducted using live rock with the wet weight of 20 (A), 65 (B),
and 95 g (C), respectively. Since the decrease in NH
4+
concentration
was negligible in the absence of the live rocks (less than 5% of the
initial), the depletion of NH
4+
in the aquaria with the live rocks can
be ascribed to their removal activities. We exclusively observed
NH
4+
removal activity of the live rock in all the samples tested
under either light or dark conditions. It is important to note that
the activities observed under the light conditions were always
higher than those measured under the dark conditions (ANOVA,
P<0.001). In comparisons between multiple and single live rocks,
a higher NH
4+
uptake was observed in multiple samples (larger
surface area) than that of single sample (smaller surface area)
(ANOVA, P<0.001). These results suggest that surface area of the
live rock determines overall NH
4+
removal activity of the live rock,
especially under dark conditions.
3.2. N producer experiment
We next examined more practical situation to simulate actual
coral aquaculture. Unlike the experiments with chemical supple-
mentation as shown in Fig. 2, various N compounds can be
continuously inputted into the aquaculture system. To explore the
effectiveness of the live rock on coral culture, biotic input of N
compounds were needed. For this purpose holothurians have been
studied as model organisms (Uthicke and Klumpp, 1998). In the
present study, the sea cucumbers H. leucospilata and H. atra were
used as biotic sources that continuously input N compounds into
seawater. We verified that H. leucospilota and H. atra excreted N
Fig. 2. Effects of weight and surface area of live rock on percent of NH
4+
removed.
Single live rock and multiple live rocks with similar weight but different surface
areas were used for both light (open bar) and dark (filled bar) treatments. Numbers
with g indicate the total weight of the live rock used. The values are presented as
percent of NH
4+
removed after 24 h incubation with 100
m
MNH
4
Cl (n=3,
SD = standard deviation).
Y.S. Yuen et al. / Aquacultural Engineering 41 (2009) 35–43
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compounds and in fact the concentrations of NH
4+
,NO
3
and NO
2
in the aquaria increased constantly (Fig. 3A–F). A faster increase in
NH
4+
concentration was observed in the aquaria containing H.
leucospilota than that containing H. atra (Fig. 3A and B). The initial
rate of the increase in NH
4+
concentration was 3.3
m
Mh
1
and
1.5
m
Mh
1
for H. leucospilata and H. atra, respectively. After
placing the live rock in the aquaria, all concentrations of NH
4+
,
NO
3
and NO
2
decreased drastically (Fig. 3A–F, indicated as gray
Fig. 3. Sea cucumber-dependent increase in NH
4+
,NO
3
and NO
2
concentrations of seawater. The sea cucumbers H. leucospilota and H. atra were used as nitrogen producing
source. The grey sections represent the period when the live rock was present.
Fig. 4. Suppressive effects of the live rock on the elevation of NH
4+
and NO
3
+NO
2
concentrations in seawater. The filled circles indicate NH
4+
and NO
3
+NO
2
concentrations in the control aquarium contained the sea cucumber H. atra only. The filled triangles represent concentrations in the aquarium including H. atra and the coral
A. digitifera. The open circles show the concentrations in the aquarium containing H. atra,A. digitifera and the live rock (n= 3, SD = standard deviation).
Y.S. Yuen et al. / Aquacultural Engineering 41 (2009) 35–43
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area). It is notable that the effect was temporal and those
concentrations increased again just after removal of the live rock
from the aquaria (Fig. 3B, D and F).
3.3. Combination experiment
Fig. 4 represents changes in the concentrations of NH
4+
and
total nitrates (NO
3
+NO
2
) throughout the experimental
period. H. atra was used as an N production source instead of
H. leucospilota since its smaller size fit into the aquarium. In
the aquarium with sea cucumber only (control) and those
without live rock (sea cucumber + coral), the concentration of
NH
4+
increased significantly, suggesting that corals do not
contribute to the removal of NH
4+
from seawater (Fig. 4A). In
fact, there was no substantial difference in the increasing rate
regardlessofthepresenceofcorals.Inthepresenceofliverock,
the NH
4+
concentration of seawater remained at a low level
(<50
m
M) even though sea cucumbers and corals were present
(Fig. 4A). Fig. 4B shows changes in total nitrate concentration.
Similar to the results of NH
4+
, the control treatment containing
only sea cucumbers displayed higher values in total nitrate
concentration. Unlike the case of NH
4+
, the total nitrate
concentration decreased in the presence of corals, suggesting
that corals uptake NO
3
. The presence of the live rock
dramatically decreased the total nitrate concentration to
0.5
m
M that was maintained throughout the experimental
period (Fig. 4B).
Fig. 5 shows effects of the live rock on the pH stability of
seawater. In the aquarium where the live rock was present (open
circle), higher pH values ranging from 7.75 to 8.00 were obtained
whereas the values in the control (contained H. atra only) and
coral aquaria (contained H. atra and A. digitifera) were 7.60–7.80
and 7.40–7.70, respectively (ANOVA, P<0.01). It is interesting to
note that the live rock functioned as pH stabilizer in the aquarium
even in the presence of coral and sea cucumber. To ascertain
whether this phenomenon is due to the dissolution of calcium
carbonate from the live rock, coral skeleton was added into
seawater and pH was monitored daily. A gradual decrease in
seawater pH was observed within 5 days incubation as shown in
inset in Fig. 5.
During the experimental period, we observed coral bleaching
and coral mortality for both treatments with the live rock and
without the live rock as shown in Fig. 6A. At the end of the
experiment, the percentage of survived coral was 92% and 67% for
treatments with and without live rock, respectively.
We further investigated effects of live rock on coral
physiology using the pulse-amplitude modulation (PAM) chlor-
ophyll afluorometry technique. The technique allows us to
monitor photosynthetic activity of the symbiotic alga in a non-
invasive way (Takahashi et al., 2004; Nakamura et al., 2005).
Fig. 5. Stabilizing effects of the live rock on pH of seawater. The control aquarium
contained H. atra only (filled circle). One set of treatment contained H. atra and A.
digitifera (filled triangle) while another set of treatment contained H. atra,A.
digitifera and live rock (open circle). A gradual decrease in seawater pH was
observed when coral skeleton was used as a control as shown in inset (open square)
(n= 3, SE = standard error).
Fig. 6. Effects of the live rock on the physiology and survivorship of the coral A.
digitifera. Open circles or bars, in the presence of the live rock; closed circles or bars,
in the absence of the live rock. The nitrogen producer H. atra was included in both
aquaria. (A) Percentage of bleached sample and survived sample. Bars indicate the
percentage of bleached coral whereas lines indicate the percentage of survived coral
throughout the experimental period (n= 12, SE = error bars); (B) maximum
quantum yield, F
v
/F
m
; (C) electron transport rate of photosynthesis, ETR. To
obtain the F
v
/F
m
values, the samples were pretreated in darkness for 15 min (n= 12,
SE = standard error).
Y.S. Yuen et al. / Aquacultural Engineering 41 (2009) 35–43
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Fig. 6B shows changes in F
v
/F
m
throughout the experimental
period. Higher F
v
/F
m
values were observed in the treatments
with live rock compared to the ones without live rock (ANOVA,
P<0.001).
Fig. 6C represents light saturation curves for the electron
transport rate (ETR) of the corals, another measure of physiological
status. The ETR was saturated at 500
m
mol photons m
2
s
1
and
400
m
mol photons m
2
s
1
for the corals in treatments with live
rock and without live rock, respectively. A steep decline at a super
saturation light suggests the light-induced inhibition of photo-
synthesis, i.e. photodamage. Higher values of ETR were obtained
for corals cultured with live rock compared to those without them
(ANOVA, P<0.001).
3.4. Bacteria associated with live rock
To explore the mechanism for N compound removal activity
of the live rock, we examined possible association of bacteria
colonized inside of the live rock. Two functional genes
were investigated: amoA gene encoding the ammonia mono-
oxygenase (AMO) enzyme in ammonia oxidizing bacteria and
nirS gene encoding the cytochrome cd
1
of the nitrite reductase
(nir) enzyme in denitrifying bacteria. PCR products correspond-
ing to amoA and nirS were successfully detected in the DNA
isolated from the carbonate substrate of the live rock as shown
in Fig. 7B, indicating the occurrence of ammonia oxidizing
bacteria and denitrifying bacteria within the live rock. Although
a bacterial gene (16S rRNA) was detected in seawater and CCA
live tissue, both amoA gene and nirS gene were not detected
(Fig. 7B).
To ascertain whether these bacteria are active inside the live
rock throughout the experimental period, expression of amoA gene
and nirS gene was investigated using RT-PCR. Both genes were
being expressed in either NO
3
or NH
4+
incubated live rocks as
shown in Fig. 7C.
4. Discussion
4.1. Live rock improves water quality
The present study has shown that live rock covered with CCA is
capable of preventing eutrophication by the removal of inorganic N
compounds (Figs. 2–4). The results of the experiment with coral
nubbins also suggest that the presence of the live rock suppresses
the photoinhibition of coral under eutrophic conditions (Fig. 6B
and C). To ascertain beneficial effects of the live rock in biofiltration
process, we have evaluated the health of coral by assessing their
photosynthetic performance using a PAM fluorometer. Low values
of F
v
/F
m
and ETR of corals in the eutrophic treatments (high level of
N compounds) clearly indicate that photosynthetic activity of the
symbiotic algae was inhibited by sub-saturating light when
concentrations of N compounds were kept at high level (ETR
saturated at >400
m
mol photons m
2
s
1
in light saturation
curves) (Yamasaki, 2000). In a previous study of sediment-based
biological filtration systems, Toonen and Wee (2005) found that
nitrate concentration was significantly lower in aquaria that
contained ‘‘live rock’’. However, there has been no further
explanation for such lower nitrate concentration. In our best
knowledge, the results presented in this study have provided the
first experimental evidence supporting the idea that live rock is an
effective biofilter in coral reef aquaria. It should be noted that with
the presence of live rock in aquaria, water quality can be
maintained even without water filtering system or circulating
system for a short duration (5–7 days). This is important when
circulating or filtration systems fail in aquaria.
The presence of live rock provided pH stability. The pH value of
seawater in the aquaria with live rock was higher than that in the
absence of live rock and the value was maintained at suitable level
(Fig. 5). This effect is mainly ascribed to the photosynthetic activity
of CCA that increases pH through the removal of carbon dioxide due
to carbon assimilation process. Inhibition of CCA photosynthetic
activity using DCMU (N
0
-[3,4-dichloropheny]-N,N-demethylurea)
resulted in a decrease in seawater pH (data not shown). One may
argue that dissolution of calcium carbonate from the live rock
resulted in the increasein seawater pH. However, this is not the case
in this study since the presence of coral skeleton in seawater did not
increase the pH (inset in Fig. 5). Live rock could act as a buffering
agent due to itscalcium carbonate nature butthe dissolution process
of calcium carbonate may be very slow. Calcification of corals is
highly sensitive to the changes in seawater pH that affect the CO
2
/
HCO
3
ratio (Barnes and Chalker, 1990). The calcification rate of the
coral Porites compressa growing in seawater at pH 7.2 was reported
to be half of that in corals cultured at pH 8.0 (Marubini and Atkinson,
1999). In addition to the N compound removal, we consider that the
pH stabilizing effect could contribute to maintaining water quality
suitable for the growth of reef-building corals.
4.2. Possible association of anaerobic bacteria
The presence of endolithic microorganisms such as cyano-
bacteria has been reported in the carbonate substrate of CCA
Fig. 7. Occurrence of ammonia oxidizing bacteria and denitrifying bacteria in the
carbonate substrate of the live rock. (A) Cross-section of the live rock displaying a
green layer underneath the CCA tissue in whitish carbonate skeleton. The circle
indicates the part where DNA and RNA samples were extracted; (B) PCR
amplification of the fragment of the amoA gene, nirS gene and 16S rRNA gene
from carbonate substrate, CCA live tissue and seawater; (C) expression of amoA
gene and nirS gene in the live rock detected using reverse-transcription PCR. RT (+)
indicates that reverse transcriptase enzyme was added whereas () indicates that
reverse transcriptase enzyme was not added. (For interpretation of the references to
color in this figure legend, the reader is referred to the web version of the article.)
Y.S. Yuen et al. / Aquacultural Engineering 41 (2009) 35–43
41
Author's personal copy
(Tribollet and Payri, 2001; Chaco
´n et al., 2006). When a
substratum of the live rock is entirely covered with CCA, its
internal space is expected to be hypoxic or anoxic, a condition
where anaerobic bacteria can colonize. Micro-diversity of
oxygen tension in rhizosphere (land soils) enables the coex-
istence of nitrifying and denitrifying bacteria (Arth and Frenzel,
2000). Likewise, live rock can be considered as a consortium of
CCA and bacteria that functions similar to the combination of
land plants and soil bacteria.
Until recently, various types of biological filtration systems
utilizing microbes or algae have been applied to remove
nitrogenous wastes in the facilities of large-scale aquaculture
(Gutierrez-Wing and Malone, 2006; Herna
´ndez et al., 2002; Van
Rijn et al., 2006) and wastewater treatment (Gracı
´a et al., 2000;
Lydmark et al., 2007). Detection of key genes expression (amoA
and nirS) for ammonia oxidizing bacteria and denitrifying
bacteria suggests that the removal activity of N compounds
can be partly ascribed to nitrification and denitrification
processes of bacteria (Fig. 7C). In this context, we consider that
live rock offers a unique bioremediation system in which N
compounds are processed by the alga (CCA) on the surface and
internal bacterial communities.
5. Conclusions
The laboratory experiments conducted in this study have
confirmed that live rock (encrusted by CCA) provides positive
effects on reef-building coral by: (1) maintaining NH
4+
,NO
3
and
NO
2
concentrations in seawater at low level; (2) stabilizing the
pH of seawater; (3) preventing the inhibition of photosynthetic
activity of the coral under eutrophic conditions; and (4) harboring
nitrifying and denitrifying bacteria under the CCA layer that are
active under eutrophic conditions. These results support the
application of ‘‘live rock’’ as biofiltrator or bicompensator for the
maintenance of coral reef aquaculture systems.
Acknowledgements
We thank Dr Michael Cohen of Sonoma State University for
critical reading of our manuscript. This work was supported by the
Grant-in-Aid for the Basic Research (B) to H.Y. and by the 21st
century COE program of the University of the Ryukyus from the
Ministry of Education, Culture, Sports, Science and Technology,
Japan. S.S.Y and T.N. are thankful for the support from JSPS research
fellowships for young scientists. Y.S.Y. is financially supported by
the Ministry of Education, Culture, Sports, Science and Technology,
Japan.
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Yeong Shyan Yuen is a Ph.D. candidate in the Faculty of
Science, University of the Ryukyus, Japan. She holds an
M. Sc. degree in marine biology from Malaya University
in Malaysia. Her study interest is in the response of reef
organisms including reef-building corals and macro-
algae to eutrophication. Her current research has been
mainly on crustose coralline algae (CCA) in subtropical
coral reefs, focusing on nutrient uptake and bioreme-
diation potential of CCA under eutrophication. In
addition, she is also working on the biofiltration
mechanisms of live rock in aquariums in relation to
CCA and microorganisms involved.
Y.S. Yuen et al. / Aquacultural Engineering 41 (2009) 35–43
42
Author's personal copy
Seitaro S. Yamazaki is a Ph.D. candidate in the Faculty
of Science, University of the Ryukyus, Japan. He is a
recipient of the JSPS (Japan Society for the Promotion of
Science) Research Fellowship for Young Scientists. He
holds an M.Sc. degree in marine biology from the
University of the Ryukyus. His research has been mainly
on the endolithic microorganisms residing inside the
skeleton of reef-building coral, focusing on the protec-
tive role of endolithic microorganisms to the coral host
under stress conditions. His recent study includes the
changes of endolithic microorganisms’ diversity under
eutrophication and its implication on coral reef ecology.
Takashi Nakamura is a postdoctoral fellow of JSPS
(Japan Society for the Promotion of Science) Research
Fellowship for Young Scientists. He holds a Ph.D. degree
from the University of the Ryukyus, Japan. His main
research interest is in stress responses of reef-building
corals at various scales and times. Dr. Nakamura has
published several papers on coral bleaching inhibition
and recovery in relation to water flow. He is actively
involved in the conservation of coral reefs as a reef-check
volunteer as well as educator for public awareness.
Gaku Tokuda is an Assistant Professor in Tropical
Biosphere Research Center at University of the Ryukyus.
His main research interest is molecular biology of
nutritional symbiosis between invertebrates and
microorganisms. He is also actively involved in
biochemistry of lignocellulolytic enzymes, especially
in termites. Dr. Tokuda has published several papers
and reviews on these subjects.
Hideo Yamasaki is a Professor of Biology at the
University of the Ryukyus in Okinawa, Japan. From
1997 to 1998, he was a visiting fellow at the Research
School of Biological Sciences (RSBS) in the Australian
National University. An invited speaker at seven
international congresses from 2000 to 2008, Dr
Yamasaki has written many peer-review papers, seven
book chapters, three recent journal reviews and
numerous article citations. In addition to antioxidant
research, he has led the field of nitric oxide (NO) biology
for 10 years.
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