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Inuence of carbohydrate addition on nitrogen transformationsand greenhouse gas emissions of intensive aquaculture system
Zhen Hu a, Jae Woo Lee b, Kartik Chandran c, Sungpyo Kim b, Keshab Sharma d, Samir Kumar Khanal e,a Shandong Key Laboratory of Water Pollution Control and Resource Reuse, School of Environmental Science and Engineering, Shandong University, Jinan 250100, Chinab Department of Environmental Engineering, College of Science and Technology, Korea University, Sejong-ro 2511, Sejong 339-700, South Koreac Department of Earth and Environmental Engineering, Columbia University, 500 West 120th Street, New York, NY 10027, USAd Advanced Water Management Centre, University of Queensland, St. Lucia, QLD 4072, Australiae Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, Honolulu, HI 96822, USA
H I G H L I G H T S
Addition of soluble starch to intensive aquaculture system enhanced heterotrophic bacterial growth and denitrication.
Soluble starch addition minimized the nitrous oxide emissions from intensive aquaculture system by 83.4%.
Soluble starch addition had signicant adverse effects in controlling GHG emissions from aquaculture systems.
a b s t r a c ta r t i c l e i n f o
Article history:
Received 12 July 2013
Received in revised form 12 September 2013
Accepted 17 September 2013
Available online xxxx
Editor: Prof. P. Kassomenos
Keywords:
Intensive aquaculture system
Nitrogen transformations
Greenhouse gases
Nitrous oxide
Carbohydrate addition
Aquaculture is one of the fastest-growing segmentsof the food economy in modern times. It is alsobeing consid-
eredas an important source of greenhouse gas(GHG) emissions. To date, limited studieshave beenconducted on
GHG emissions from aquaculture system. In this study, daily addition ofsh feed and soluble starch at a carbon-
to-nitrogen (C/N) ratio of 16:1 (w/w) was used to examine the effects of carbohydrate addition on nitrogen
transformationsand GHG emissions in a zero-water exchange intensive aquaculture system. The addition of sol-
uble starch stimulated heterotrophic bacterial growth and denitrication, which led to lower total ammonia ni-trogen, nitrite and nitrate concentrations in aqueous phase. About 76.2% of the nitrogen output was emitted in
the form of gaseous nitrogen (i.e., N2and N2O) in the treatment tank (i.e., aquaculture tank with soluble starch
addition), while gaseousnitrogen accounted for33.3% of the nitrogen output in the controltank (i.e., aquaculture
tank without soluble starch addition). Although soluble starch addition reduced daily N2O emissions by 83.4%, it
resulted in an increaseof daily carbondioxide (CO2) emissions by 91.1%. Overall,starch addition did not contrib-
ute to controlling the GHG emissions from the aquaculture system.
2013 Elsevier B.V. All rights reserved.
1. Introduction
Global food sh supply has increased dramatically in the last ve
decades, with an average growth rate of 3.2% per year in the period of
19612009, to meet therising world demands for protein. In 2010, cap-
ture sheries and aquaculture supplied about 148 million metric tons
ofsh globally (FAO, 2012). Because world shery productions have
leveled off since the 1970s, aquaculture plays a crucial role in meeting
the increasing global demands for sh.
Since the expansion of aquaculture is restricted by land and water
requirements, intensive aquaculture, which aims at raising sh at
mass densities as high as 30 kg/m3 in closed or semi-closed systems
with sufcient oxygen, fresh water and feed, has been increasingly
popular to overcome space and resource limitations (Delong et al.,
2009). However, the development of intensive aquaculture has created
serious environmental problems.
Themost importantissueduringthe management of intensiveaqua-
culture is avoiding the accumulation of toxic inorganic nitrogen species
(especially, NH3 and NO2) in the aqueous phase (Durborow et al., 1997;
Hargreavesand Tucker, 2004). In intensive aquaculturesystems,share
fed a high protein diet with protein levels varying from 25 to 55% (Pillay
and Kutty, 2005). Protein is digested by sh, producing mainly ammo-
nia, and is excreted to the surrounding aqueous phase. In the aqueous
phase, ammonia exists in two forms: un-ionized ammonia (NH3) and
ionized ammonium (NH4+).Thesum ofNH4
+ and NH3 is usually referred
to as total ammonia nitrogen (TAN). Ammonia can further be oxidized
to NO2 and NO3
by nitrifying bacteria present in the aquaculture sys-
tem. Inorganic nitrogen species, especially NH3and NO2, are toxic to
sh. High concentrations of NH3and NO2 can stimulate the release of
corticosteroid hormones into the venous circulation, which may inhibit
Science of the Total Environment 470471 (2014) 193200
Corresponding author. Tel.: +1 808 956 3812; fax: +1 808 956 3542.
E-mail address:[email protected](S.K. Khanal).
0048-9697/$ see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.scitotenv.2013.09.050
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sh growth and cause a variety of physiological dysfunctions (Tomasso,
1994).
One of the most common approaches to prevent excess nitrogen
buildup is through water exchange. This approach, however,has sev-
eral environmental issues, e.g., requires a perpetual supply of fresh
water and the generation of nitrogen-rich efuent, among others
(Avnimelech, 2009). Anotherapproachis to enhance nitrication to fa-
cilitate the conversion of ammonia and NO2 into relatively non-toxic
NO3
by using nitrifying bio
lters. Although nitrifying bio
lters havebeen successfully employed in aquaculture systems and are effective
in ammonia and NO2 removal, they are costly and most importantly,
do not provide an opportunity for nutrients recycling ( Avnimelech,
2006; Crab et al., 2007). This is particularly signicant because protein
insh feed is the most expensivecomponent of aquaculture operational
costs especially when taking into consideration the fact that only about
25% of the nitrogen consumed by sh is converted to sh mass
(Hargreaves, 1998).
An additional strategy, namely carbohydrate addition, has been
demonstrated to be effective in achieving low or zero-water ex-
change intensive aquaculture systems (Gao et al., 2012). By adding
carbohydrate to aquaculture systems and regulating the carbon-to-
nitrogen (C/N) ratios, the growth of heterotrophic bacteria can be
stimulated, which results in the removal of inorganic nitrogen
through assimilation. As bacterial biomass increase, they tend to
form noticeable aggregates (i.e., bioocs), which serve as a potential
source of food for sh. Thus, the utilization of protein in the sh feed
is enhanced due to nutrients recycling by heterotrophic bacteria. Re-
cently, this approach has become increasingly popular for closed-
water shrimp and tilapia cultivation (Asaduzzaman et al., 2010;
Widanarn et al., 2012).
Besides water pollution, the contribution of aquaculture systems to
global greenhouse gases (GHG) emissions has aroused great attention
in recent years (Williams and Crutzen, 2010; Hu et al., 2012). In aqua-
culture systems, major GHG emissions include carbon dioxide (CO2),
methane (CH4) and nitrous oxide (N2O). CO2is produced from the de-
composition of organic materials and through respiration by sh. High
levels of CO2 can be detected in aquaculture systems with high feed
loading rates and relatively slow water turnover (Good et al., 2010).CH4 is produced when organic materials are broken down under anaer-
obic conditions (Adams et al., 2012). In conventional aquaculture sys-
tems, however, signicant concentrations of CH4are typically unlikely
(Rakocy et al., 2006). N2O is an important GHG which has a global
warming potential (GWP) that is 296 times higher that of CO2 on a
100-year timescale (IPCC, 2007).Hu et al. (2012)estimated that aqua-
culture could contribute up to 5.72% of global anthropogenic N2ON
emission by 2030 if it continues to increase at the present annual
growth rate. However, the mechanisms for N2O generation in an aqua-
culture system are highly inuenced by environmental conditions and
are not well known. Both nitrication and denitrication can contribute
to the emission of N2O from aquaculture systems (Beaulieu et al., 2011;
Hu et al., 2013a).
The addition of carbohydrate to aquaculture systems could de-crease the abundance of nitrifying bacteria, and thus may minimize
nitrication-driven N2O production (Avnimelech, 1999). Furthermore,
the supplementation of external organic carbon could provide electron
donors for denitrication and may reduce N2O generation through de-
nitrication (Hu et al., 2013b). However, carbohydrate addition might
increase the concentration of organic materials, which may lead to
higher CO2and CH4 emissions. Nonetheless, there has been a dearth
of studies thus far on the emissions of GHGs from zero-water exchange
intensive aquaculture system.
The purpose of this study was to investigate the effect of soluble
starch addition on GHG emissions from a zero-water exchange tila-
pia intensive aquaculture system. Nitrogen transformations in aqua-
culture system with and without soluble starch addition were also
examined.
2. Materials and methods
2.1. System setup
Two aquaculture systems were placed side by side in an air-
conditioned room at a temperature around 25 C and exposed to 24-h
lighting. The schematic diagram of aquaculture system is shown in
Fig. 1. The system was mainly composed of a plastic tank (KMT85, Tuff
Stuff, Terra Bella, CA, USA), with a working volume of 200 L. Air wassupplied continuously by an air pump through three diffusers placed
at the bottom of the tank, and desired DO concentrations were obtained
by controlling the air ow rate. Aeration also provided mixing for the
system. In each tank, one biolter composed of mesh nylon biolter
media bags lled with 1.5 kg of biomedia (Kaldnes @ media, Aquatic
Eco-System, Apopka, FL, USA), was placed adjacent to the air diffusers
to facilitate the growth of nitrifying bacteria. Semi-transparent acrylic
plastic lids were used to minimize water loss by evaporation and to pre-
vent algal growth. Freshwater was occasionally added to the system to
compensatewater lossthrough evaporation,whennecessary. The aqua-
culture tank was maintained at pH of around 7.0 by periodic dosing of
sodium bicarbonate (NaHCO3).
2.2. Experiment design
It generally requires about 4 weeks to establish the required micro-
bial community in a biolter of an aquaculture system (Avnimelech,
2009). However, since heterotrophic bacteria typically have a maxi-
mum growth rate ve-fold faster and biomass yields two to three-fold
greater than that of nitrifying bacteria, bioocscould be established rap-
idly in a matter of days (Grady and Lim, 1980). To overcome the time
difference, efuent from a stable operating aquaculture system was
used as the initial tank water for the experiment, and the biolters
were inoculated in the stable operating aquaculture system for two
weeks prior to the start of the experiment. This facilitated the establish-
ment of nitrifying bacteria in the biolters, which were used in subse-
quent experiments.
Each tank was stocked with mixed sex tilapia sh (Oreochromis
niloticus) with an average weight of 120.5 27.3 g, to obtain an initialstocking density of around 23.5 kg/m3. The sh were obtained from
Windward Community College (Honolulu, Hawaii, USA). Fish were
grown without water exchange for 8 weeks, and were fed once daily
at 10:00 A.M. with 42% protein commercial aquatic feed pellets (Silver
Cup Trout Feed, Tooele, UT, USA). The amount of feed per feeding time
was determined based on sh response to previous feeding (Casillas-
Hernndez et al., 2006). Ten minutes after feeding, all the feed pellets
remaining above water surface were collected, dried and weighed. The
feeding rate was adjusted in the subsequent days so that the leftover
(un-consumed) feed 10 min after feeding was no more than 5% of the
total feed added. In the treatment tank, soluble starch was added daily
alongwithfeed to maintain a C/N ratio (w/w) of 16:1; while thecontrol
tank was supplied with sh feed only (Nootong et al., 2011). The
amount of daily starchaddition was calculated as per theequations pro-posed by Avnimelech (1999), and about 1.4 g of soluble starch was
added for each gram of formulatedsh feed. The pre-weighed soluble
starch was completely mixed with tank water in a beaker and was
then uniformly sprayed over the tank surface water after each feeding.
2.3. Analytical method
Water samples from each tank were obtained after feeding every
other day and were analyzed immediately for TAN, NO2, NO3
, total
phosphate (TP), and chemical oxygen demand (COD) concentrations
using HACH reaction kits (Loveland, CO, USA), namely Ammonia
TNTplus (TNT 830), Nitrite TNTplus (TNT 839), Nitrate TNTplus (TNT
836), Phosphorus TNTplus (TNT 845), and COD Reagent TNTplus (TNT
822), respectively. Dissolved oxygen(DO) concentrations, temperature,
194 Z. Hu et al. / Science of the Total Environment 470471 (2014) 193200
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pH, and salinity were measured in situ daily using the HQ40d Portable
Water Quality Lab Package (HACH, Loveland, CO, USA). Bioocs concen-
tration in water, expressed as total suspended solids (TSS), was deter-
mined according to the Standard Methods (APHA, 2005). The bioocs
attached to the biomedia of the biolter were washed out with pure
water at the end of the experiment, ltered, dried at 105 C and
weighed. A LECO TruSpec CN analyzer (LECO Corp., St. Joseph, MI,
USA) was used to measure the total nitrogen (TN) contents of sh
mass, sh feed and bioocs.
Diurnal variations of GHG emission rates were monitored once a
week during the last three weeks of the study period, after attaining
fairly constant daily feed consumption. Dissolved N2O concentrations
in the aqueous phase were determined using a Clark type electrode
N2O sensor (Unisense, Aarhus N, Denmark) (Andersen et al., 2001).
N2O concentrations in the gas phase were calculated based on the fol-
lowing equation (Rassamee et al., 2011),
CN2O;air kLaSN2OVr
= QairVr kLa=H 1
where CN2O;air is the gas phase N2O concentration (mol/L); kLais the
volumetric mass transfer coefcient (1 / h), which varied with different
air ow rates and is determined according to equation provided by
Foley et al. (2010); SN2O is the concentration of N2O in the liquid
phase (mol/L);Vris the working volume of the tank (L);Qairis the air
ow rate (L/h); H is the Henry's constant, which is 1.303 for N2O at
25 C (Sander, 1999).
The gas phase concentrations of CO2and CH4were determined by
using a Varian CP-4900 micro gas chromatograph (Varian Inc., Walnut
Creek, CA, USA), equipped with thermal conductivity detector and a
10 m PPQH BF column. Helium maintained at 30 mL/min was used as
a carrier gas, and the temperature of the column and injector were set
at 80 C and 100 C, respectively.
2.4. Statistical analysis
Experimental data were presented as a mean of three replicates. Sta-
tistical analyses were performed using SPSS v16.0 for Windows soft-
ware (SPSS v16.0 IBM Corporation, Somers, NY, USA). The signicance
for all p-values was 0.05 unless otherwise stated.
3. Results
3.1. Growth performance of tilapia
The growth performances of tilapia in both tanks are shown in
Table 1. During the 8-week study period, nosh mortality was observed
in both treatment and control tanks. Also, no signicant differences in
sh growth performance were found between the treatment and con-
trol tanks. Nearly the same average sh weight increase was recorded
in both tanks. However, the use of mixed sex tilapia as the test subject
resulted in unexpected breeding during the study period, particularly
in the treatment tank. The larvae were collected with a ne mesh net
and weighed after counting. During the study period, about 246 and
37 tilapia larvae were collected from treatment and control tanks, re-
spectively. The wet weight of the collected larvae in treatment and con-
troltanks were 19.6 g and 3.5 g, respectively. The same feed conversion
ratio (FCR) of 2.2 was obtained in both treatment and control tanks.
3.2. Water quality variation
The ranges of pH, DO concentrations, and temperature in the treat-
ment and control tanks are presented inTable 2. All of the parameters
were similar and were not suspected to be growth limiting. It is worth
noting that the aeration rates of treatment and control tanks were
different. The addition of soluble starch to the treatment tank resulted
in a decrease of DO concentrations due to the enhanced growth of het-
erotrophic bacteria. In order to maintain a comparable DO concentra-tion in both tanks, the control tank was supplied with 4.7 L/min of air,
whereas theaeration rate in the treatment tankwas graduallyincreased
from 5.1to 9.8 L/min to compensate forincreasing oxygenconsumption
resulting from higher bioocs concentration in the tank.
The variations of TAN, NO2, and NO3
concentrations during the
study period are presented inFig. 2. The variations of TSS, COD, and TP
concentrations in the treatment and control tanks are presented in
Fig. 3. Accumulation of TAN was observed following sh stocking in
both tanks. The highest TAN concentrations of 6.78 mg N/L and
2.84 mg N/L were observed on day 26 and day22 in treatment and con-
trol tanks, respectively. In addition, TAN accumulation lasted for a lon-
ger period of time in the treatment tank. It required 36 and 28 days
for TAN concentrations to decrease to less than 1.0 mg N/L in the treat-
ment and control tanks, respectively. The TAN concentrations in thetreatment tank were, however, consistently lower than that in the con-
trol tank at the later stage of the study period.
Signicant increases in NO2 and decreases in NO3
concentrations
were observed in the treatment tank after the start of the experiment.
Fig. 1.Schematic diagram of aquaculture system.
Table 1
Growth performances ofsh in control and treatment tanks.
Parameters Treatment Control
A verage initial weight (g) ( n = 4 0) 120.3 24.6 1 20 .6 3 0.2
Average nal weight (g) (n = 40) 137.3 29.1 137.3 32. 8
Fish biomass increasea (g) 672.7 654.5
Fish feed consumption (g) 1448.0 1459.1
Feed conversion ratio 2.2 2.2
a
Includes the weight of tilapia larvae collected during the research period.
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Both NO2
increases and NO3
decreases continued until day 14. Thehighest NO2 concentration of 7.2 mg N/L was observed on day 14 in
the treatment tank. The NO2 concentration then decreased rapidly to
less than 0.4 mg N/L after day22. In the control tank, a relatively constant
accumulation of NO2 and a linear increase in NO3
concentrations were
observed. Similar to TAN proles, the treatment tank showed lower
NO2 and NO3
concentrations during the later stage of the study period.
The TSS concentrations in the treatment tank increased from
52.5 7.3 mg/L to 796.5 18.8 mg/L during the study period, while
it remained fairly constant at around 47.5 9.2 mg/L in the control
tank. In addition, although the nitrogen contents of bioocs in both
tanks were initially around 6.1 0.2%, the nitrogen content in the
treatment tank decreased to 5.3 0.3%, but remained fairly constant
in the control tank following the 8-weeks study period. The COD con-
centration increased at a much faster rate in the treatment tank than
in the control tank. At the end of the study period, the COD concentra-
tion in the treatment tank was 2.8-fold higher than that in the control
tank. Statistical analysis showed that starch addition had no signicant
effect on TP removal. Similar TP concentration proles were found in
both the treatment and control tanks.
3.3. Daily GHG emissions
Fig. 4shows diurnal variations of CO2and N2O emission rates in the
treatment and control tanks. The time-weighted GHG emissions from
treatment and control tanks are listed in Table 3. CH4concentrations
were consistently below the detection limits during the study period
(data not shown), and was not included in the calculation of GHG
emissions in this study. The treatment tank had higher CO2emission
rates and lower N2O emission rates than the control tank. The addition
of soluble starch increasedthe daily CO2 emission by 91.1%, andreduced
the daily N2O emission by 83.4%. Slight increases in the CO2emission
rate was observed after feeding in both tanks.
Table 2
Physical parameters of water in treatment and control tanks.
Parameters Treatment (n = 56) Control (n = 56)
pH 7.08 0.07 7.04 0.06
DO (mg/L) 4.49 0.33 4.53 0.38
Temperature (C) 24.6 0.3 24.4 0.3
Fig. 2.Variation of total ammonia nitrogen (A), nitrite (B), and nitrate (C) concentrations
in treatment andcontrol tanks during thestudy period.Valuesare mean of triplicatesam-
ples analyzed for each parameter.
Fig. 3. Variation of total suspended solids (A), chemical oxygen demand (B), and total
phosphate (C) concentrations in treatment and control tanks during the study period.
Values are mean of triplicate samples analyzed for each parameter.
Fig. 4.Diurnal variation of CO2(A) and N2O (B) emission rates in treatment and control
tanks. Each data point is the mean of three different measurements on different days.
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To investigate the major factors inuencing GHG emissions, the di-
urnal variation of TAN, NO2, NO3
, and COD concentrations in both
treatment and control tanks were also determined (Fig. 5). The control
tank showed higher TAN, NO2, and NO3
concentrations than the treat-
ment tank, whereas higher COD concentrations were observed in the
treatment tank. All of the water quality parameters were relatively
stable within a short time span of 24 h.
4. Discussion
4.1. Effect of soluble starch addition on sh performance
In order to minimize inorganic nitrogen accumulation and to accel-
erate bioocsformation, organic carboncan be added eitheras an exter-
nal organic carbon source or by changing the feed composition to
increase its organic carbon content (Avnimelech, 1999). Both ap-
proaches were reported in the literature without identifying their in-
herent merits (Azim and Little, 2008; Asaduzzaman et al., 2010). In
this study, we used the former approach because it is easier to imple-
ment. Adouani et al. (2001) reported that compared with shorter
chain carbon compounds (e.g., ethanol, acetate), long chain carbon
compounds (e.g., peptone, starch) could produce less N2O when used
as the carbonsourcefor denitrication,mainly becauseof itslower elec-
tron supply rate. Soluble starch is a cheap and widely used carbon
source and was thus employed in this study.
Hargreaves (1998)reported that about 11 to 36% of the nitrogen in
sh feed could be recovered in an aquaculture system. In this study, ti-lapia in both the treatment and control tanks exhibited the same feed
conversion ratio (FCR) of 2.2, meaning that about 18% of the nitrogen
in the feed was recovered as sh mass. Interestingly, no signicant dif-
ference was found in the FCR between treatment and control tanks.
Studies reported that the addition of carbohydrates resulted in the pro-
duction of microbial proteins that could serve as sh food, thereby in-
creasing the FCR (Avnimelech, 1999; Crab et al., 2012). In this study,
signicant increase in reproductive activity was observed in the treat-
ment tank as evidenced from the spawning ofsh. During the breeding
process, most of the energy obtained from feeding was reported to be
utilized in gonad development (Widanarn et al., 2012). Thus, the avail-
ability of additional feed through bioocs formation might be compen-
sated by their enhanced consumption during breeding. Several studies
reported the enhancement effect of bioocs on the reproduction of tila-
pia and shrimp (Emerenciano et al., 2011; Widanarn et al., 2012).
It is also important to note that the high TSS concentrations in the
treatment tank may also have a negative impact on sh growth. Severe
growth inhibition and increased mortality of tilapia were reported
when the suspended solids concentration exceeded 850 mg/L (Little
Table 3
Greenhouse gases emissions and N2ON conversion ratio in treatment and control tanks.
Parameters Treatment Control
CO2emission (g/d) (n = 3) 60.59 7.21 31.70 4.53
N2ON emission (mg N/d ) ( n = 3) 3.83 2.0 5 2 3.12 5 .13
Feed nitrogen input (mg N/d) (n = 3) 1888.69 96.51 1890.23 84.76
N2O conversion ratio (%)a 0.20 1.22
a N2O conversion ratio = (N2ON emission / feed nitrogen input) 100.
Fig. 5.Diurnal variations of total ammonia nitrogen (A), nitrite (B), nitrate (C), and chemical oxygen demand (D) concentrations in treatment and control tanks. Each data point is the
mean of three different measurements on different days.
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et al., 2008). It is recommended that the maximum suspended solids
concentration in an aquaculture system should not exceed 500 mg/L
(Azim and Little, 2008; Little et al., 2008). This could be another reason
for the same FCR in both treatment and control tanks. The integration of
a clarier with the aquaculture tank may help to maintain a low TSS
concentration in aquaculture tank.
Daily addition of soluble starch inuenced the formation of bioocs,
as shown in Fig. 3A. During the 8-week study period, the TSS concentra-
tion (representing bio
ocs) in the treatment tank increased by 14-folds.The TSS concentration in the control tank, however, remained relatively
constant. The accumulation of bioocs in the treatment tank wasattrib-
uted to the enhancement effectof added starchon the growthof hetero-
trophic bacteria, and the uptake of bioocs by sh was apparently not
sufcient to prevent its buildup. This nding was in close agreement
with that ofNootonget al. (2011), whoreported an increase in TSS con-
centration from 52 to 1180 mg/L in their tilapia aquaculture tank that
received feed and tapioca starch, while the TSS concentration remained
relatively constant at 99 mg/L in control tanks which received feed only.
Based on statistical analyses, the nitrogen contents of bioocs in the
treatment and control tanks were signicantly different at the end of
the study period. Bioocs in the treatment tank showed lower nitrogen
contents than that in the control tank. This is mainly attributed to the ac-
cumulation of soluble microbial products (SMP) andunconsumed starch.
During substratemetabolism, bacterial growth, and bacterial decay, SMP
is produced (Barker and Stuckey, 1999). The production rate of SMP is
proportional to the concentration of bacteria and their retention time
(or age) (Jarusutthirak and Amy, 2006). Since there were no water ex-
change during the study period and high concentration of bioocs was
observed in the treatment tank, it is reasonable to believe that high
accumulation of SMP occurred in the treatment tank. Furthermore, solu-
ble starch was added to the treatment tank according to the theoretical
quantity calculated based on equations reported by Avnimelech
(1999).However, the added starch may not have been completely uti-
lized for microbial metabolism.Gao et al. (2012)reported that the opti-
mumcarbohydrate addition was75% of itstheoretical quantityin a zero-
water exchange system cultured with Whiteleg shrimp (Litopenaeus
vannamei). The accumulation of SMP and starch was evident from the
higher COD concentrations in the treatment tank (Fig. 3B). Bioocs iscomposed of micro-organisms and a mixture of non-living detritus ma-
terials. Micro-organisms only account for 220% of the organic fraction
of bioocs (Wilen et al., 2003). Part of SMP and unconsumed starch
could be absorbed by bioocs, thus reducing its nitrogen content.
4.2. Effect of soluble starch addition on nitrogen transformations
After sh stocking, TAN concentration started to accumulate follow-
ed by decrease and remained fairly constant at the later stages in both
the treatment and control tanks (Fig. 2A). However, the treatment
tank showed higher peak of TAN concentrations and required a longer
period of time to decrease to a relatively stable TAN concentration.
This was caused by the addition of soluble starch. In aquaculture sys-tems, most of the TAN produced by themetabolism ofsh are removed
through nitrication. During the initial stages of the study period, there
was not enough nitrifying bacteria in the system to remove all of the
TAN excreted bysh, and part of the TAN was accumulated in the sys-
tem. However, the TAN concentration decreased slowly and was able
to reach low stableconcentrationswith thegrowthof nitrifying bacteria
at the later stages. In the treatment tank, while the addition of soluble
starch stimulated the growth of heterotrophic bacteria, it inhibited the
growth of nitrifying bacteria, due to competition for nutrients(especially
nitrogen) between heterotrophic and nitrifying bacteria. Michaud et al.
(2006) also reported the inhibition effect of organic carbon on nitrifying
bacteria in aquaculture systems. The lower growth rate of nitrifying
bacteria in the treatment tank led to higher TAN accumulation and
more time was needed to attain stable concentrations.
Although the DO concentrations in both tanks were maintained
above 4.0 mg/L throughout the study period, the existence of micro-
anoxic zones waslikely in theinner part of thebioocs where denitri-
cationcould have occurred (Zenget al.,2003). In thetreatmenttank, the
presence of nitrates and the addition of soluble starch stimulated
denitrication during the initial stages of the study period. High nitrate
reduction rates were observed, which also led to an internal accumula-
tion of nitrite (Fig.2). Relatively lowconcentrationsof nitrateand nitrite
were achieved in the treatment tank three weeks after
sh stocking,which was most likely associated with denitrication.
Nitrogen mass balances of the treatment and control tanks through-
out the study period were conducted and the results are presented in
Fig. 6. The composition of nitrogen input for both treatment and control
tanks were similar. Fish feed contributed to over 80% of the nitrogen
input, and about 15% of the nitrogen inputs were from inorganic nitro-
gen species, which were already present in the tank water before the
start of the experiment.
A FCR of 2.2was obtained in both tanks, indicating that about14% of
the nitrogen output was associated with sh mass increase. In the treat-
ment tank, only about0.4%of the nitrogen output wasdistributed as in-
organic nitrogen species in thetank water,whileit accountedfor as high
as 48.5% of thenitrogen output in thecontrol tank. There was also a sig-
nicant difference in unaccounted nitrogen (mainly gaseous nitrogen
loss, including N2, N2O and NH3) between the treatment and control
tanks. Since the pH in both tanks was around 7.0, and themajor fraction
of TAN was in ionized form (i.e., NH4+), ammonia volatilization was
likely to be negligible. The N2O emissions during the study period
were also considered to be insignicant compared to N2(as discussed
inSection 4.3). Thus, N2produced through denitrication was consid-
ered to be the major source of unaccounted nitrogen.
Gaseous nitrogen loss in the treatment tank accounted for about
76.2% of the nitrogen output. This is because the addition of soluble
starch provided sufcient organic carbon for denitrication. The nitro-
gen loss in this study was much higher than that of Nootong et al.
(2011) whoreported a nitrogen gasloss of 32% in zero-water exchange
tilapia cultivation tanks supplied with tapioca starch at a C/N weight
ratio of 16:1. Thiscould beattributedto a highinitialNO3 concentration
in the tank water at the beginning of the experiment. Onthe other hand,about 33.3% of nitrogen output was distributed as gaseous nitrogen loss
in the control tank. This was similar to the results ofThakur and Lin
(2003)who reported gaseous nitrogen loss of 36% in their closed aqua-
culture system without carbohydrate addition.
Fig. 6.Nitrogen balance for treatment and control tanks.
198 Z. Hu et al. / Science of the Total Environment 470471 (2014) 193200
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4.3. Effect of soluble starch addition on GHG emissions
Soluble starch additionsignicantly increased the daily CO2 emissions
from the treatment tank. This was mainly due to the following reasons:
(i) starch addition stimulated the growth of heterotrophic bacteria,
which could convert nearly 50%of theorganic matter (stoichiometrically)
into CO2 under non-limiting oxygen conditions; (ii) part of the starch
added to the treatment tank was unconsumed and could be absorbed
by bio
ocs (the unconsumed starch was converted to CO2by heterotro-phic bacteria); and (iii) higher aeration rates in the treatment tank (to
maintain similar DO concentrations with the control tank) led to higher
stripping effects.
On the other hand, the addition of soluble starch reduced the N2O
emissions from the treatment tank. In aquaculture systems, both nitri-
cation and denitrication are responsible for the emission of N2O (Hu
et al., 2013b). The abundance and activity of nitrifying bacteria in the
treatment tank was inhibited by the addition of soluble starch, which
was evident from the higher TAN accumulation and lower TAN removal
rate (Fig. 2A). With no signicant differences in operational parameters
(i.e., DO, pH and temperature) and TAN concentrations, less nitrifying
bacteria led to lower nitrication-driven N2O production. Furthermore,
the addition of soluble starch could have stimulated the occurrence of
denitrication, which was apparent from the high nitrate reduction
rate in the treatment tank (Fig. 2C). N2O is an important intermediate
during denitrication (Hu et al., 2012). However, the addition of soluble
starch provided enough electron donors for denitrication, thus
avoiding the accumulation of N2O and consequently reducing N2O
emissions from denitrication (Hu et al., 2013b). It is worth noting
that at the end of the experiment, the control tank water contained
much higher NO3 concentrations than the treatment tank water. The
NO3 in the efuent may contribute to the N2O emissions of the sur-
rounding environments (Wang et al., 2009).
Soluble starchreduced thedaily N2O emissionsby 5.7 g CO2,eq, while
it increased the daily CO2emissions by 28.9 g, with respect to the con-
trol tank. Thus, the addition of soluble starch actually increased the
daily GHG emissions from the aquaculture system by 23.2 g CO2,eq,
or as much as 60.2% more than the control tank. In addition, higher aer-
ation wasneeded for thetreatment tank to maintain the DO concentra-tions similar to the control. At the end of the study period, the aeration
rate for the treatment tank was more than twice that for the control
tank.Taking higher energy consumptionfor aeration into consideration,
soluble starch addition had an overall adverse effect in controlling GHG
emissions from the aquaculture system.
Although the same FCR was obtained in treatment and control
tanks in this study, several studies reported that the uptake of
bioocs by aquatic animals could improve their production perfor-
mance (Avnimelech, 2007; Kuhn et al., 2009; Nootong et al., 2011). Fur-
ther studies using monosex species and lower TSS concentrations are
necessary to closely determine the economic and environmental im-
pacts of carbohydrate addition on minimizing GHG emissions from
aquaculture systems.
5. Conclusions
Solublestarchaddition hadsignicant effects on thenitrogen transfor-
mations and GHG emissions in a zero-water exchange intensive aquacul-
ture system. Lower TAN, NO2, and NO3
concentrations were obtained in
an aquaculture system withsoluble starch addition, which wasattributed
to theenhancement of heterotrophic bacterial growth and denitrication.
Theaddition of solublestarch enhanced denitrication and about76.2% of
the nitrogen input was emitted as gaseous nitrogen (i.e., N2and N2O)
in the treatment tank; which was much higher than what was observed
in the control tank (33.3%). Although soluble starch addition minimized
N2O emissions from the aquaculture system, it resulted in signicantly
higher CO2 emissions. Overall, the addition of soluble starch increased
the daily GHG emissions (as CO2 equivalent)from the aquaculturesystem
by 60.2%, indicating that it may have signicantadverse effects in control-
ling GHG emissions from commercial aquaculture systems.
Conict of interest
We declare that we have no nancial and personal relationships
with other people or organizations that can inappropriately inuence
our work, there is no professional or other personal interest of any nature
or kind in anyproduct,service and/or company that could be construedasinuencing thepositionpresentedin, or thereviewof, themanuscript en-
titled Inuence of carbohydrate addition on nitrogen transformations
and greenhouse gas emissions of intensive aquaculture system.
Acknowledgments
This work is being supported by National Research Foundation
Grantfundedby the KoreanGovernment (NRF-2011-220-D00071), Na-
tionalNatural Science Foundationof China (No.21307076), andSupple-
mental Research and Extension Grant from the College of Tropical
Agriculture and Human Resources (CTAHR), University of Hawaii at
Manoa (UHM), USA. We would like to thank Dr. Clyde Tamaru for assis-
tance with experiment design and Dr. Devin Takara for his comments
on the manuscript.
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