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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:khanal@hawaii.edu(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,

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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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