Accepted Manuscript

Effect of plant species on nitrogen recovery in aquaponics

Zhen Hu, Jae Woo Lee, Kartik Chandran, Sungpyo Kim, Ariane Coelho Brotto,
Samir Kumar Khanal

PII: S0960-8524(15)00024-3
DOI: http://dx.doi.org/10.1016/j.biortech.2015.01.013
Reference: BITE 14445

To appear in: Bioresource Technology

Received Date: 13 November 2014

Revised Date: 3 January 2015
Accepted Date: 6 January 2015

Please cite this article as: Hu, Z., Lee, J.W., Chandran, K., Kim, S., Brotto, A.C., Khanal, S.K., Effect of plant
species on nitrogen recovery in aquaponics, Bioresource Technology (2015), doi: http://dx.doi.org/10.1016/
j.biortech.2015.01.013

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Title:

Effect of plant species on nitrogen recovery in aquaponics

Authors:

Zhen Hu a, Jae Woo Leeb, Kartik Chandranc, Sungpyo Kimb, Ariane Coelho Brottoc,

Samir Kumar Khanald,*

Affiliations:

a
Shandong Key Laboratory of Water Pollution Control and Resource Reuse, School

of Environmental Science and Engineering, Shandong University, Jinan 250100,

China

b
Department of Environmental Engineering, College of Science and Technology,

Korea University, Sejong-ro 2511, Sejong 339-700, South Korea

c
Department of Earth and Environmental Engineering, Columbia University, 500

West 120th Street, New York, NY 10027, USA

d
Department of Molecular Biosciences and Bioengineering, University of Hawaii at

Manoa, Honolulu, HI 96822, USA

Corresponding authors:

Phone: 1-808-956-3812; Fax: 1-808-956-3542; E-mail: khanal@hawaii.edu

1
Abstract:

Nitrogen transformations in aquaponics with different edible plant species, i.e.,

tomato (Lycopersicon esculentum) and pak choi (Brassica campestris L. subsp.

chinensis) were systematically examined and compared. Results showed that nitrogen

utilization efficiencies (NUE) of tomato- and pak choi-based aquaponic systems were

41.3% and 34.4%, respectively. The abundance of nitrifying bacteria in tomato-based

aquaponics was 4.2-folds higher than that in pak choi-based aquaponics, primarily

due to its higher root surface area. In addition, tomato-based aquaponics had better

water quality than that of pak choi-based aquaponics. About 1.5% to 1.9% of nitrogen

input were emitted to atmosphere as nitrous oxide (N2O) in tomato- and pak

choi-based aquaponic systems, respectively, suggesting that aquaponics is a potential

anthropogenic source of N2O emission. Overall, this is the first intensive study that

examined the role plant species played in aquaponics, which could provide new

strategy in designing and operating an aquaponic system.

Keywords:

Aquaponics; Nitrogen transformations; Plant species; Nitrous oxide; Ammonia

oxidizing bacteria

2
1. Introduction

During 2000-2012, global aquaculture production expanded at an average annual rate

of 6.2%, with world aquaculture production of 66.7 million tons in 2012, outpacing

world population growth rate of 1.6%. It is estimated that aquaculture would account

for 62% of world’s fish supply for human consumption by 2030 (FAO, 2014). The

large-scale application of aquaculture, however, is restricted by land and water

utilization as well as by environmental concerns (Islam, 2005; Schwitzguébel and

Wang, 2007). Thus, the further expansion of aquaculture now depends on

development and application of new technologies to intensify fish cultivation while

maximizing water and nutrients reuse, and minimizing environmental impacts.

Aquaponics, which is an integrated system that links recirculating aquaculture with

hydroponic production, is considered to be an innovative and sustainable solution

(Tyson et al., 2011). A well-managed aquaponics could improve nutrient retention

efficiency, reduce water usage and waste (mostly nutrients) discharge to the

environment, and improve profitability by simultaneously producing two cash crops

(Diver, 2006; Tyson et al., 2011). It is generally believed that aquaponics, with

concomitant nutrients recovery, will become one of the widely accepted methods of

sustainable food production in the near future (Hu et al., 2012).

One major advantage of aquaponics is its high nitrogen utilization efficiency (NUE).

Nitrogen is a vital element for all living organisms, and protein-rich fish feed which is

the major source of nitrogen for fish cultivation, representing 50-70% of fish

3
production costs (Valente et al., 2011). In aquaculture system, only about 25% of the

nitrogen input is harvested through fish biomass, and over 70% is excreted into the

surrounding environment in the form of ammonia (Hargreaves, 1998). To maintain a

good water quality for fish growth, the fish cultivation water which is rich of nitrogen

compounds, has to be exchanged with fresh water at a recirculation rate of 5% to 10%

per day. This is a waste of natural resource. Furthermore, the nitrogen-rich effluent

which is discharged to the surrounding ecosystem may lead to eutrophication and

other environmental concerns. In aquaponics, an interesting nitrogen cycle is

established to avoid the above mentioned problems. The cycle begins with the

introduction of protein in fish feed, which is ingested by fish and then excreted to the

aqueous phase in the form of total ammonia nitrogen (TAN, i.e., NH3 and NH4+).

Ammonia is firstly oxidized to nitrite (NO2-) by ammonia oxidizing bacteria (AOB),

and then converted to nitrate (NO3-) by nitrite oxidizing bacteria (NOB, mainly

Nitrobacter spp. and Nitrospira spp.). Instead of being discharged to the surrounding

ecosystem, NO3- and residual TAN and NO2- could be taken up by plants. Thus, two

crops (plants and fish) are harvested from the system by utilizing nutrient in a

sustainable way. Although a wide variety of aquaponics designs have been examined

over the past several decades; most of the studies were focused on the improvement of

cash crop yield (Lennard and Leonard, 2006; Sace and Fitzsimmons, 2013). An

in-depth study on investigation of nitrogen transformations in aquaponics is still

lacking.

4
Nitrifying bacteria, which convert NH4+ to NO3- through nitrification, play an

important role in efficient operation of aquaponics. During the nitrification process,

nitrous oxide (N2O) emission also takes place (Stief et al., 2009; Beaulieu et al., 2011).

In addition, there exist anoxic zones where denitrification could occur. N2O is an

inevitable intermediate product of denitrification. N2O is considered to be an

important greenhouse gas with the global warming potential 296 times that of CO2

(IPCC, 2007). Evidences have shown that aquaculture could be a notable source of

anthropogenic N2O emissions (Hu et al., 2013). Compared with conventional

recirculating aquaculture system, Sfetcu et al. (2008) observed lower NH4+, NO2− and

NO3− concentrations in an aquaponic system, which might lead to lower N2O

emission (Ahn et al., 2010). More thorough study is needed to further examine this.

Both leafy plant (e.g., lettuce, spinach, and pak choi) and fruity plant (e.g., tomato and

cucumber) can be used in aquaponics (Lennard and Leonard, 2006; Sace and

Fitzsimmons, 2013). To date, the selection of plant species relies mainly on

experience, depending on stocking density of fish tanks and subsequent nutrient

concentrations of aquaculture effluent (Diver, 2006). Since different plants have

different growth characteristics and nitrogen utilization capacity, nitrogen

transformations in aquaponics can be directly influenced by plant species (Jin et al.,

2010). In addition, the growth of nitrifying bacteria requires sufficient surface area

where the nitrifies can develop a coating or shield of exopolymeric substances (EPS)

that protects them. Thus, plants with larger root surface area might have unique merit

5
in an aquaponic system. However, very limited studies have been conducted to

systematically investigate the effect of plant species on the performance of an

aquaponic system.

In this study, nitrogen transformations and N2O emissions in aquaponics were

investigated. Fish production, plant growth and water quality were measured and their

dependence on plant species were examined. Furthermore, molecular techniques were

employed to identity and quantify the abundance of microbes responsible for nitrogen

transformations in the aquaponics.

2. Materials and Methods

2.1 Experimental setup and operation

Experiments were conducted in the greenhouse at Mauka campus of University of

Hawaii at Manoa (21º18´22″N, 157º48´32″W). Two aquaponics were operated side by

side for nearly 5 months. For each aquaponics, 300-L oval plastic tanks filled with

200 L water, was operated as aquaculture unit. The tank was stocked with high

density (30 kg/m3) of tilapia fish (Oreochromis niloticus), which were obtained from

Windward Community College (Honolulu, Hawaii, USA) and had been cultivated in

conventional aquaculture system for 4 months prior to the start of the experiment.

Fish were fed once per day and the feeding amount was determined based on fish

response, according to the method described in Casillas-Hernández et al. (2006). Air

pump was used to provide sufficient oxygen for fish growth by aerating the tank water

6
and dissolved oxygen (DO) concentrations were maintained above 5 mg/L. The tank

was covered with wooden board to prevent algal growth.

Peristaltic pump was used to pump the fish tank water to a clarifier, which was built

using 20-L sealed bucket filled with biomedia (Kaldnes @ media, Aquatic

Eco-System, Apopka, FL, USA). The clarifier captured majority of the suspended

solids from aquaculture tank to protect the plant roots in the grow bed. After passing

through the clarifier, tank water flowed into the grow bed. Floating raft hydroponic

system was employed in this study. Rectangle plastic tank with an effective volume of

400 L was used as the grow bed and plants were held up by a foam raft that floats on

the water. Shade cloth (50%) was installed over the grow bed to prevent excessive sun

exposure to the plants. Two different plant species, tomato (Lycopersicon esculentum),

a fruity plant and pak choi (Brassica campestris L. subsp. chinensis), a leafy plant,

were used simultaneously in different aquaponics. The plant seeds were germinated

according to method in Ako and Baker (2009). After two weeks, healthy plant

seedlings were transplanted to the grow bed at their optimum planting density as per

the seed supplier’s guidelines. The plant density of tomato and pak choi were 13 and

20 plants/m2, respectively. Nutrients present in aquaculture effluent were absorbed by

plants in grow bed and the reclaimed water was then recirculated into the fish tank.

For schematic diagram of the aquaponics, please refer to Fig. S1 (See Supporting

Information).

7
The water was recirculated continuously between fish tank and grow bed in

aquaponics and no water exchange was conducted during the study period except for

replenishing evapotranspiration losses. Calcium hydroxide and potassium hydroxide

were periodically dosed into the aquaculture tank to maintain the pH at a neutral range.

Since fish water is deficient in iron, Fe-EDTA was added to the system on a weekly

basis to maintain Fe2+ concentration above 2 mg/L (Rakocy et al., 2006).

2.2 Sampling and analytical methods

Water samples were obtained from fish tank every other day after feeding, and were

analyzed immediately for TAN, NO2−, and NO3- concentrations, using HACH reaction

kits (Loveland, CO, USA), namely Ammonia TNTplus (TNT 830), Nitrite TNTplus

(TNT 839), and Nitrate TNTplus (TNT 836), respectively. DO concentrations,

temperature, and pH were measured in situ daily using the HQ40d Portable Water

Quality Lab Package (HACH, Loveland, CO, USA). The total suspended solids (TSS)

concentration was determined according to the Standard Methods (APHA, 2005).

Daily fish feed consumption was recorded; fish and plant biomass increase were

accurately weighed using analytical balance. At the end of the experiment, samples of

fish, plants, fish feed, and suspended solids (microbial biomass) were dried, and their

total nitrogen contents were determined by using LECO TruSpec C/N analyzer

(LECO Corp., St. Joseph, MI, USA). Plant root surface area was determined using

WinRHIZO root-scanning methods (WinRhizo Pro v.2005b, Régent Instruments,

Québec, Canada) (Pang et al., 2011).

8
Diurnal variations of N2O emission from aquaculture tank was measured every other

week after stable nitrite concentration was achieved. Clark type electrode N2O sensor

(Unisense, Aarhus N, Denmark) was used to monitor dissolved N2O concentrations in

aqueous phase. Nitrous oxide concentration in the gas phase was then calculated

based on dissolved N2O concentrations, as described in Hu et al. (2014).

In this study, clarifier was fabricated of sealed bucket (airtight, without aeration) and

grow bed was closely covered with foam raft (not airtight, without aeration), and only

fish tank had aeration facilities. Considering the small water fluctuations in grow bed

(hydraulic loading rate of 4.3 cm/h) and solubility of N2O in water, it is believed that

most of the N2O emissions from aquaponics would likely have occurred in the fish

tank.

2.3 Microbial analyses

At the end of the study period, water and plant root samples were taken from

aquaponics, centrifuged and then kept at -20 °C for microbial analyses, which were

conducted in Kartik Chandran Laboratory at Columbia University. DNeasy® plant

mini kit (Qiagen, Valencia, CA, USA) was used to extract DNA, and resulting DNA

concentrations and quality were measured by NanoDrop Lite Spectrophotometer

(Thermo Fisher Scientific, Waltham, MA, USA). The abundance of AOB was

quantified via SYBR® Green chemistry quantitative polymerase chain reaction

9
(qPCR) assays targeting ammonia monooxygenase subunit A (amoA), using amoA-1F

and amoA-2R as target primers (Rotthauwe et al., 1997). Since no general molecular

probe targeting all known NOB is currently available, we chose to target and quantify

the two genera Nitrobacter and Nitrospira, which are assumed to be the major players

in nitrite oxidation (Cébron and Garnier, 2005). Nitro-1198f and Nitro-1423r were

used as target primers for Nitrobacter, while NTSPAf and NTSPAr were used as

target primers for Nitrospira (Kindaichi et al., 2006; Graham et al., 2007). qPCR

assays were conducted in triplicate on iQ5 real-time PCR thermal cycler (Bio-Rad

Laboratories, Hercules, CA. USA). Standard curves for qPCR were generated via

serial decimal dilutions of plasmid DNA and primer specificity and the absence of

primer-dimers were confirmed via melt curve analysis performed for each and every

qPCR assay conducted. qPCR data were analyzed with iCycler iQ™ software

(Bio-Rad Laboratories, Hercules, CA, USA).

2.4. Statistical analysis

Data were presented as a mean of three replicates. Since it is essential to compare

whether different forms of nitrogen are statistically significant, the results were

statistically analyzed using SPSS v16.0 for Windows software (SPSS v16.0 IBM

Corporation, Somers, NY, USA). The statistical differences were determined using a

one-way analysis of variance (ANOVA) followed by a Tukey’s comparison test at

p<0.05.

10
3. Results and Discussion

3.1 Aquaponics performance

During the study period, no fish mortality or plant disease was observed. One batch of

tomato, and three batches of pak choi were harvested, because of the short growth

period of pak choi. Table 1 shows the performance of tomato and pak choi in

aquaponics. More fish feed consumption and higher fish biomass increase were

obtained in tomato-based aquaponics, mainly because of better water quality

(discussed in section 3.2). The feed conversion ratio (FCR) of tomato- and pak

choi-based aquaponics were 1.6 and 2.0, respectively, both within the range of

conventional aquaculture system (i.e., 1-3) (Naylor et al., 2000). Both aquaponics

successfully achieved simultaneous production of two cash crops, i.e., fish and

vegetable.

It is worth noting that a large amount of fresh water was used to replenish the water

losses, which were mainly due to plant evapotranspiration. Higher water

replenishment of tomato-based aquaponics was resulted from its larger leaf surface

exposed to air. In recirculating aquaculture system (RAS); daily exchange of 5-10%

with fresh water is part of the operation routine, although some RAS have been

operated successfully without water exchange in recent years (Hu et al., 2014). With

daily water exchange of 5% as per the present study (i.e., 200 L fish tank), 1400 liters

of fresh water would be needed to run a RAS continuously for 139 days. Thus,

aquaponics doesn’t seem to have obvious advantage in fresh water conservation,

11
especially for plants with large exposed leaf surface.

3.2 Nitrogen transformations in aquaponics

DO concentrations, pH and temperatures were measured daily immediately after fish

were fed, and the results are presented in Table 2. Diurnal variation of DO, pH and

temperature were also monitored and are illustrated in Fig. S2 (See Supporting

Information). Similar DO concentrations, pH and temperatures were maintained in

both aquaponics.

Fig. 1 shows the variation of nitrogen compounds concentrations during the study

period. The study period was composed of three phases, and each phase represents a

batch of pak choi. Accumulation of TAN, followed by NO2- and NO3- was observed

right after fish stocking. In both aquaponics, the highest TAN and NO2- concentrations

were detected around day 7 and 28, respectively. This was explained by the slow

growth of nitrifying bacteria. In aquaponics, nitrogen was introduced to the system

daily through protein in fish feed and was excreted to aquaculture water in the form of

ammonia after metabolized by fish. Ammonia can be oxidized subsequently to NO2-

and NO3- through nitrification. During the startup period of aquaponics, there were

not enough nitrifying bacteria in the system to remove all of the TAN produced by

fish, and accumulation of TAN was observed. With the growth of AOB, the

accumulated TAN was gradually oxidized to NO2-. The accumulation of NO2- was

attributed to the different growth rates between AOB and NOB. The population of

12
AOB increased faster than that of NOB when temperature was above 25 °C

(Yamamoto et al., 2008).

After phase Ⅰ, significant difference in TAN concentrations was observed between

tomato- and pak choi-based aquaponics. In pak choi-based aquaponics, plant root had

to be removed after each harvest to transplant the new seedlings. The removal of plant

root would cause the loss of nitrifying bacteria in the system. Microbial analysis

(Section 3.3) showed that most of the nitrifying bacteria were attached on plant root

surface. Thus, increase and decrease of TAN concentrations were observed repeatedly

during both phase Ⅱ and Ⅲ after each root removal. In tomato-based aquaponics,

with only one harvest at the end of the experiment and there was no disturbance to the

growth of nitrifying bacteria. Thus, perpetual decrease in TAN concentrations was

observed. Interestingly, the NO2- concentrations were maintained below 4 mg/L and

no significant fluctuation was observed after phaseⅠin both aquaponics. This is

because AOB were responsible for the rate-limiting step of nitrification (Kowalchuk

and Stephen, 2001). The disturbance caused by root removal had more significant

influence on removal of TAN compared to NO2-.

Another noteworthy phenomenon is that NO3- concentrations were consistently low

during the study period. A 200-L recirculating aquaculture system stocked with same

fish density as the aquaponics was operated without water exchange during the study

period. NO3- concentration in the RAS increased linearly and quickly reached over

13
100 mg/L in one month (See Supporting Information, Fig. S3). This indicated the

important role of plant in avoiding NO3- accumulation in aquaculture tank. The

fluctuation of NO3- concentration during phase Ⅱ and Ⅲ in pak choi-based

aquaponics was attributed to the growth period of pak choi. At the beginning of each

phase, the newly transplanted pak choi had low N absorption ability, and the NO3-

formation rate by nitrifying bacteria outpaced the NO3- uptake rate of plants. With the

growth of pak choi, the plant uptake rate increased and decrease of NO3-

concentration in the aquaculture tank was observed. Tomato-based aquaponics had

more plant biomass (Table 1), indicating higher plant uptake rate, resulting in lower

NO3- concentrations. The increase of NO3- concentrations in tomato aquaponic at the

later part of the experiment was attributed to the decrease of NO3- uptake rate was

caused by the maturation of tomato.

Overall, significant lower TAN and NO3- concentrations were observed in

tomato-based aquaponics during most of the study period. Better water quality in

tomato aquaponics resulted in better fish performance. This also explains the higher

feed consumption and lower FCR obtained in tomato aquaponics (Table 1).

The nitrogen mass balance analysis of tomato- and pak choi-based aquaponics

throughout the study period was conducted and the findings are presented in Fig. 2.

Fish feed was the major nitrogen source in both aquaponics, accounting for over 99.5%

of nitrogen input. Fresh water replenishment only accounted for no more than 0.5% of

14
nitrogen input in both aquaponics. About 24.3% and 19.4% of nitrogen input were

recovered as fish biomass in tomato- and pak choi-based aquaponics, respectively.

This difference was the result of different FCR between the two aquaponics, as

elucidated in Table 1. About 17.0% and 15.0% of nitrogen input were retained by

tomato and pak choi biomass, respectively. Thus, the total NUE of tomato- and pak

choi-based aquaponics were 41.3% and 34.4%, respectively. These values are higher

than that of aquaculture system, with an average NUE of 25.0%, indicating that

aquaponics could significantly improve NUE and avoid the loss of nutrients in

conventional aquaculture system (Hargreaves, 1998; Hu et al., 2012). However,

according to the data from Food and Agriculture Organization (FAO), on a global

scale, the recovery of fertilizer N through crop production is around 50%. Further

research is needed to improve the NUE of aquaponics.

Gaseous nitrogen losses (NH3, N2O and N2) accounted for 51.2% and 57.3% of total

N output in tomato- and pak choi- based aquaponics, respectively. pH was maintained

around neutral in both aquaponics (Table 1), and most of the TAN was in ionized form

(i.e., NH4+), so ammonia volatilization was likely to be negligible. Nitrous oxide

emissions from aquaponics were also considered to be insignificant compared with N2

emission (as shown in section 3.4). Thus, most of the nitrogen losses to the

surrounding atmosphere were in the form of N2, which was the end-product of

denitrification. Hu et al. (2014) reported nitrogen loss of 32.0% of nitrogen input,

while Thakur and Lin (2003) claimed that gaseous nitrogen losses accounted for 5.2

15
to 36.0% of nitrogen input in an intensive aquaculture system. Compared with

aquaculture system, higher nitrogen losses were observed in aquaponics, mainly

because more anoxic zone existed in aquaponics. The component ratio (i.e., the

effective volume of fish tank to grow bed) of aquaponics was commonly designed as

1:2 (Diver, 2006). Since aeration was only required in fish tank, significant part of

grow bed could have pockets of anoxic zone that provided favorable conditions for

the occurrence of denitrification.

3.3 Effect of plant species on abundance of nitrifying bacteria

To elucidate the significant difference in TAN concentrations between tomato and pak

choi aquaponics, abundance of AOB, indicated by amoA gene copy numbers, were

detected by using qPCR, and the results are presented in Table 3. The abundance of

AOB in tomato-based aquaponics were 3.97±1.18×1011 copies, exceeded that of pak

choi-based aquaponics by 4.4 folds. The abundance of nitrifying bacteria is correlated

with the potential nitrifying activities. More AOB led to higher ammonia oxidation

rate, thereby resulting in lower TAN concentrations in tomato-based aquaponics.

The abundance of NOB were also detected (Table 3) and results showed that the

abundance of NOB in tomato-based aquaponics was about 4.4 times higher than that

in pak choi-based aquaponics. The predominant NOB species were Nitrobacter spp.

This is because Nitrobacter spp. are fast-growing r-strategist. Under high NO2-

concentration, they grow faster than Nitrospira spp., which are K-strategists

16
(Matsumoto et al., 2010). During the startup period, high accumulation of NO2-

occurred in both aquaponics, and Nitrospira spp. could out-compete Nitrospira spp.

Even though the NO2- concentrations decreased to relatively low values during phase

Ⅱ and Ⅲ, the dominance of Nitrobacter spp. over Nitrospira spp. could not be

reverted. Similar irreversible process was also observed in wastewater treatment

system (Nogueira and Melo, 2006).

About 99.8% of AOB and 99.9% of NOB were attached to root surface in

tomato-based aquaponics, while about 96.3% of AOB and 75.4% of NOB were

attached to root surface in pak choi-based aquaponics. The microbial analyses were

conducted at the end of the study period, when tomato-based aquaponic had been

operated continuously for 139 days (one batch) and pak choi-based aquaponics had

completed three batches, due to the different growth characteristic between tomato

and pak choi. Plant root were removed from the system at the end of each batch. It

seems that NOB is more tolerated to the disturbance of root removal than AOB. This

explained the variation of TAN concentrations and relative stable NO2- concentrations

during phase Ⅱ and Ⅲ in pak choi-based aquaponics (Fig. 1).

A large surface area is optimum for the growth of nitrifying bacteria. At the end of the

study period, root surfaces area of both tomato- and pak choi-based aquaponics were

analyzed. The total root surface area of tomato was 830.2±108.9 m2, while pak choi

had a total root surface area of 131.3±19.2 m2. With longer growth period, the root

17
surface area of tomato was nearly 6.3 folds higher than that of pak choi. The large

surface area facilitated the growth of nitrifying bacteria.

3.4 N2O emissions from aquaponics

Monitoring of N2O emissions from aquaponics were conducted during phase Ⅱ and

Ⅲ after stable NO2- concentrations were reached. Fig. 3 shows the diurnal variation

of dissolved N2O concentrations in tomato and pak choi aquaponics. The

time-weighted N2O-N emissions from aquaponics are listed in Table 4.

About 1.5% and 1.9% of nitrogen input were emitted to the atmosphere as N2O in

tomato- and pak choi-based aquaponics, respectively. Previous research showed that

N2O conversion ratio of intensive aquaculture system was about 1.3% (Hu et al.,

2013). Higher N2O conversion ratios were observed, mainly because of higher fish

stocking density in the present study. In our previous research on intensive

aquaculture systems, fish stocking density was around 20 kg/m3, while in present

study fish stocking density of 30 kg/m3 was used and this value eventually reached

over 50 kg/m3 at the end of the experiment (Hu et al., 2013). Higher fish density led to

more TAN excretion, which explained the higher TAN concentrations in the present

aquaponics. It is believed that nitrification plays an important role in N2O emissions

from fresh water system (Ni et al., 2013). High TAN concentration could increase the

nitrification-driven N2O emission. Overall, the present results showed that aquaponics

could be an important source of anthropogenic N2O emission, and further study is

18
needed to verify whether effective approaches exist to minimize N2O emissions from

aquaponics.

The variations of water quality parameters (i.e., DO, TAN, NO2-, NO3- concentrations,

pH, and temperature) were also monitored during N2O measurement. Results showed

that dissolved N2O concentrations were significantly correlated with NH4+ and NO2-

concentrations (data not shown) and negatively correlated with DO concentrations (r

=-0.603, p < 0.05). This is consistent with our previous research on aquaculture

system, and indicated that nitrification plays an important role in N2O emissions from

aquaponics (Hu et al., 2013).

4. Conclusions

Nitrogen uptake by plants played an important role in avoiding the accumulation of

NO3- in aquaponics. The present study showed that plant species had significant

influence on nitrogen transformations in aquaponics. Higher NUE were obtained in

tomato-based aquaponics, due to its higher abundance of nitrifying bacteria, which

was associated with its higher root surface area. The N2O conversion ratios of tomato-

and pak choi-based aquaponics were 1.5% and 1.9%, respectively, suggesting that

aquaponics could also be an important source of anthropogenic N2O emission. Further

research is necessary to improve NUE and minimize N2O emissions of aquaponics.

19
Acknowledgements

This work is being supported by National Research Foundation Grant funded by the

Korean Government (NRF-2011-220-D00071), National Natural Science Foundation

of China (No. 21307076), Supplemental Research and Extension Grant from the

College of Tropical Agriculture and Human Resources (CTAHR), University of

Hawaii at Manoa (UHM), USA, and Fundamental Research Funds of Shandong

University (No. 2014TB003). We would like to thanks Dr. Clyde Tamaru for

providing the fish, Dr. Travis Idol for maintaining the greenhouse, and Dr. Hye-Ji Kim

for helping us with root analysis. Without their support, this study would not have

been possible.

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Figure captions

Fig. 1 Variation of TAN, Nitrite, and Nitrate concentrations during the study period.

(A) TAN; (B) Nitrite; (C) Nitrate

Fig. 2 Nitrogen distributions in different aquaponics.

Fig. 3 Diurnal variation of dissolved N2O and DO concentrations in aquaponics. (TA:

tomato-based aquaponics; PA: pak choi-based aquaponics)

Fig. S1 Schematic diagram of experiment setup.

Fig. S2 Diurnal variation of DO, pH and temperature in tomato- and pak choi-based

aquaponics.

Fig. S3 Variation of NO3- concentration in recirculating aquaculture system.

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 Table 1 Performance of different aquaponics
Tomato-based Pak Choi-based
Parameters*
aquaponics aquaponics
Water replenishment (L) 1863.2 569.5
Fish feed consumption (g) 6950.0 6582.4
Fish biomass increase (g) 4345.0 3285.6
Feed conversion ratio (FCR) 1.6 2.0
Saleable part (g) 13858.2 24897.4
Plant yield**
Unsaleable part (g) 21194.4 1429.7
*
Fish feed consumption is the dry weigh, while fish biomass increase and plant yield is the wet
weight.
**
The saleable parts of tomato and pak choi are fruit and leaf, respectively.

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 Table 2 Physical parameters of water in aquaponics.
Parameters Tomato-based aquaponics Pak Choi-based aquaponics
DO concentration (mg/L) 5.6±0.6 5.4±0.6
pH 7.1±0.5 7.2±0.6
Temperature (ºC) 26.0±0.9 26.2±1.0

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 Table 3 Abundance of nitrifying bacteria in different aquaponics (unit: copies)
Tomato-based aquaponic Pak Choi-based aquaponics
Nitrifying bacteria
Root surface Water Root surface Water
AOB 3.97±1.18×1011 6.85±0.85×108 8.67±0.71×1010 3.26±0.79×109

Nitrobacter
4.13±0.27×1012 4.49±1.57×108 7.39±0.26×1011 2.47±0.05×1011
spp.
NOB
Nitrospira
1.31±0.06×1011 3.66±0.65×108 3.59±0.15×1010 4.55±0.08×109
spp.

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 Table 4 N2O emissions from different aquaponics.
Parameters Tomato-based aquaponics Pak Choi-based aquaponics
N2O-N emission (mgN/d) 58.3±14.9 72.5±13.2
Fish feed (g/d) 60.6±1.5 59.3±2.3
N2O conversion ratio (%)* 1.5 1.9
*
N2O conversion ratio = (N2O-N emission / (Fish feed × nitrogen content of fish feed)) × 100, and
the nitrogen content of fish feed in 6.5±0.3%.

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Highlights

Plant species had significant influence on nitrogen transformations in aquaponics.

Aquaponics could be important anthropogenic N2 O emission source.

Tomato-based aquaponics showed higher NUE than pak choi-based aquaponics,

mainly attributed to its higher abundance of nitrifying bacteria associated with

higher root surface area.

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