Nitrogen Transformations

in Aquaponic SystemsSamir Kumar Khanal, University of Hawaii at ManoaKartik Chandran, Columbia UniversityClyde Tamaru, University of Hawaii at ManoaHye-Ji Kim, Purdue University

USDA-AFRI PD Meeting, Washington, D.C., October 12-13, 2016

What is aquaponics?Aquaponics is a soilless system in which recirculating aquaculture is integrated with a hydroponic system.

N2O

3

Research objectives

1. Quantify the impact of physical and chemical variables on nitrogen transformations in an aquaponic system.

2. Evaluate the transformations of different forms of nitrogen in an aquaponic system under different conditions.

3. Examine the ecology of functionally important living species and assess microbial contributions to nitrogen transformations in an aquaponic system.

4. Investigate the greenhouse gases emissions from an aquaponic system, with particular emphasis on nitrous oxide (N2O) emission.

4

Pak choi Lettuce

Tilapia

5

Tomato Chive

2-stage biofilter (20 L)

Aquaponic system

pH was controlled in a range of 6.8-7.2 by adding Ca(OH)2 and KOH solution.

Two -stage biofilterGrow bed

Fish tankAir

supply

Feed

Down-flow with partial

aeration

Up-flow

Sediment

Fish tank (330 L)

Grow bed (300 L)

DO in fish tank was about 6-7 mg/L.

7

0.004.00

8.0012.00

16.0021.00

25.0029.00

33.0037.00

2

4

6

8HLR 1.0 m/d

Fish tankBiofilters (outlet)Grow bed (outlet)

Day

DO (m

g/L)

0.004.00

8.0012.00

16.0021.00

25.0029.00

33.0037.00

2

4

6

8HLR 1.5 m/d

Fish tankBiofilters (outlet)Grow bed (outlet)

Day

DO (m

g/L)

0 7 14 21 28 352

4

6

8HLR 2.0 m/d

Fish tankBiofilters (outlet)Grow bed (outlet)

Days

DO (m

g/L)

0 7 14 21 28 352

4

6

8HLR 2.5m/d

Fish tankBiofilters (outlet)Grow bed (outlet)

DaysDO

(mg/

L)

DO dropped significantly in biofilters, due to sediment accumulations

0 7 14 21 28 352345678

HLR 2.0 m/d

Fish tankBiofilters (outlet)Grow bed (outlet)

Days

DO (m

g/L)

0 7 14 21 28 352

4

6

8

HLR 1.5 m/d

Fish tankBiofilters (outlet)Grow bed (outlet)

Days

DO (m

g/L)

Significantly different (Growbed outlet)

Not significantly different

Not significantly different

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Feed Fish

Fece

s

Sedi

men

t

NO3-

Wat

er

Root

s

NO3-

Root

s

Stem

s

NO3-

Stem

s

Leav

es

NO3-

Leav

es

Frui

ts

NO3-

Frui

ts0.05.0

10.015.020.025.030.035.0

Pak choiLettuceChiveTomato

δ15N

(‰)

Natural abundance δ15N of bulk nitrogen and NO3-

Σ (N x δ15N)before = Σ (N x δ15N)after

- Assimilation and redox pathways were identified by the isotopic fractionations. However, The results were isotopically impossible. There should be at least one output that has δ15N below the δ15N value of the feed (< 5.2 ‰) (Robinson, 2001).

- Gaseous nitrogen loss via denitrification caused the δ15N of gas loss below 5.2 ‰.

fN.Mf = (CTAN + CNO2-N + CNO3-N + Corg-N)V + Nveg/T + Nfish/T + Nsed/T + Nloss/TMass balance:

Isotopic mass balance:

0 3 6 9 1217.017.518.018.519.019.520.020.5

Days

δ15N

(‰)

DO affected denitrification and nitrogen loss via denitrification

Low DO

High DO

0 3 6 9 1215.016.017.018.019.020.021.022.023.0

Days

δ15N

(‰)

Actual

No feeding

No denitrification

Note:1. Denitrification caused the enrichment in 15N and increase in δ15N of nitrate.2. Feed lowered δ15N of nitrate during denitrification , but denitrification was identified.

ParametersConditions

Feed 25g/d Feed 20 g/d Feed 15 g/d1.5 m/d 1.0 m/d 1.5 m/d 0.25 m/d 1.5 m/d 0.5 m/d

TKN (mgN/L) 8.2 (2.9)* 10.6 (2.7) 9.4 (0.8) 8.9 (0.9) 10.3 (1.4) 11.5 (0.9)

TAN (mgN/L) 0.69 (0.37)

1.10 (0.29)

0.52 (0.16)

0.59 (0.15)

0.69 (0.09)

0.89 (0.10)

NO2- (mgN/L) 0.155

(0.061)0.255

(0.087)0.303

(0.052)+0.616

(0.161)+0.227

(0.062)0.284

(0.054)NO3

-

accumulation rate

(mgN/L/d)

0.723 (0.129)

0.534 (0.074)

0.814 (0.011)

0.996 (0.173)

0.905 (0.080)

0.897 (0.056)

COD (mg/L) 89.0 (8.5) 91.3 (6.4) 58.6 (4.0) 65.2 (4.5) 61.6 (4.3) 64.2 (4.2)

This is mean value of 15 samples; *represents standard deviation and + represents significant difference. Statistical analyses of the collected data were carried out using an analysis of variance (one-way ANOVA) at a confidence level of α = 0.05

Nitrite oxidation rate dropped at low HLR (below 0.25 m/d), but ammonia oxidation was still active

HLR (< 0.1 m/d) decreased TAN oxidation rates

HLR (< 0.25 m/d) decreased nitrite oxidation rates

0 7 14 21 280.0

0.1

0.2

0.3

0.1 m/d1.5 m/d

Days

NO

2- (m

g N

/L)

0 7 14 21 280.0

0.2

0.4

0.6

0.25 m/d1.5 m/d

Days

NO

2- (m

g N

/L)

0 7 14 21 280.00.20.40.60.81.01.21.41.61.8

0.1 m/d1.5 m/d

Days

TAN

(mg

N/L

)

0 7 14 21 280.00.20.40.60.81.01.21.41.61.8

0.25 m/d1.5 m/d

Days

TAN

(mg

N/L

)

Not significant differentSignificant different

Significant different Significant different

pH (<6.0) decreased TAN oxidation rates. pH 5.2 inhibited TAN oxidation

At low pH (<6.0), decrease in TAN oxidation rate caused the accumulation of TAN and lowered nitrite substrate in nitrite oxidation. This does not mean nitrite oxidation was improved at low pH.

0 7 14 21 28 350

1

1

pH 5.2pH 6.8

Days

TAN

(mg

N/L

)

0 7 14 21 28 350123456

pH 6.0pH 6.8

Days

TAN

(mg

N/L

)

0 7 14 21 28 350.0

0.1

0.2

0.3

0.4

pH 5.2pH 6.8

Days

NO

2- (m

g N

/L)

0 7 14 21 28 350.0

0.1

0.2

0.3

0.4

pH 6.0pH 6.8

Days

NO

2- (m

g N

/L)

Significant different

Significant differentSignificant different

Significant different

0 5 10 15 20 25 30 35 40130

140

150

160

170

180

Days

NO

3- (m

g N

/L)

weekly drained

monthly drained

Sediment draining improved NO3- accumulation and reduced N loss in aquaponics

0 15 30 45 60 75 900

50

100

150

200

ChiveLettucePak choiTomato

Days

Nitr

ate

conc

entr

ation

(m

gN/L

)

Nitrate accumulation/consumption for different plant species in aquaponicsTwo -stage biofilterGrow bed

Fish tankAir

supply

Feed

Down-flow with partial

aeration

Up-flow

Sediment

Root system

Root surface area Pak choi Lettuce Chive Tomato

cm2/plant 724(251)

474(109)

227(104)

6.01 x 104

(1.71 x 104)

This is mean value of 24 samples; *represents standard deviation

Pak choi Lettuce Chive Tomato

15

First month (NO3- accumulation)

16

Third month (NO3- depletion)

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Nitrite Oxidation

Nitrogen loss (denitrification)

Nitrate accumulation& depletion rate

DO

Plant NUE

Feed consumption

Feed Feed DO

DO ≤ 3.5 mg/L

Balance input and output

HLR ≤ 0.25 m/d

DO ≤ 3 mg/L

TANOxidation

TANAccumulation

pH ≤ 6.0pH ≤ 5.2

Redu

ced

N lo

ss

HLR ≤0.1 m/d

Balance between input and outputs

Plant species

Tomato > Lettuce & Pak choi > Chive

pH DrainingHLR

Objective #1: Impact of physical and chemical variables

Increase nitrate

accumulation

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Pak choi Lettuce Chive Tomato Input Input20%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Nitrate inputFish feedN lossPlant biomassNitrate accumulatedSedimentFish biomass

Contribution of nitrogen products by mass balance in aquaponic systems, operated at HLR of 1.5 m/d, feeding rate of 35 g/d and high DO (~7 mg/L).

- NO3- accumulation served as nitrogen output (NO3

- uptake rate < NO3- generation rate) and

nitrogen input (NO3- uptake rate > NO3

- generation rate).- Nitrogen loss was found in aquaponic systems.

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TAN Nitrite

NitrateN2O & N2

(N loss)

Org N in fish

Org N in sediment

Org N in plants

Fish

Nitrate accumulation

Feed

DO

Plant species

pH

HLR

Objective #2 Transformations of different forms of nitrogen

Output

Parameters affecting N transformations

Other N forms

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Feed Fish

Fece

s

Sedi

men

t

NO3-

Wat

er

Root

s

NO3-

Root

s

Stem

s

NO3-

Stem

s

Leav

es

NO3-

Leav

es

Frui

ts

NO3-

Frui

ts0.05.0

10.015.020.025.030.035.0

Pak choiLettuceChiveTomato

δ15N

(‰)

3. Ecology of functionally important living species

• Nitrate reductase could occur in the plant organs and the nitrate reduction occurs after the translocation from recirculating water to leaves (Black et al., 2002).

• High efflux of NO3- occurred in the root zone of plants, and NO3

- concentration exceeded the plant requirements (Evans, 2001); resulting to the accumulation of NO3

-.

• This nitrate in recirculating water could subsequently cause the high nitrogen loss via denitrification by nitrifiers (Ryabenko, 2013).

• Microbial results are still required to support the ecology and microbial contributions to nitrogen transformations.

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Nitrifying bacteria

Types of rootPak choi Lettuce Chive Tomato

EU bacteria 7.57 x 109 2.91 x 1011 1.96 x 1010 2.32 x 1010

Nitrobacter spp. 7.02 x 106 4.09 x 106 6.88 x 106 8.84 x 106

Nitrospira spp. 1.16 x 109 7.74 x 108 9.70 x 108 3.22 x 109

Abundances of bacteria in different root systems(Condition: HLR 1.5 m/d and pH 6.8)

(unit: copies).

High relative abundances of EU bacteria over Nitrobacter spp. and Nitrospira spp. suggested the high abundance of denitrifiers, which contributed to nitrogen loss in aquaponic systems.

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Nitrifying bacteria

Tomato-based aquaponics Pak choi-based aquaponicsRoot surface Water Root surface Water

AOB 3.97 ± 1.18x 1011

6.85 ± 0.85x 108

8.67 ± 0.71x 1010

3.26 ± 0.79x 109

NOBNitrobacter spp.

4.13 ± 0.27x 1012

4.49 ± 1.57x 108

7.39 ± 0.26x 1011

2.47 ± 0.05x 1011

Nitrospira spp. 1.31 ± 0.06x 1011

3.66 ± 0.65x 108

3.59 ± 0.15x 1010

4.55 ± 0.08x 109

Abundance of nitrifying bacteria in different aquaponics

(unit: copies)

Higher NUE were obtained in tomato-based aquaponics, due to higher abundance of nitrifying bacteria on root surface.

(Hu et al., 2015)

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4. Nitrous oxide (N2O) emission from fish tanks

Aquaponic types N2O emission (mgN/d)

N2O conversion (%) References

Tomato-based aquaponics 58.3 (14.9) 1.5 (Hu et al., 2015)

Pak choi-based aquaponics 72.5 (13.2) 1.9 (Hu et al., 2015)

Chive-based aquaponics 29.6 (0.4) 1.2 Our study

Tomato-based aquaponics 17.2 (7.7) 0.7 Our study

Aquaponics without plants 11.9 (10.2) 0.5 Our study

We found that higher N2O emission from biofilters than that in the fish tanks.

* denotes standard deviation

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Summary• The NO3

- accumulation in recirculating water occurred when NO3- exceeded

the amount that the plants could utilize. NO3- depletion suggested the

insufficient nitrogen input.

• When the NO3- accumulation occurs, reducing the feeding rate can increase

NUE and decrease the denitrification in the systems.

• The growth of plants was dependent on HLR of 0.25 to 2.5 m/d; however, NO2

- and TAN oxidizing rates were significantly dropped at HLR of 0.25 m/d and 0.10 m/d, respectively.

• Low pH (< 5.2) inhibited ammonia oxidation, leading to TAN accumulation.

• The nitrogen mass balance and the isotopic mass balance suggested that denitrification, affected by DO at the inlet of biofilters, was the major factor of nitrogen loss in the floating-raft aquaponic systems.

• To reduce the nitrogen loss in aquaponic systems, higher rate of sediment draining and higher plant-to-fish ratio are recommended.

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On-going research

3. Examine the ecology of functionally important living species and assess microbial contributions to nitrogen transformations in an aquaponic system

4. Investigate the greenhouse gases emissions from an aquaponic system, with particular emphasis on nitrous oxide (N2O) emission

• Quantitative polymerase chain reaction (qPCR) targeting:

• Eubacteria, ammonia monooxygenase subunit A (amoA), Anaerobic ammonia oxidizing bacteria (AMX), Nitrospira spp., Nitrospira spp. and Nitrobacter spp.

• 16S rRNA

• Measurement of N2O emissions from aquaponic systems

• Development of strategies to minimize N2O emission

2. Evaluate the transformations of different forms of nitrogen in an aquaponic system under different conditions

• Labeling isotope study using ammonium 15N sulfate

Student training/Extension/Dissemination activities • One Ph.D., one undergraduate and three high school students

have been trained.• Aquaponic facility tour for farmers, students from Environ.

Science, Nagasaki University (Japan) and staffs from Kapiolani Community College (Hawaii).

Symposium:Wongkiew, S. and Khanal. S.K. “Nitrogen transformations in floating-raft aquaponic systems”, Poster presentation, 28th Annual CTAHR Symposium, University of Hawaii at Manoa, April 8th, 2016. BEST POSTER AWARD.Publications: • Wongkiew, S., Hu, Z., Chandran, K., Lee, J.W., and Khanal, S.K.

Nitrogen transformations in aquaponic systems: A review. Aquacultural Engineering (submitted).

• Wongkiew, S., Popp, B.N., Kim, H.J., and Khanal, S.K. Nitrogen transformations in aquaponics: evaluation of physical and chemical factors (ready for submission).

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Acknowledgments

• Ryan Kurasaki• Bradley Kai Fox, Mari’s Gardens

• This project is being supported by Agriculture and Food Research Initiative Competitive Grant no. 2013-67019-21376 from the USDA National Institute of Food and Agriculture.