sustainability
Article
pH Dynamics in Aquaponic Systems: Implications for Plant
and Fish Crop Productivity and Yield
Yi-Ju Wang 1,2 , Teng Yang 1,3
Citation: Wang, Y.-J.; Yang, T.; Kim,
H.-J. pH Dynamics in Aquaponic
Systems: Implications for Plant and
Fish Crop Productivity and Yield.
Sustainability 2023, 15, 7137. https://
doi.org/10.3390/su15097137
Academic Editors: Pantazis Georgiou,
Mike Spiliotis, Christos Tzimopoulos
and George Papaevangelou
Received: 28 February 2023
Revised: 19 April 2023
Accepted: 23 April 2023
Published: 24 April 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Sustainability 2023, 15, 7137. https://doi.org/10.3390/su15097137
and Hye-Ji Kim 1, *
1 Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47907, USA
2 Kula Bio, Technology and Development Center, 6 Mercer Rd, Unit 1, Natick, MA 01760, USA
3 School of Applied and Interdisciplinary Studies, Kansas State University, Olathe, KS 66506, USA
* Correspondence: heidikim219@hotmail.com
Abstract: The pH range of 7.0 to 7.2 is recommended to ensure reasonable nitrification rates in
aquaponics; however, this range is conducive neither to nitrification, a critical process that oc-
curs at pH 8.0, nor to plant growth. To determine the effects of pH in an aquaponic system,
Swiss chard (Beta vulgaris L.), kale (Brassica oleracea L.), mustard green (Brassica juncea L.), cilantro
(Coriandrum sativum L.), lettuce (Lactuca sativa L.), and arugula (Eruca vesicaria L.) were cultured with
tilapia (Oreochromis niloticus) in 5-year-old coupled aquaponic systems at three pH levels, 6.0, 6.5,
and 7.0, in comparison with hydroponics. Morphological and physiological growth parameters of
vegetable and fish crops were measured regularly, and the ammonia-oxidizing bacteria (AOB) in
the aquaponic system were analyzed by qPCR at the end of the production. This study found that
feed conversion ratio, fish biomass, and copy number of AOB were not affected by different pH, but
similar to hydroponic systems, lower pH in aquaponic systems increased fresh and dry mass and
nutrient levels of all plant species tested. This study suggests that pH has a significant impact on plant
performance and yield in both aquaponic and hydroponic systems and that, similar to hydroponics,
a pH of 6 is desirable for aquaponic systems to improve plant crop yield without compromising
nitrification activity and fish yield.
Keywords: aquaponics; hydroponics; pH effect; indoor farming; ammonia oxygen bacteria
1. Introduction
Facing the challenge of food demand and sustainable agricultural practices, aquapon-
ics has emerged as an important concept in global food production. Therefore, it is necessary
to adjust a suitable environment for three organisms (aqua-organisms, microorganisms,
and plants) to increase yield and quality in aquaponics. It has been reported that many
aquaponics-grown fish release approximate 70–80% of nitrogen waste into the water [1,2].
This waste has the potential to serve as a valuable nutrient source for crop growth. Aquatic
organism waste is filtered through microbial tanks in aquaponic systems, where ammonia
(NH4 + ) is converted to nitrate (NO3 − ) for plant uptake [3,4]. While plants can assimilate
nitrogen in either nitrate or ammonium form, excessive accumulation of NH4 + has been
proven to be toxic and can significantly reduce plant productivity [5]. Besides nitrogen, the
presence of other essential macronutrients, such as phosphorus (P) and potassium (K), along
with several micronutrients, significantly affects the yield of plants in aquaponic systems.
The major source of P in aquaponics is from fish feeds. Fish use only 15% of the P
in fish feeds, and plants have different abilities to absorb P from recycling aquaculture
wastewater based on different aquaponic designs [3,4]. Fish feeds also contain K and
other micronutrients, but the amount of K and other micronutrients, such as iron (Fe),
magnesium (Mg), manganese (Mn), and copper (Cu), is limited [5,6]. Therefore, some
aquaponic systems apply synthetic salts into the aquaponic nutrient solution or apply a
foliar spray to prevent the deficiency in K, Cu, Ca, Mg, Mn and Fe [5,6]. Moreover, it is
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Sustainability 2023, 15, 7137 2 of 20

common to observe some nutrient deficiency in plants when aquaponics relies on fish feed
for plant nutrients [5,7,8]. Additionally, Quagrainie et al. [9] point out that it is hard for
aquaponic growers to make a profit from vegetables compared to hydroponic growers
due to lower yield. Therefore, it is necessary to find a balance among three organisms in
aquaponics to have higher plant yields.
Aquaponic nutrient solutions are not as easy to maintain as hydroponic solutions.
The aquaponic solution is affected by many factors, such as fish feeding rate, hydraulic
loading rate, and pH [10,11]. Yang and Kim [2] analyzed three different feeding rates and
found that a uniform feeding rate can enhance crop yield and/or quality in aquaponics by
increasing initial nutrient availability and overall nitrogen-use efficiency of the system [2].
According to Yang and Kim [12], a flow rate of 3.3 m3 /m2 /day resulted in an increase of
50% and 80% in NO3 -N concentration, in comparison to flow rates of 2.2 m3 /m2 /day and
1.1 m3 /m2 /day, respectively. The pH is another important factor in regulating the nutrient
level in aquaponics [10] and must be balanced for three different organisms in aquaponics
at the same time. The critical step in aquaponics is nitrification, which converts toxic
NH3 to NO3 − for plant uptake [10,13]. The optimal pH for ammonia-oxidizing bacteria
(AOB) and nitrite-oxidizing bacteria (NOB) is 7.2 to 8.2, while their growth is restricted
at pH 5.8 and 6.5, respectively [9,12,14]. The activity ceases typically below pH 5.5 in
liquid pure culture [9,12,14]. As a result, aquaponic systems are set at pH 7.0 to get the best
performance of nitrification. However, the recommended pH for hydroponics is 5.5–5.8 [15].
The available P for plants depends on the pH [16]. As pH increases above 7.0, most P
converts to insoluble complexes and 30–65% of the P remains in solid fish sludge, which is
unavailable to plants. Moreover, it is hard for plants to absorb Fe, Cu, zinc (Zn), boron (B),
and Mn when pH is higher than 6.5 [16].
In this study, we established deep-water coupled aquaponic systems to investigate
the interactive effects of pH and nutrient uptake on the growth of six plant species in
comparison to hydroponic systems. The results led to a better understanding of how
pH affects nitrification and plant yields and to a potential solution for limited nutrients
in aquaponics.

2. Materials and Methods

2.1. System Design
Experiments were repeated three times between February and July 2019. Three iden-
tical aquaponic systems and three identical hydroponic systems were assembled in a
greenhouse at Purdue University in West Lafayette, USA (40◦ 250 26.400 N, 86◦ 550 44.400 W).
Each aquaponic system unit consisted of a fish tank or a nutrient reservoir (350 L), a clarifier
(20 L), a two-stage biofilter (40 L) [2,10], and a deep-water hydroponic grow bed (1.0 m2
and 350 L) (Figure 1). The biofilter was connected to peristaltic pumps (MasterflexLive™
Cole-Parmer L/S Digital Drive, Vernon Hills, IL, USA) to enable the recirculation of the
nutrient solution within the unit. The determination of water flow rate and nutrient man-
agement in the aquaponic and hydroponic systems in this study was made based on prior
investigations conducted by the research team [2,17]. The photoperiod for the aquaponic
and hydroponic systems in the greenhouse was established according to the previous
design of Teng and Kim [2,17,18].

2.2. Plant and Fish Materials

Arugula (Eruca vesicaria L.), cilantro (Coriandrum sativum L.), kale (Brassica oleracea L.),
mustard green (Brassica juncea L.), lettuce (Lactuca sativa L.) and Swiss chard (Beta vulgaris L.)
were used in this study. Seeds (Johnny’s Selected Seeds, Winslow, ME) were sown in
commercial growing media (Jiffy Preforma Plugs, Lorain, OH, USA). Seeds were irrigated
with tap water, and the electrical conductivity (EC) was gradually increased with a half-
strength fertilizer solution after germination. Seedlings were irrigated with full fertilizer
after true leaves developed [19]. (The nutrient composition of the fertilizer is described in
Sustainability 2023, 15, 7137 3 of 20

Table 1). Then, 21-day-old seedlings were transplanted to the aquaponic and hydroponic
experiment units. There were 24 plants per m2 for each experiment unit [19].

Figure 1. (A) Aquaponic experiment units. (B) Hydroponic experiment units. (C) Photograph of the
aquaponic and hydroponic systems utilized in this study.

Table 1. Nutrient composition and some macro- and micro-nutrient contents in hydroponic nutrient
solutions and aquaponic fish feed.

Parameter Hydroponic Fertilizer a Aquaponic Fish Feed b

Macronutrient (%)
Total nitrogen (N) 0.043 >6.88
Phosphorus pentoxide (P) 0.093 >1.10
Potassium oxide (K) 0.035 0.99
Sulfate(S) – 0.43
Calcium (Ca) 0.075 2.25–2.75
Magnesium (Mg) 0.039 0.23
Micronutrient (ppm)
Boron (B) 2 –
Copper (Cu) 1.05 10
Iron (Fe) 21 40
Manganese (Mn) 1.9 80
Molybdenum (Mo) 0.42 –
Zinc (Zn) 2.1 153
Data obtained from product description. The symbol “–” is used to indicate that no related information was avail-
able or was not included. a Sample collected was a 1:100 diluted solution of commercial fertilizer. b Information
was calculated based on daily fish feeding rate.

Nile tilapia (Oreochromis niloticus L.) were sourced from the Animal Sciences Research
and Education Center (ASREC) at Purdue University. The fish weights were measured and
subsequently equitably allocated to three distinct fish tanks with a density of 20 kg m−3 .
In this study, the fish were fed a complete diet (AquaMax Sport Fish 500, Purina Mills, St.
Louis, MO, USA) consisting of 4.8-mm floating pellets, which contained 41% protein and
1.1% phosphorus. The determination of the fish feeding rate was carried out according
to the methodology proposed by Teng and Kim [2]. Water temperature was maintained
at optimized level of 26–28 ◦ C for tilapia by aquarium thermostat heaters (Eheim Jager
TruTemp, Deizisau, Germany).
Sustainability 2023, 15, 7137 4 of 20

2.3. Measurement of Water Quality Parameters

The electrical conductivity was 1.5 mS cm−1 and was maintained by adjusting the
feeding rate in the aquaponic and hydroponic systems. The pH of the aquaponic and
hydroponic systems was maintained at pH 6.0, 6.5, and 7.0 by 10% H2 SO4 or a combination
of base solutions (0.02 mM Mg(OH)2 and 0.02 mM Ca(OH)2 with 1:1 v:v). Air stones
were supplied for each aquaponic system to maintain optimized dissolved oxygen (DO)
concentrations at full saturation as per the recommendation. Water quality parameters
such as EC, pH, water temperature, and DO were measured daily before feeding using the
HQ40d portable meters (HACH Corp., Loveland, CO, USA).

2.4. Measurement of Water Quality Parameters Quantitative PCR

The abundances of AOB were quantified via SYBR Green chemistry qPCR using
specific primers targeting amoA. The primer sequences and qPCR program utilized in
this study followed the methodology described by Cristina et al. [20]. Duplicate qPCR
runs were performed on an iQ5 real-time PCR thermal cycler, and the resulting data was
analyzed using iCycler iQTM software (BioRad Laboratories, Hercules, CA, USA). To
generate standard curves for qPCR, plasmid DNA was serially diluted by a factor of ten,
and primer specificity was ensured.

2.5. Measurement of Fish Growth Rate and Feed-Conversion Ratio

At the commencement and conclusion of the experiments, fish specimens were ob-
tained from each aquaponic system and weighed to determine the total fish biomass. The
fish stocking density was determined by dividing the total fish biomass in each aquaponic
system by the volume of the fish tank. The specific growth rate (SGR) and feed-conversion
ratio (FCR) were calculated by the following formula [21]:

SGR = (ln final weight of fish − ln initial weight of fish) × 100/days.

FCR = total weight of fish feed applied/total fish biomass increase (wet weight).

2.6. Measurement of Plant Biomass and Photosynthetic Properties

All plant tissues were harvested at 30 days after transplanting into the aquaponic and
hydroponic systems. The plants were then carefully divided into roots, stems, and leaves
for fresh weight measurements. Then, the plant samples were subjected to desiccation in
an oven set to 70 ◦ C for a period of 72 h to eliminate all moisture content. Subsequently, the
dry weight of the plant samples was assessed by measurement.
The Fv/Fm (maximum photochemical efficiency of PSII) is widely used for early stress
detection in plants [22]. The Fv/Fm value of healthy C3 plants is above 0.8 [23–25]. The
values were recorded by a Handy chlorophyll fluorescence meter (Handy PEA+, Hansatech
Instruments, Norfolk, UK). Five leaves per plant were taken and averaged, and four
replicate plants were taken and averaged from each treatment every 3 days. A young fully
expanded leaf was shielded by dark adaptation leaf clips (PEA, Hansatech Instruments,
Norfolk, UK) for 20 min. The SPAD-502 Chlorophyll Meter (Minolta Camera Co., Ltd.,
Osaka, Japan) was used to measure the SPAD value, which indicates the chlorophyll
content per unit leaf area. SPAD values were determined using the method outlined by
Dong et al. [26]. Gas exchange measurements were conducted in this study using a portable
gas exchange system (LI-6400XT; LI-COR Biosciences, Lincoln, NE, USA) according to the
methodology described by Chang et al. [27].

2.7. Nutrient Analysis

The oven-dried plant samples as described in Section 2.6 were ground and filtered
through a 40-mesh sieve with a Wiley mini-mill (Thomas Scientific, Swedesboro, NJ, USA).
Subsequently, two milligrams of each sample were placed into an empty 2-microliter tube
and meticulously wrapped to prevent any loss of the sample. The dry samples were sent to
Sustainability 2023, 15, 7137 5 of 20

Midwest Laboratories (Midwest Laboratories, Omaha, NE, USA) for nutrient analysis. Total
nitrogen was measured by thermal conductivity/IR detection (LECO TruMac and TruSpec
CN Analyzers), and other mineral nutrients were analyzed by ICP-AES Analysis [28].

2.8. Mediation Analysis

Mediation analysis is a statistic model to illustrate the observed relationship between
an independent variable and dependent variable with a third hypothetical variable [29–31].
Figure 2 illustrates the relationship between pH effects, dependent variable (such as Fv/Fm,
and shoot and root ratio), and plant yield (harvest index). The harvest index is the ratio
of harvested shoot fresh weight to total plant fresh weight and can be used to measure
the reproductive efficiency [32]. The function is shoot fresh weight/total plant fresh
weight × 100% [32]. Considering the nonlinear version of the mediation model, as depicted
in Figure 2, the corresponding structural equations would have the form [33–35]:

X = F1(e1) z = F2(x, e2) y = F3(x, z, e3) (1)

where X, Y, Z are discrete or continuous random variables, F1, F2, and F3 are arbitrary
functions, and e1, e2, e3 represent omitted factors. With the above formula, we next
determine total effect, T E0,1 which measures the change in harvest index produced by a
unit change in pH effects, by the formula [33–35]:

TE0,1 (e2, e3) = F3[1, F2(1, e2), e3] − F3[0, F2(0, e2), e3] (2)

Then, we determine the natural effects by the following formula [33–35]:

NDEx,x0 (Y ) = ∑ [ E (Y X = x 0 , Z = z) − E(Y X = x 0 , Z = z)] P( Z = z | X = x ) (3)

z

NDEx,x0 (Y ) is defined as the expected change in Y induced by changing X from x to x0

while keeping all mediating factors constant at whatever value they would have obtained
under X = x, before the transition from x to x0 [33–35].

Figure 2. A schematic outline of mediation analysis.

2.9. Data Analysis

The experiment was conducted using three aquaponic systems and three hydroponic
systems. Each system has ability to provide space for 24 plants to grow. The pH was set
as 6, 6.5 and 7 for three aquaponic systems and three hydroponic systems accordingly.
The sequence of steps employed to assess statistical models is demonstrated in Figure 2.
This study focused on the pH effects in plant yields associated with Fv/Fm in soilless
production systems. For the analysis of water conditions, water quality parameters, and
fish performance in aquaponic and hydroponic systems, randomized complete block design
and two-way ANOVA were used. The plant biomass and nutrient concentrations were
tested by using Tukey’s Honestly Significant Difference. The statistical analysis used
post-hoc pairwise comparisons (R 3.6.1, Comprehensive R Archive Network, USA) at a
significance level of 0.05.
Sustainability 2023, 15, 7137 6 of 20

3. Results
3.1. Water Quality
Solution pH was maintained at an average of 6.0, 6.5, and 7.0 across all aquaponic and
hydroponic systems in the different pH treatments, which led to a significant difference in
the usage of pH correction solution (Table 2). Nutrient input was different in the aquaponic
and hydroponic systems (Table 1), but the average EC was not significantly different
between systems and pH levels (Table 2). DO and water temperature were maintained
above 7 mg L−1 and 22 ◦ C, respectively, and there was no difference between the systems
and pH levels (Table 2). Similarly, there was no significant difference in NH4 + -N, NO3 − -N,
and NO2 –N in the aquaponic solution between different pH treatments. AOB was also not
affected by pH treatment in the aquaponic system (Table 3).

Table 2. Average values of water quality in plant growth bed for 4 weeks.

pH Correction Solution Electrical Conductivity Dissolved

System pH Temperature (◦ C)
(mL day−1 ) (mS cm−1 ) Oxygen (mg L−1 )
Aquaponic 6.0 39.5 1.57 7.51 22.8
6.5 80.6 1.61 7.32 23.3
7.0 91.3 1.59 7.25 23.1
Hydroponic 6.0 2.0 1.54 7.75 23.2
6.5 0.8 1.50 7.80 23.0
7.0 0.0 1.51 7.83 23.6
ANOVA
System *** ns ns ns
pH *** ns ns ns
System × pH *** ns ns ns
Each value in the table is the mean. ns, *** mean no significant or significant at p ≤ 0.001, respectively. Significantly
different based on ANOVA (α = 0.05).

Table 3. Average concentrations of ammonia-oxidizing bacteria (AOB), ammonia, nitrite, nitrate in

plant growth bed for 4 weeks.

AOB
System pH (Copy Numbers g−1 Ammonia (mg L−1 ) Nitrite (mg L−1 ) Nitrate (mg L−1 )
Biomedia)
Aquaponic 6.0 5.3 × 105 1.62 0.12 29
6.5 1.1 × 106 1.45 0.09 30.4
7.0 3.2 × 106 1.13 0.12 30.2
p ns ns ns ns
Abbreviations: AOB, ammonia-oxidizing bacteria. Each value in the table is the mean. ns means not significant.
Significant difference is based on ANOVA (α = 0.05).

3.2. The pH Effect on Fish Production

Optimizing pH can prevent metabolic stress and avoid the mortality of stocked fish in
ponds. The optimal pH range for Nile tilapia is between 5 to 8, and Nile tilapia can survive
in acidic water as low as pH 4 [36–38]. In our study, we set up the aquaponic system at pH
6, 6.5, and 7, which was within the optimal pH range for Nile tilapia. Our results show that
there were no significant differences in fish biomass gain, SGR, and FCR between different
pH treatments (Table 4).

3.3. Plant Biomass

In this study, we repeated the experiments three times, and all the results showed a
similar trend in plant biomass. The average biomass of most plant species was affected by
the cropping system and pH treatments. Except for lettuce, plants had higher fresh weight
and dry weight in the hydroponic system than in the aquaponic system regardless of pH
Sustainability 2023, 15, 7137 7 of 20

treatment (Table 5). In general, the total fresh weight of arugula, cilantro, kale, mustard
green, and Swiss chard in the hydroponic system was significantly increased by 142.5%,
50.6%, 52%, 48.7%, and 52.2% compared to aquaponic plants (Table 5).

Table 4. Fish production in aquaponics.

Fish Feed Initial Stocking Final Stocking Fish Biomass

Treatment SGR FCR
Applied (g) Density (kg m−3 ) Density (kg m−3 ) Gain (kg m−3 )
pH 6 2100 19.9 27.0 2.5 8.3 0.84
pH 6.5 2100 20.3 27.5 2.5 8.4 0.83
pH 7 2100 20.4 27.9 2.6 8.7 0.80
p ns ns ns ns ns
Abbreviations: SGR, Specific growth rate. FCR, Feed conversion ratio. ns means not significant. Significant
difference is based on ANOVA (α = 0.05).

Table 5. Average biomass of arugula, cilantro, mustard green, kale, lettuce, and Swiss chard in
aquaponic and hydroponic systems.

Fresh Biomass (g plant−1 ) Dry Biomass (g plant−1 )

Treatments
Total Shoots Roots Total Shoots Roots
Arugula
System
Aquaponic 111.5 b 96.2 b 15.4 b 8.5 b 7.8 b 1.0 b
Hydroponic 270.4 a 270.8 a 22.8 a 19.5 a 20.2 a 1.9 a
pH
6 253.7 a 238.0 a 25.6 a 18.2 a 18.0 a 1.9 a
6.5 181.2 b 188.2 b 20.2 ab 14.1 b 14.0 b 1.7 a
7 137.9 c 124.4 c 11.5 b 9.6 c 9.9 c 0.8 b
Significance
System *** *** * *** *** **
pH *** *** ** *** *** **
System × pH ns ns ns ns ns ns
Cilantro
System
Aquaponic 72.5 b 55.0 b 23.0 b 6.6 b 4.8 b 2.0
Hydroponic 109.2 a 80.8 a 27.9 a 9.7 a 7.3 a 2.2
pH
6 124.7 a 87.7 a 37.0 a 11.3 a 8.1 a 3.2 a
6.5 87.6 b 65.0 b 22.6 b 7.7 b 5.8 b 1.9 b
7 60.3 c 51.0 b 16.9 c 5.4 c 4.4 b 1.1 b
Significance
System *** *** ns ** *** ns
pH *** *** *** *** *** **
System × pH * ns * ns ns ns
Mustard green
System
Aquaponic 218.0 b 195.6 b 17.5 17.9 b 16.4 b 1.1 b
Hydroponic 324.2 a 300.1 a 19.4 26.7 a 25.1 a 1.5 a
pH
6 323.9 a 296.2 a 23.2 a 27.8 a 25.9 a 2.0 a
6.5 275.6 b 249.3 ab 16.6 b 22.3 ab 20.9 b 1.4 b
7 213.7 c 198.1 b 15.5 b 16.9 b 15.6 c 0.7 c
Significance
System *** *** ns *** *** *
pH *** *** * *** *** ***
System × pH *** *** ns *** ** *
Sustainability 2023, 15, 7137 8 of 20

Table 5. Cont.

Fresh Biomass (g plant−1 ) Dry Biomass (g plant−1 )

Treatments
Total Shoots Roots Total Shoots Roots
Kale
System
Aquaponic 177.3 b 160.3 b 19.1 b 16.0 b 14.5 b 1.6 b
Hydroponic 269.5 a 244.4 a 27.9 a 22.8 a 21.4 a 2.0 a
pH
6 247.1 a 223.4 a 22.6 22.4 a 20.3 a 1.9
6.5 246.9 a 228.2 a 25.6 20.9 b 19.7 a 1.9
7 176.1 b 155.4 b 22.3 15.0 c 14.0 b 1.6
Significance
System *** *** *** *** *** *
pH *** ** ns *** *** ns
System × pH ** ** ns * ** ns
Lettuce
System
Aquaponic 303.9 286.5 17.2 10.4 a 8.9 1.3
Hydroponic 290.0 289.6 14.4 9.1 b 8.4 1.0
pH
6 334.8 a 315.4 a 20.4 a 11.6 a 9.7 a 1.5 a
6.5 270.4 b 277.9 b 13.6 b 8.8 b 8.3 b 1.1 ab
7 285.5 ab 270.9 b 13.5 b 8.8 b 7.9 c 0.9 b
Significance
System ns ns ns * ns ns
pH * * ** ** ** *
System × pH ns ns ns ns * ns
Swiss chard
System
Aquaponic 342.9 b 311.7 b 30.3 b 28.4 26.7 2.5
Hydroponic 521.9 a 419.2 a 50.0 a 33.5 30.4 3.0
pH
6 526.6 a 393.9 a 53.8 a 38.8 a 35.4 a 3.3
6.5 435.0 b 402.9 a 33.7 b 32.7 a 31.0 a 2.6
7 335.7 c 299.6 b 33.1 b 21.3 b 19.3 b 2.3
Significance
System *** * * ns ns ns
pH ** ns ns *** *** ns
System × pH ** ns ns ** ** ns
Each column is a mean followed by a letter. The different letters show significant difference based on Tukey’s
HSD test (α = 0.05). Each value in the table is the mean of 9 replicates of system and 6 replicates of pH treatment.
“ns” means not significant, “*” means significant at a level of p ≤ 0.05, “**” means significant at a level of p ≤ 0.01,
and “***” means significant at a level of p ≤ 0.001.

Furthermore, high pH levels had a negative impact on the fresh weight and dry weight
of shoots across all plant species (Table 5). In both the aquaponic and hydroponic systems,
plants grown in pH 7 conditions exhibited lower total fresh weight, shoot fresh weight,
total dry weight, and shoot dry weight compared to those in other pH treatments (Table 5).
Notably, the total fresh weights of arugula, cilantro, kale, lettuce, mustard green, and Swiss
chard were substantially lower at pH 7 than at pH 6, with reductions of 45.6%, 51.6%,
28.7%, 14.7%, 34%, and 36.3%, respectively (Table 5). In addition, the total biomass of
arugula, cilantro, mustard green, kale, lettuce, and Swiss Chard in a pH 6 environment
were significantly increased by 184%, 207%, 152%, 140%, 117%, and 157% over those grown
in pH 7 treatments (Table 5).
Sustainability 2023, 15, 7137 9 of 20

3.4. Leaf Chlorophyll and Photosynthetic Parameters

The effects of pH on SPAD and Fv/Fm values of plants grown in aquaponic and
hydroponic systems were investigated in this study. Table 6 shows that there were no
significant differences in these values among the different pH treatments at day 7 after
transplanting into the aquaponic or hydroponic systems. However, the pH affected the
SPAD value of most plant species at day 21 and 28 after transplanting, except for lettuce and
Swiss chard (Figure 3). Specifically, the SPAD value of arugula, cilantro, kale, and mustard
green decreased in pH 7 aquaponics (Figure 3). In general, SPAD value was not affected by
growing systems (Table 6). The Fv/Fm of six plant species decreased after transplanting
into the aquaponic and hydroponic systems but most of them recovered by day 14 after
transplanting (Figure 4). However, the Fv/Fm of arugula, cilantro, and mustard green
in pH 7 aquaponic systems was significantly lower than other treatments at day 21 after
transplanting (Figure 4). Nonetheless, photosynthetic parameters, such as photosynthetic
rate (Pn), stomatal conductance (gs), transpiration rate (Tr), intercellular CO2 concentration
(Ci), were not affected by pH when measured at day 28 after transplanting (Figure 5).

Table 6. SPAD value and Fv/Fm of crops grown in aquaponic and hydroponic systems with different
pH. The values are photosynthetic parameters measured at day 7 after transplanting.

Arugula Cilantro Mustard Green Kale Lettuce Swiss Chard

Systems pH SPAD Fv/Fm SPAD Fv/Fm SPAD Fv/Fm SPAD Fv/Fm SPAD Fv/Fm SPAD Fv/Fm
AQU 6.0 38.1 0.76 36.9 0.70 30.6 0.80 37.2 0.75 28.4 0.79 34.0 0.75
6.5 31.8 0.71 29.5 0.62 31.7 0.74 32.7 0.70 26.5 0.75 33.2 0.73
7.0 43.6 0.75 34.9 0.71 27.8 0.76 35.8 0.73 28.9 0.73 33.9 0.76
ns ns ns ns ns ns ns ns ns ns ns ns
HYD 6.0 45.9 0.80 35.4 0.76 32.7 0.80 37.9 0.75 27.2 0.77 31.4 0.76
6.5 38.4 0.78 38.7 0.81 30.1 0.81 43.0 0.74 30.1 0.77 31.0 0.78
7.0 39.7 0.76 34.3 0.69 30.4 0.75 32.4 0.69 26.2 0.71 32.8 0.77
ns ns ns ns ns * ns ns ns ns ns ns
Systems ns * ns * ns ns ns ns ns ns ns ns
pH ns ns ns ns ns * ns ns ns ns ns ns
Systems × pH * ns ns ns ns ns ns ns ns ns ns ns
The value is the mean of 9 replicates of system and 6 replicates of pH treatment. “ns” means not significant,
“*” means significant at a level of p ≤ 0.05, based on the ANOVA test.

3.5. The Nutrient Analysis

The study analyzed the impact of different production systems and pH treatments on
the accumulation of macronutrients and micronutrients in arugula, cilantro, kale, mustard
green, and Swiss chard. In general, both the pH levels and production systems had
significant effects on the macronutrient concentrations in most of the crops tested (Table 7).
In this study, significant interactions were observed between the production system and
nutrient levels in pH in most of the crops. However, the nutrient accumulation in mustard
greens was less affected by the production system and the pH, and little interaction was
observed between these variables (Table 7).
Likewise, the accumulation of micronutrients was significantly affected by the pro-
duction system and the pH (Table 7). Mn concentrations were significantly higher when
the crops were grown in the aquaponic system, but the pH had inconsistent effects on Mn
accumulation based on each crop. Similarly, Fe concentration was higher in the aquaponic
system in most of the crops except for cilantro, in which Fe concentration was higher in the
hydroponic system, and a lower pH increased the concentration (Table 7). Zn concentration
was also higher in the aquaponic system, except for mustard greens, but the pH effect
varied with the crop species.
Sustainability 2023, 15, 7137 10 of 20

Figure 3. Dynamic changes in SPAD value of arugula, cilantro, mustard green, kale, lettuce, and Swiss
chard in aquaponic and hydroponic systems set at pH 6, 6.5, or 7 over 28 days of production period.

3.6. The Mediation Analysis of pH Effect on Nutrient Level in Plant Tissues and Total
Fresh Weight
Mediation analysis is a commonly employed statistical modeling approach that aims
to investigate the relationship between an independent and dependent variable. It does
so by introducing a hypothetical third variable that acts as a mediator or an explanatory
mechanism between the independent and dependent variable [29–31]. The mediation
model is shown in Figure 2. The data should fit three linear regulation models to fit the
mediation analysis. We used casual mediation analysis to examine the mediation effect
of nutrients.
Total fresh weight = b0 + b1* pH effect + e (4)

Dependent variable = b0 + b2* pH effect + e (5)

Total fresh weight = b0 + b4* pH effect + b3* dependent variable + e (6)

Except for Mn, the p-value of the average casual mediation effect (ACME) of other
plant nutrients was not significant, which indicated that those variables were not mediation
effects between pH treatment and plant yields. The p-value of the average direct effect
(ADE) of all nutrients was lower than 0.05, which showed that pH treatment had direct
effects on all nutrients and plant yields. Meanwhile, the p-value of both ACME and ADE of
Mn were lower than 0.05, which indicated the partial effect of pH treatment on Mn and
plant yield in the aquaponic and hydroponic systems (Table 8).
Sustainability 2023, 15, 7137 11 of 20

Figure 4. Dynamic changes in Fv/Fm value of arugula, cilantro, mustard green, kale, lettuce, and Swiss chard in aquaponic and hydroponic systems set at pH 6, 6.5,
or 7 over 28 days of production period.
Sustainability 2023, 15, 7137 12 of 20

Figure 5. (A) Pn (Plant photosynthetic rate), (B) gs (stomatal conductance), (C) Ci (intercellular CO2 concentration), and (D) E (transpiration rate) of 28-day-old crops
in aquaponic systems with different pH.
Sustainability 2023, 15, 7137 13 of 20

Table 7. Average mineral nutrient concentrations of arugula, cilantro, mustard green, kale, lettuce, and Swiss chard in aquaponic and hydroponic systems.

Macronutrient Concentration (%) Micronutrient Concentration (ppm)

Treatments
N P K Mg Ca S Fe Mn B Cu Zn
Arugula
System
Aquaponic 5.1 0.9 a 9.2 a 0.7 a 2.2 b 0.9 b 48.4 a 135.6 a 42.4 b 12.4 a 275.6 a
Hydroponic 5.7 0.8 b 8.0 b 0.6 b 2.5 a 1.1 a 42.1 b 62.1 b 44.5 a 7.8 b 164.8 b
pH
6 5.7 a 0.8 b 7.8 c 0.5 b 2.8 a 1.3 a 42.5 b 53.8 c 43.1 6.8 133.5 c
6.5 5.3 b 0.8 b 9.8 a 0.8 a 2.2 b 1.0 b 51.3 a 112.9 b 46.5 10.7 187.6 b
7 5.3 b 1.0 a 8.5 b 0.7 a 1.9 c 0.9 c 41.9 b 129.8 a 40.8 13 339.5 a
Significance
System ns ** *** *** ** *** *** *** * * ***
pH * * ** ** ** *** ** *** ns ns ***
System × pH * ns ** ** ** *** ns *** * ** **
Cilantro
System
Aquaponic 4.0 b 0.6 b 10.1 0.4 2.1 0.4 43.7 b 751.1 a 78.5 a 15.4 284.8 a
Hydroponic 4.7 a 0.8 a 9.1 0.5 2.2 0.4 80.3 a 192.3 b 61.1 b 14.2 171.3 b
pH
6 4.9 a 0.8 a 8.7 b 0.4 b 1.8 0.4 95.3 a 533.0 b 70.4 b 14.5 214.7
6.5 4.2 b 0.7 b 10.1 a 0.5 a 2.3 0.4 56.5 b 619.8 a 58.5 c 15.3 263.7
7 3.9 b 0.6 c 10.0 a 0.5 a 2.3 0.4 34.2 c 262.3 c 80.4 a 14.7 205.8
Significance
System ** ** ns ns ns ns *** *** ** ns ***
pH ** ** ** * ns ns *** *** ** ns ns
System × pH * ** *** ns ns ns ** *** ns ns ns
Mustard green
System
Aquaponic 4.7 0.7 7.9 0.5 2.8 0.8 b 40.9 83.4 a 36.3 5.4 165.8
Hydroponic 4.8 0.7 8.3 0.5 3.1 1.2 a 35 62.0 b 40.3 6.3 156.8
pH
6 4.3 0.7 7.8 0.4 2.9 0.9 b 35.2 64.3 35.7 5 152.8
6.5 4.9 0.8 8.6 0.5 3.2 1.2 a 37.5 59.8 39 6.5 142.5
7 5.1 0.7 7.7 0.6 2.8 0.9 b 41.2 84.2 40.3 6.2 188.7
Sustainability 2023, 15, 7137 14 of 20

Table 7. Cont.

Macronutrient Concentration (%) Micronutrient Concentration (ppm)

Treatments
N P K Mg Ca S Fe Mn B Cu Zn
Significance
System ns ns ns ns ns * ns * ns ns ns
pH ns ns ns ns ns *** ns ns ns ns ns
System × pH * ns ns * ns *** ns ** * ns *
Kale
System
Aquaponic 5.4 0.7 7.4 a 0.5 2.9 1.3 a 66.1 a 105.3 a 41.7 a 7.8 247.6 a
Hydroponic 5.5 0.7 7.0 b 0.6 3 1.1 b 51.0 b 71.1 b 38.3 b 6.1 116.4 b
pH
6 5.5 a 0.7 a 6.8 c 0.7 a 4.2 a 1.6 a 41.5 c 93.7 a 51.5 a 6.2 b 192.7 b
6.5 5.3 b 0.6 b 7.7 a 0.4 b 2.0 b 0.9 b 81.2 a 89.2 b 31.7 b 7.7 a 241.7 a
7 5.6 a 0.6 b 7.3 b 0.5 b 2.6 b 1.1 b 53.0 b 81.8 c 36.8 b 7.0 b 111.7 c
Significance
System ** ns *** ns ** *** *** ns * ns ***
pH ** *** *** *** *** *** *** *** *** * ***
System × pH *** ** *** *** *** *** *** ns *** * **
Lettuce
System
Aquaponic 4.5 1.2 a 9.9 a 0.5 2 0.4 253.1 386.9 a 33.8 13.6 156.9 a
Hydroponic 4.5 1.0 b 9.0 b 0.5 1.5 0.3 231.1 294.1 b 29.8 11.4 102.2 b
pH
6 4.4 b 1.2 a 8.8 0.5 1.7 b 0.4 265.2 b 392.5 a 29.5 12.5 b 143.8 a
6.5 4.2 c 1.1 b 8.5 0.5 1.5 b 0.4 334.2 a 362.3 b 30.7 14.0 a 106.2 b
7 4.9 a 1.0 c 11.1 0.5 2.0 a 0.4 127.0 c 266.7 c 35.2 11.0 b 138.7 a
Significance
System ** *** *** ns ns *** *** *** ns ns ***
pH *** *** ns * *** *** *** *** ns *** **
System × pH * *** *** *** *** *** *** *** *** *** ***
Swiss chard
System
Aquaponic 5.1 b 0.6 a 10.7 a 1.0 b 1.5 a 0.3 53.1 a 191.9 a 49.7 b 9.4 116.2 a
Hydroponic 5.4 a 0.5 b 9.5 b 1.1 a 1.1 b 0.4 49.8 b 34.9 b 51.1 a 6.1 49.9 b
Sustainability 2023, 15, 7137 15 of 20

Table 7. Cont.

Macronutrient Concentration (%) Micronutrient Concentration (ppm)

Treatments
N P K Mg Ca S Fe Mn B Cu Zn
pH
6 5.5 0.6 a 9.2 b 0.9 b 1.1 0.3 52.5 105.3 b 45.0 b 7.8 b 67.3 c
6.5 5.2 0.5 b 9.2 b 1.2 a 1.3 0.4 56 97.0 c 52.8 a 8.0 a 72.0 b
7 5.1 0.6 a 11.7 a 1.1 a 1.5 0.3 45.8 138.0 a 53.3 a 7.5 b 109.8 a
Significance
System ** *** ** * *** ns ** *** ** ns ***
pH ns *** ** ** ns ns ns *** ** ** ***
System × pH ** *** *** *** *** ns ns *** * ns ***
Each column is a mean followed by a letter. The different letters show significant difference based on Tukey’s HSD test (α = 0.05). Each value in the table is the mean of 9 replicates of
system and 6 replicates of pH treatment. “ns” means not significant, “*” means significant at a level of p ≤ 0.05, “**” means significant at a level of p ≤ 0.01, and “***” means significant at
a level of p ≤ 0.001.
Sustainability 2023, 15, 7137 16 of 20

Table 8. Causal mediation modeling results.

Dependent Variable ACME (p-Value) ADE (p-Value) Result

N 0.79 <0.05 Direct effect
P 0.16 <0.05 Direct effect
K 0.94 <0.05 Direct effect
Mg 0.28 <0.05 Direct effect
Ca 0.91 <0.05 Direct effect
S 0.48 <0.05 Direct effect
Na 0.83 <0.05 Direct effect
Fe 0.45 <0.05 Direct effect
Partial mediation
Mn <0.05 <0.05
effect
B 0.67 <0.05 Direct effect
Cu 0.14 <0.05 Direct effect
Zn 0.38 <0.05 Direct effect
Abbreviations: ACME, average causal mediation effect. ADE, average direct effect.

4. Discussion
4.1. Water Quality of Aquaponic System
Water physical parameters, such as EC, DO, and water temperature, have direct im-
pacts on plant yield in soilless culture systems. In this study, EC level was maintained above
1.5 mS cm−1 for plant growth in the aquaponic and hydroponic systems [18,39,40]. That
the EC level increased with time in the aquaponic system may be due to the accumulated
mineral nutrient in the aquaponic solution [18]. The dissolved oxygen was maintained
above 6 mg L−1 to supported nitrification and fish growth in the aquaponic system [41,42].
The water temperature can also affect plant crop yield in soilless culture systems. Thomp-
son et al. [43] indicated that root temperatures of around 24 ◦ C can support better market
quality and production. In this study, a water temperature of 22–25 ◦ C was better for plant
yield and fish feed conversion ratio [43–45].

4.2. The pH Effect on Nitrification Activity

In the process of nitrification, both AOB and NOB had great influences on the NH4 + –N
removal, NO2 — N, and NO3 — N production. The growth rate of nitrifiers was significantly
affected by different temperatures and pH values, due to the energy requirement of cell
maintenance. The most efficient nitrification activity in aquaculture biofilters was reported
from pH 7.0 to 9.0 [12,14]. Both AOB and NOB are important in nitrogen fixation, but
AOB are typically considered more important as they initiate the process of nitrification,
which is a key step in the conversion of atmospheric nitrogen to a form that can be used by
plants [46]. Without AOB, NOB would not have a source of nitrite to convert to nitrate, and
nitrogen fixation would not occur efficiently [46].
Tyson et al. [39] examined the effect of pH on nitrogen transformations in the biofilters
in the aquaponic system. They found that no nitrification occurred at pH 5.5, and it
took 12, 20, and 20–24 days at pH 8.5, 7.5, and 6.5 to decrease total ammonia nitrogen
from 5 to 0 mg L-1, respectively [42]. Additionally, Wongkiew et al. [4] found there was
higher total ammonia nitrogen and lower nitrate at a lower pH (6.0 and 5.2) aquaponic
solution than in a neutral aquaponic solution. Even though lower pH decreases nitrification
activity in aquaponic biofilters, the nitrogen utilization efficiency can reach a maximum
at lower pH due to the ability of nutrient uptake in plants. Zou et al. [46] found that the
maximum nitrogen utilization efficiency reached 50.9% at pH 6.0, followed by 47.3% at
pH 7.5 and 44.7% at pH 9.0 in media-based aquaponics. Moreover, the nitrogen level of
plant tissues was 34.8%, 30.3%, and 28.5% at pH 6.0, 7.5, and 9.0, respectively, which led to
Sustainability 2023, 15, 7137 17 of 20

significantly higher plant yields at pH 6.0 [47]. They also found that the nitrogen level in
water was not significantly different between pH 6.0, 7.5, and 9.0 [47]. The recommended
pH for aquaponics is 7.0–7.5 for better nitrification activity [13,48]. However, there was
no significant difference of AOB between aquaponic systems at different pH, which was
similar to a previous study [49]. Day et al. [48] compared the effect of two different
nitrifying bacteria sources, commercial nitrifying bacteria and their own biofilter medium,
with nitrogen-limited aquaponics. They did not find a difference in nitrifying bacteria
populations in aquaponics in both treatments, even though treating commercial nitrifying
bacteria led to a higher plant yield [49].
Our results were similar to their finding. There was no significant difference of
nitrogen concentration between different pH treatment aquaponics, but plant growth was
significantly better at pH 6.0. Additionally, there was no significant difference in AOB
populations between different pH treatments in our study.

4.3. The pH Effect on Plant Yield

Numerous studies have compared plant yields in aquaponic and hydroponic sys-
tems [50]. Due to different aquaponic designs, some studies showed aquaponics can
have higher or similar plant yields compared to hydroponics, while some studies showed
lower plant yields in aquaponics [18,51–53]. Roosta and Afsharipoor found that growing
strawberries with 100% of perlite in an aquaponic system had higher fruit numbers and
yields than in a hydroponic system, although strawberry accumulated less N, P, K in the
aquaponic system [53]. Meanwhile, Yang and Kim compared nitrogen and phosphorous
use efficiency for lettuce-, basil-, and tomato-based aquaponic and hydroponic systems,
and found that the nitrogen and phosphorous use efficiency were significantly higher
in the aquaponic than the hydroponic system even though plant yields were low in the
aquaponic system [18]. Various environmental factors can affect plant yields in aquaponics,
such as fish feeding rate, water flow rate and pH, since those factors can affect nitrogen
transformation in aquaponics [10]. In our lab’s previous study, Yang and Kim found that
nitrogen-use efficiency and crop production were increased in aquaponic systems with a
uniform fish feeding rate [2]. Moreover, Yang and Kim compared the effect of the hydraulic
loading rate in aquaponic systems, and they found not only improved plant performances
from a higher flow rate (3.3 m day−1 ) but also improved fish growth rate and biomass [17].
In this study, we grew six different plant species in aquaponic and hydroponic systems with
different pH treatments and found that the plants had the lowest total fresh weight at pH 7
regardless of production system. This result is similar to previous findings. Anderson et al.
grew butterhead lettuce at different pH in hydroponics, and pH 7 hydroponic systems
produced 26% less shoot fresh weight than pH 5.8 hydroponic systems [51].

4.4. The pH Effect on Nutrient Level in Plant Tissues and Total Fresh Weight
The higher pH levels in aquaponics may lead to lower plant yields with lower nutrient
uptake, as demonstrated in previous studies [2,51,54]. Meanwhile, the pH level of a
hydroponic solution can significantly influence the availability of minerals such as P, Ca,
and Mg [16]. In this study, we used mediation analysis to check the relation between pH,
nutrient level in plant tissues, and plant total fresh weight. We found that most nutrients,
except Mn, are directly affected by system pH and correlate with plant yield in aquaponic
and hydroponic systems. Our findings are consistent with prior studies on plant yield in
hydroponic systems, wherein low levels of nitrogen, copper, and molybdenum in lettuce
grown under hydroponic conditions with a pH of 7 were found to be associated with
reduced plant growth [51]. Mn is considered a crucial micronutrient for plant growth,
particularly in the photosynthesis process [55,56]. However, the impact of Mn on plant
yield is not direct, as the photosynthetic rate is more significantly influenced by N and
Mg [57]. Based on our study, a lower pH level creates an optimal growth environment with
increased iron levels, leading to higher crop yields. Hence, it can be inferred that Mn’s
contribution to crop yield is less pronounced than N, Mg, and pH levels.
Sustainability 2023, 15, 7137 18 of 20

5. Conclusions
This study revealed that reducing the pH level from 7 to 6 enhanced the fresh and dry
mass of all plant species in aquaponic systems, without any negative impact on fish yield.
In addition, lower pH increased tissue nutrients in arugula, cilantro, lettuce, and Swiss
chard in both the aquaponic and hydroponic systems. Findings from this study provide
guidelines for aquaponic growers to increase profit margins by optimizing plant crop yields
and quality through pH manipulation.

Author Contributions: Y.-J.W. and T.Y. conducted the experiment, collected and analyzed the data,
and drafted the manuscript. H.-J.K. supervised the research, provided critical feedback, and com-
pleted the final version of the manuscript. All authors have read and agreed to the published version
of the manuscript.
Funding: This research was funded by Indiana State Department of Agriculture (ISDA) Specialty
Crop Block Grant, under grant number A337-19-SCBG-19-005; USDA National Institute of Food and
Agriculture, Multistate Hatch project NE 1835 Resource Optimization in Controlled Environment
Agriculture; Purdue University Research Funds.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author.
Acknowledgments: We are grateful to Nathan Deppe and Dan Little for their help with crop manage-
ment; Seunghyun Choi, and Gaotian Zhu for their help with crop management and data collection;
and Lori Hoalgand and Bob Rode for their helpful discussions during the research.
Conflicts of Interest: The authors declare no conflict of interest.

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