New BIOTECHNOLOGY 41 (2018) 15–24

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New BIOTECHNOLOGY
journal homepage: www.elsevier.com/locate/nbt

Full length article

Nutrient removal from hydroponic wastewater by a microbial consortium

and a culture of Paracercomonas saepenatans
Ju Yeon Leea,d, Arifur Rahmanb, Juliana Behrensc, Conor Brennanc, Baknoon Hama,d,
⁎
Hyung Seok Kima, Chu Won Nhoa, Seong-Taek Yund,e, Hossain Azamc, Man Jae Kwond,e,
a
Korea Institute of Science and Technology, Gangneung, Republic of Korea
b
Civil and Environmental Engineering, The George Washington University, DC, USA
c
Civil and Environmental Engineering, Manhattan College, NY, USA
d
Graduate School of Energy and Environment, Korea University, Seoul, Republic of Korea
e
Department of Earth and Environmental Sciences, Korea University, Seoul, Republic of Korea

A R T I C L E I N F O A B S T R A C T

Keywords: The potential of microbial processes for removal of major nutrients (e.g., N, P) and inorganic cations (e.g., Ca2+,
Algae Mg2+, and Fe2+) from hydroponic systems was investigated. Microbial consortium- and axenic culture-based
Hydroponic system experiments were conducted in a waste nutrient solution (WNS). A microbial consortium grown in the WNS and
Nitrogen and phosphorous uptake selected microalgae species of Paracercomonas saepenatans were inoculated in two different synthetic media
Microbial community
(Bold’s Basal Medium (BBM) and synthetic WNS) in batch systems, and the microbial growth characteristics and
Mineral precipitation
the rate and extent of nutrient removal were determined for each system. No toxicity or growth inhibition was
observed during microbial growth in either media. Both the waste-nutrient-grown microbial consortium and
Paracercomonas saepenatans can be grown effectively in BBM and WNS, and both remove most ions from both
media (e.g., > 99% removal of NO3− and 41–100% removal of PO43−) within 16 days. Significant nutrient
removal was observed during the growth phase of the microbial communities (4–10 days period), indicating
major nutrient utilization for microbial growth as well as chemical mineral precipitation. Furthermore, MINEQL
+ 4.6 modeling showed higher PO43− removal in WNS during microbial growth (compared to BBM) due to
precipitation of phosphate minerals (e.g., hydroxyapatite, vivianite). The dominant microbial species in both
systems were also identified. DNA sequencing showed that Vorticella (58%) and Scenedesmus (33%) in WNS and
Scenedesmus (89%) in BBM were the predominant species. This study demonstrates the potential application of
microbial consortium (predominantly algae and protozoan)-based treatment techniques for hydroponic systems.

Introduction contains highly concentrated nitrogen (N) (150–500 mg L−1) and

Over the last few decades, cultivation techniques for vegetables and
ornamentals have increasingly shifted toward open- and closed-loop
hydroponic systems. These involve a soilless cultivation technique ap-
propriate for greenhouse or plant factory systems, which are used to
conserve arable land in many countries in Europe and Asia [1–3]. In
such systems, solutions of essential nutrients must be provided to
achieve adequate plant growth; however, frequent reuse/recycling of
the nutrient solution without replenishment may result in a gradual
nutrient imbalance [4–6] in closed hydroponic systems. Hence, the
depleted nutrient solution is periodically discarded and replaced with a
new nutrient solution. The total area in use for hydroponic systems has
increased significantly, from 23 ha in 1993 to 1107 ha in 2008 [7], and
by now it has probably at least doubled. Hydroponic wastewater
phosphorous (P) (30–100 mg L−1) [8–10], resulting in increasing con-
cern about the enormous amounts of untreated waste nutrient solution
(WNS) being discharged from hydroponic systems into surrounding
environments including streams, rivers, and lakes.
WNS discharged from hydroponic systems in South Korea represents
a major source of N and P in aqueous environments. In general, WNS
contains relatively high concentrations, up to 100 s of mg L−1, of major
ions (e.g., K+, Mg2+, Ca2+, NO3−, PO43−, and SO42−) compared to
concentrations found in municipal and industrial wastewater [11].
Thus, the discharge of WNS into natural ecosystems can result in the
accumulation of excess nutrients (especially N and P), leading to con-
tamination or eutrophication of water bodies. This has significant ne-
gative impacts on aquatic life, water quality and agricultural develop-
ment, as well as human quality of life [12]. Although no specific

⁎
Corresponding author at: Korea University, 145 Anam-ro, Seongbuk-gu, Seoul 02841, Republic of Korea.
E-mail address: manjaekwon@korea.ac.kr (M.J. Kwon).

https://doi.org/10.1016/j.nbt.2017.11.003
Received 27 July 2017; Received in revised form 20 November 2017; Accepted 20 November 2017
Available online 21 November 2017
1871-6784/ © 2017 Elsevier B.V. All rights reserved.
J.Y. Lee et al.
regulations on WNS discharged from hydroponic systems have yet been
set in South Korea, general water-quality guidelines for total N and total
P recommend less than 60 mg L−1 (4.28 mM as N) and 8 mg L−1
(0.26 mM as P), respectively, in Korea [7]. It could be also reasonably
assumed that the WNS discharge limits in South Korea are similar to
those in the European Union (EU), as hydroponic systems are widely
used in Europe. The EU wastewater discharge limits for N are
10 mg L−1 as N (0.71 mM as N) to 15 mg L−1 as N (1.1 mM as N), while
those for P are 1 mg L−1 as P (0.03 mM as P) to 2 mg L−1 (0.065 mM as
P) [13]. In addition, to prevent significant deterioration of water
quality, many point sources have very low N and P limits under Na-
tional Pollutant Discharge Elimination System (NPDES) permits in the
US. It is therefore desirable to achieve very low levels of N and P in
WNS effluents [14].
Though different treatment processes (e.g., enhanced biological
phosphorus removal (EBPR), chemical (aluminum or iron)-based re-
moval, and algae-based techniques) have been studied extensively for
nutrient removal or recovery in municipal and industrial wastewater
systems [15–19], no such technologies have yet been applied for nu-
trient treatment of WNS. Different effective nutrient removal techni-
ques applicable in WNS systems must be developed for the wide range
of types of such systems as well as the hydroponic processes employed.
Several conventional wastewater treatment processes (e.g., biological
nutrient removal) are relatively expensive; however, algae-based
treatment, with demonstrated success for domestic wastewater treat-
ment, may be applicable to hydroponic wastewater treatment alongside
chemical mineral precipitation. Microbial (bacterial, microalgal and
protozoan) treatment has potentially high growth rates, with lower
operational and maintenance costs and better environmental and en-
ergy footprints compared to traditional EBPR and other chemical-based
removal techniques (e.g. alum, ferric chloride, etc.) [20]. Therefore, a
microbiological treatment in combination with chemical precipitation
provides an efficient, eco-compatible strategy for reducing water pol-
lution from hydroponic systems, including plant factories.
Photosynthetic microorganisms including microalgae have been
extensively investigated for N and/or P removal in industrial, muni-
cipal, and conventional agricultural wastewaters [17–19,21,22]. How-
ever, there have been few investigations into the use of microalgae for
hydroponic wastewater treatment. In addition, the combination of
microbial together with chemical mineral precipitation has not been
demonstrated for nutrient removal. Supersaturated chemicals could be
precipitated as chemical minerals, whereas undersaturated nutrients
could be used for microbial growth, together with other trace metals
and chemicals. In addition to the nutrient removal capabilities of
photosynthetic microbes, the biomass harvested after microbial culti-
vation can potentially provide valuable resources (added income
streams) including biodiesel, food supplements, and cosmetics [23–25].
Plant factories have recently drawn substantial public attention
because they are considered a sustainable resource for production of
valuable foods and medicines [26,27]. They provide controlled en-
vironmental conditions (e.g., temperature, light intensity, and hu-
midity) for plant growth, thereby avoiding daily and seasonal fluctua-
tions found in exterior environments [27]. The types and
concentrations of nutrients in WNS needed to support the growth of
photosynthetic microorganisms are well known [28]. Cultivation of
microalgae is usually performed in closed photobioreactors that are
similar to plant factory systems [22,29]; it is therefore very likely that
plant factories also provide the optimal atmospheric and nutrient con-
ditions for growing microalgae and other microbes.
Biological treatments for nutrient removal from municipal and in-
dustrial wastewater have been implemented successfully; however,
systems of this type have rarely been investigated for treating effluent
wastewater produced from hydroponic systems within plant factories.
In this study, the feasibility of using microbes adapted to the environ-
mental conditions of a plant factory for removal of excess nutrients in
WNS from plant factory effluent has been investigated. It had three
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New BIOTECHNOLOGY 41 (2018) 15–24
specific objectives: 1) to investigate the growth characteristics of a
microbial consortium and a strain of the flagellate protozoan
Paracercomonas saepenatans under plant factory conditions; 2) to ex-
amine the removal rate and efficiency of major and minor nutrients
during the growth phase of the microbes; and 3) to determine the types
of dominant microbial species applicable to WNS-based nutrient re-
moval systems.
Material and methods
Microbial taxa, culture conditions, and cultivation
A waste-nutrient-grown microbial consortium was originally ob-
tained from WNS arising from lettuce cultivation in a pilot-scale plant
factory system. The plant factory, which had approximately 100 m2 of
space equipped with vertical cultivation beds, a closed-loop nutrient
solution circulating system, an automatic climate control system, and a
light-emitting diode (LED) lighting system, was operated at the Korea
Institute of Science and Technology, Gangneung, South Korea.
Paracercomonas saepenatans strain DPS-1 was originally isolated from
CO2-rich spring water in the vicinity of Daejeon, South Korea.
The growth of the microbes (i.e., the microbial consortium and P.
saepenatans) and the nutrient removal rate and efficiency were ex-
amined in the WNS from the commercial nutrient solution used for
lettuce cultivation (Gafatech, S. Korea) and Bold’s Basal medium
(BBM), which is highly enriched and has been used for many green
algae, under two different experimental conditions (i.e., general la-
boratory conditions and plant factory conditions). The composition of
the WNS was as follows (mg L−1) KNO3 (405), Ca(NO3)2·4H2O (296),
MgSO4·7H2O (124), NH4H2PO4 (69), K2PO4 (44), and trace elements.
The composition of the BBM was as follows (mg L−1): KH2PO4 (175),
CaCl2·H2O (25), MgSO4·7H2O (75), NaNO3 (250), K2HPO4 (75), NaCl
(25), H3BO3 (11), and trace elements. Detailed chemical compositions
of the WNS and BBM are provided in Table S1. Culture bottles were
sterilized by autoclaving at 121 °C under pressure for 20 min, and batch
experiments were conducted using 250 mL serum bottles.
Approximately 4 mL of microbes (i.e., microbial consortium and P.
saepenatans) from the original culture stock were inoculated into
250 mL serum bottles with 200 mL of BBM or WNS. To prevent mi-
crobial contamination during aeration of the medium, ambient air was
passed through 0.2-μm nylon membrane filters (GE, Germany) at a rate
of 1.875 mL min−1, and then injected into the medium. The tops of the
bottles were covered with sterilized cotton. WNS was synthesized based
on the chemical composition of the effluent from the plant factory
system.
The experiments were conducted under two different conditions:
incubation inside the plant factory and incubation in a laboratory for 16
d, both under aerobic conditions. The control experiment (WNS without
microbial inoculum) was conducted under the same conditions. A
summary of the experimental matrix under each condition is provided
in Table 1.
Sampling
A liquid sample (2.5 mL) was taken from each bottle (total 24
bottles) with sterilized pipette tips. A 0.5 mL aliquot of the suspension
was used to measure the pH and optical density at 680 nm (OD680) up
to Day 16. The remaining 2 mL aliquot of suspension was immediately
filtered through a 0.2 μm nylon membrane filter (GE, Germany) to re-
move microorganisms and fine suspended particles and used for mea-
suring anions and cations. Subsamples (5 mL) were collected at the end
of the experimental period for chemical and microbial community
analysis. No more than six samples were taken from any incubation
batch; thus, the final volume of each incubation batch was approxi-
mately 90% of the original volume. The final volume in each incubation
batch was further affected by evaporation during bubbling.
J.Y. Lee et al. New BIOTECHNOLOGY 41 (2018) 15–24

Table 1
Experimental matrix performed in this study.

Conditions Culture Source of microbe(s) Medium Initial pH of media T (°C) Humidity (%) Light intensitya (μmol m−2 s−1)

Plant Factory Microbial consortium WNS of lettuce cultivation WNS 6.8 25.5 ± 0.5 47.5 ± 2.5 50
Laboratory Microbial consortium WNS 5.9 22.5 ± 1.5 25 ± 5 500 ± 100
Plant Factory Paracercomonas saepenatans CO2-rich spring water BBM 6.8 25.5 ± 0.5 47.5 ± 2.5 50
Laboratory Paracercomonas saepenatans BBM 5.9 22.5 ± 1.5 25 ± 5 500 ± 100

Note: WNS: waste nutrient solution; BBM: Bold's Basal medium; T: temperature.
a
Light to dark period: 12:12 h.

Fig. 1. Variation in pH (a, b) and optical density (c, d) during batch incubations in the plant factory (a, c) and laboratory (b, d).

Table 2
Removal rate of inorganic anions (PO43−-P, NO3−-N and SO42−) under laboratory and plant factory conditions from Days 0 to 4, Days 4 to 10, and Days 10 to 16 of operations.

Condition Matrix PO43−- P (mM d−1) NO3− −N (mM d−1) SO42− (mM d−1)

0–4 4–10 10–16 0–4 4–10 10–16 0–4 4–10 10–16

Lab Microbial consortium + BBM 0.02 0.09 0.05 0.55 0.30 0.01 0.01 0.04 0.01
Paracercomonas + BBM 0.01 0.20 −0.03 0.51 0.28 0.01 0.03 0.03 0.01
BBM (Control) 0.12 0.01 0.14 0.03 0.06 0.10 0.03 0.00 0.01
Microbial consortium + Waste nutrient solution (WNS) 0.06 0.08 0.00 1.26 0.86 0.03 0.00 0.06 0.00
Paracercomonas + WNS 0.00 0.11 0.00 1.31 0.65 0.01 0.00 0.00 0.03
WNS(Control) 0.03 −0.02 0.03 −0.21 −0.06 0.08 −0.03 0.00 0.01
Plant Factory Microbial consortium + BBM 0.07 0.10 −0.01 0.03 0.38 0.00 −0.04 0.05 0.00
Paracercomonas + BBM 0.09 0.06 0.02 0.23 0.22 0.00 0.01 0.02 0.01
BBM (Control) 0.13 −0.01 −0.01 −0.05 0.04 −0.03 −0.01 0.02 0.00
Microbial consortium + Waste nutrient solution (WNS) 0.06 0.08 0.00 −0.05 0.52 0.39 −0.03 0.03 0.04
Paracercomonas + WNS 0.10 0.06 0.00 0.06 0.61 0.23 −0.02 0.08 0.03
WNS (Control) 0.05 −0.02 0.01 −0.16 −0.02 −0.03 −0.06 0.05 0.01

Note: Negative sign indicates no removal; Paracercomonas indicates Paracercomonas saepenatans

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J.Y. Lee et al. New BIOTECHNOLOGY 41 (2018) 15–24

Table 3
Removal rate of inorganic cations (Ca2+, Mg2+, and Fe2+) under laboratory and plant factory conditions on Days 0 to 4, Days 4 to 10, and Days 10 to 16 of operations.

Condition Matrix Ca2+ (mM d−1) Mg2+ (mM d−1) Fe2+ (mM d−1)

0–4 4–10 10–16 0–4 4–10 10–16 0–4 4–10 10 v 16

Lab Microbial consortium + BBM −0.0126 0.0160 0.0045 0.0074 0.0211 0.0054 0.0003 0.0023 0.0003
Paracercomonas + BBM −0.0071 0.0195 −0.0001 0.0121 0.0249 −0.0016 0.0005 0.0015 −0.0002
BBM (Control) −0.0014 0.0030 0.0066 0.0203 0.0000 0.0091 0.0009 0.0002 −0.0006
Microbial consortium + Waste nutrient solution (WNS) −0.0632 0.2518 −0.0090 0.0009 0.0486 −0.0174 0.0079 0.0033 −0.0034
Paracercomonas + WNS 0.0045 0.1455 0.0140 0.0215 0.0467 0.0055 0.0139 0.0017 −0.0005
WNS (Control) −0.0359 0.0293 0.0294 0.0064 0.0079 0.0021 0.0085 0.0022 0.0003
Plant Factory Microbial consortium + BBM 0.0033 0.0247 −0.0042 0.0164 0.0368 −0.0125 0.0008 0.0026 −0.0003
Paracercomonas + BBM −0.0028 0.0142 0.0065 0.0249 0.0185 0.0066 0.0010 0.0014 0.0007
BBM (Control) −0.0099 0.0041 0.0031 0.0066 0.0025 0.0028 −0.0002 0.0004 0.0003
Microbial consortium + Waste nutrient solution (WNS) −0.0488 0.1087 0.1028 0.0086 0.0285 0.0113 0.0064 0.0077 −0.0004
Paracercomonas + WNS −0.0272 0.0861 0.0596 0.0157 0.0247 0.0183 0.0057 0.0072 0.0005
WNS (Control) −0.0121 0.0195 0.0121 0.0176 0.0052 0.0051 0.0068 0.0035 0.0015

Note: Negative sign indicates no removal; Paracercomonas indicates Paracercomonas saepenatans.

Analytical methods
The solution pH was measured using an Orion Star A325 pH Meter
(Thermo Scientific, USA). Microbial growth was measured via optical
density using a spectrophotometer at 680 nm (Hach DR/2800,
Loveland, CO, USA). Light intensity was analyzed using a Light Meter
LI-250A. The concentrations of nitrate (NO3−-N), phosphate (PO43−-
P), and sulfate (SO42−) were determined using single-column ion
chromatography (Metrohm 850 Professional, IC, Switzerland). Cations
were analyzed using a Varian 730-ES inductively coupled plasma-op-
tical emission spectrophotometer (ICP-OES, PerkinElmer SCIEX, USA)
after acidification with 1% (v/v) nitric acid. Ammonium was analyzed
using an ammonium assay [30].
temperature changes for each experimental matrix. Temporal variations
in the concentrations of nitrate, phosphate, and other chemicals for
each experimental matrix were plotted using the color-fill contour from
OriginPro 8 to confirm the Pearson correlation coefficients. The p-value
at the 5% significance level was used to determine the statistical sig-
nificance of nutrient removal efficiency for each experimental matrix.
In addition, the precipitation potentials (saturation indices) of the mi-
nerals in BBM and WNS were calculated using the water quality mod-
eling program MINEQL+ version 4.6. The saturation index (a loga-
rithmic value of the ratio between the ion product of the solid and
solubility constant of a particular solid) was used to indicate the sa-
turation condition of BBM and WNS. Each mineral phase can poten-
tially be precipitated when its saturation index (SI) is > 0.

Microbial community analysis

Results and discussion

DNA extraction
Subsamples (2 mL) were collected for microbial community analysis
at the end of the experimental period. The subsamples were dispensed
into a sterile micro-centrifuge tube and immediately stored at −80 °C
to inactivate biological activity before the laboratory analysis. Total
genomic DNA was extracted using an i-genomic Soil DNA Extraction
Mini Kit (iNtRON, South Korea) with a bead-beating apparatus ac-
cording to the manufacturer’s instructions. The DNA concentration was
quantified using a Qubit fluorometer (Invitrogen, USA) according to the
manufacturer’s instructions.
Clone library, sequencing, and sequence analysis
Clone library construction, sequencing, and sequence analysis were
conducted following a previous study [31], with minor modifications.
Briefly, for PCR amplification of bacterial DNA, 8F primer (5′-AGAGT
TTGATCMTGGCTCAG-3′) (10 μM) and 1492R primer (5′-TACGGYTAC
CTTGTTACGACTT-3′) (10 μM) were used, whereas primers 109F
(5′-ACKGCTCAGTAACACGT-3′) and 912R (5′-CTCCCCCGCCAATTCCT
TTA-3′) were used for amplification of eukaryotic DNA. Clone libraries
were prepared using the T-Blunt™ PCR Cloning Kit (with DH5a Com-
petent Escherichia coli; Solgent, Korea); ∼50 colonies per plate were
chosen randomly. The sequencing was performed by Marcrogen (Seoul,
South Korea). The sequences were trimmed using DNA Baser (Heracle
BioSoft, Romania) and also screened with the Mallard program [32].
The sequences screened were blasted against publicly available se-
quences within the Ribosomal Database Project (RDP) Release 11 [33].
Statistical analysis, contour plot, and saturation index calculation
The Pearson correlation coefficient was employed (using SYSTAT
10.2) to determine potential relationships of nutrient concentrations,
including micronutrients on each operational day, with pH and
Dynamics of pH and nutrient removal during microbial growth
The pH of WNS and BBM increased in the presence of the microbial
consortium and P. saepenatans (Fig. 1), as expected, due to microbial
growth. After 2 days, the solution pH value rapidly increased from 6.1
to 10 during the WNS treatment with the microbial consortium (Fig. 1a
and b). Increases in pH might be due to microbial photosynthesis, re-
sulting in consumption of inorganic carbon such as HCO3− and accu-
mulation of OH− in the solution [34].
OD680 measurements showed that the microbes grew well in both
WNS and BBM (Fig. 1c and d). The growth rates varied, and they were
strongly dependent on the media type, whether a microbial consortium
or a strain of P. saepenatans. In general, P. saepenatans grew faster, but
the microbial consortium grew more. The growth of the microbial
consortium ended on Day 10 and subsequently the OD680 of P. saepe-
natans decreased. No significant change in pH value from Day 10 to Day
16 was observed, as the growth of the microbes was limited during that
period. Based on these microbial activity changes, three distinct phases
of microbial growth were observed: 0–4 days (lag phase), 4–10 days
(exponential growth or log phase), and 10–16 days (stationary or decay
phase).
The removal rates of major anions and cations from the WNS and
BBM were calculated, along with the specific growth period of P. sae-
penatans and the microbial consortium (Tables 2 and 3) during the three
phases. Though Paracercomonas growth started after 2 days, the
0–4 days lag phase was employed for removal comparison with the
microbial consortium. Both synthetic media have a number of major
ions (e.g., NO3̄N, PO4̄3– P, SO42−, Ca2+, Mg2+, and Fe2+) (Table S1) that
are nutrients essential for cell growth, synthesis, cell repair and other
cell maintenance. After a 4 days adaptation period, P. saepenatans and
the microbial consortium grew exponentially to Day 10 (maximum cell

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J.Y. Lee et al. New BIOTECHNOLOGY 41 (2018) 15–24

Fig. 2. Variation in NO3−-N concentrations in BBM (a, b) and waste nutrient solutions (WNS) (c, d) during batch incubations in the plant factory or laboratory. Variation in PO43−-P
concentrations in BBM (e, f) and WNS (g, h) during batch incubations in the plant factory or laboratory.

density of 1–2 OD680) after which the cells entered a static or death
phase. In general, the removal trends of NO3−-N matched well with the
microbial growth phases, suggesting that most of the N-removal me-
chanism was actually used for microbial growth. The SO42− and Ca2+
removal trends also matched closely with the microbial growth rates.
Meanwhile, the removal rates of PO43-P, Mg2+, and Fe2+ did not
clearly match the growth phase.
Nutrient removal by microbial uptake and chemical precipitation
The nutrient removal kinetics and efficiency of the microbial con-
sortium and P. saepenatans in the plant factory and under laboratory

19
J.Y. Lee et al.
Fig. 3. Removal efficiency of dissolved anions (a) and cations (b) in the plant factory (P)
and laboratory (L) using a microbial consortium and Paracercomonas saepenatans in BBM
and WNS media (including control medium). The nutrient removal efficiency was cal-
culated based on the data of Days 0 and 16.
conditions are shown in Figs. 2 and 3. Fig. 2 shows the removal char-
acteristics of NO3− and PO43− under different conditions. The nutrient
removal efficiency differed significantly between the two media (WNS
and BBM) as well as between the two environmental conditions (plant
factory and laboratory conditions). However, comparison between the
microbial growth experiments and the control experiments (the absence
of microbes) clearly showed that microbial uptake was a dominant
mechanism for nutrient removal from WNS and BBM (Fig. 2). NO3−-N
was completely removed within 16 days in the presence of microbes
(Fig. 2a–d), and the low concentration of ammonium (NH4+;
∼0.2 mM) was also rapidly removed within 8 days in WNS in the
presence of microbes (data not shown). Furthermore, Fig. 3 shows the
removal efficiencies of NO3−, PO43−, SO42−, Ca2+, Mg2+, and Fe2+ in
the plant factory and under laboratory conditions. In the case of SO42−,
39–95% removal was observed by both the microbial consortium and P.
saepenatans in both WNS and BBM (Fig. 3).
Interestingly, the concentrations of PO43−-P, Ca2+, Mg2+, and Fe2+
decreased over time, even in the absence of microbes. For instance,
PO43−-P in BBM and WNS decreased by 18–45% and 19–22%, re-
spectively, in the absence of microbes (Fig. 2). These results suggest
that the removal of PO43− from BBM and WNS occurred both due to the
microbial consortium or P. saepenatans and by precipitation of PO43−.
This is likely to be due to differences in the composition of the medium,
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New BIOTECHNOLOGY 41 (2018) 15–24
as well as to changes in the physical and chemical conditions in the
aqueous solution during incubation. Previous studies have reported P-
recovery or removal from wastewater through crystallization of struvite
(MgNH4PO4·6H2O), hydroxyapatite (Ca10(PO4)6(OH)2), and vivianite
(Fe3(PO4)2·8H2O) [35–37]. Chemical equilibrium modeling with
MINEQL+ 4.6 showed that hydroxyapatite could be the dominant sink
for P within the experimental pH range during microbial uptake in both
BBM and WNS (SI > 0). Several other phosphate minerals (e.g., vivia-
nite) could be supersaturated in WNS (compared to levels in BBM) and
thus could contribute to the higher percentage of P removal in WNS
during microbial growth.
As with PO43−-P, the removal efficiency of Ca2+ from WNS was
significantly higher than that from BBM, suggesting that some of the
Ca2+ was removed by the precipitation of hydroxyapatite
(Ca10(PO4)6(OH)2). This was confirmed by MINEQL+ modeling.
Battistoni et al. [35] also reported that NH4+, Ca2+, Mg2+, and PO43−
in aqueous solution in the pH range 8–10 can produce insoluble pre-
cipitates (e.g., struvite and hydroxyapatite). Hydroxyapatite is formed
preferentially over struvite if sufficient Ca2+ coexists [38]. This phe-
nomenon coincides with the results of an equilibrium simulation.
MINEQL+ modeling confirmed that hydroxyapatite was the dominant
calcium and phosphorus sink (SI > 0), whereas struvite was under-
saturated (SI < 0) in both BBM and WNS. The results also showed that
the microbes may utilize Mg2+ directly, but it is also possible that some
amount of Mg2+ was removed by the precipitation of minerals such as
magnesium phosphate, dolomite, or huntite.
The Fe2+ concentration in WNS, in the absence of microbes, rapidly
and remarkably decreased over time (70–72% removal). This suggests
that the physical and chemical conditions in WNS favored iron pre-
cipitation. In general, iron in the medium is provided in the form of
chelated complexes such as ethlenediaminetetraacetic acid (FeEDTA)
because Fe2+ is easily oxidized to Fe3+, which is then precipitated in
the form of iron hydroxides [39]. MINEQL+ modeling of the current
study showed that several iron minerals (i.e., vivianite and siderite)
were supersaturated (SI > 0) under the pH conditions in WNS and
could potentially have precipitated during the microbial uptake ex-
periment.
In addition to the removal of these dissolved trace elements by
chemical precipitation, removal could also have occurred by different
mechanisms, including cell wall sorption and intracellular accumula-
tion [40,41].
Correlation of nutrient concentrations with pH and temperature
The Pearson correlation coefficients of nutrient concentrations with
pH and temperature are summarized in Tables S2 and S3. The pH was
strongly negatively correlated with ion concentration regardless of the
specific microbial and nutrient (medium) composition. This suggests
that microbial growth resulted in an increase in pH. Interestingly, the
temperature showed poor correlation with ion concentrations under
plant factory conditions and was positively correlated with ion con-
centrations (except PO43−) under laboratory conditions. The relatively
low and variable temperature in the laboratory compared to conditions
in the plant factory suggests that microbial growth was affected by
temperature in the laboratory and that microbes removed nutrient ions
accordingly. The poor correlation between PO43− and other ions sug-
gests that PO43− was removed both by microbes and by the precipita-
tion of phosphate mineral(s), as described in the previous section.
Contour plots were created to better understand the relationship of
removal of specific nutrient ions with pH and temperature under plant
factory and laboratory conditions (Figs. 4 and 5). Positive correlations
were found between pH value and decreased nutrient ion concentra-
tions in successive time intervals. P. saepenatans strain grew more and
with a limited change in pH over time compared to the microbial
consortium under plant factory conditions. The pH changes for the
microbial consortium showed greater variation during the growth
J.Y. Lee et al.
Fig. 4. Temporal variation in NO3− and PO43− concentrations (in mg L−1 as N and P) with pH under plant factory conditions for a microbial consortium (‘Micro’) and Paracercomonas
saepenatans (‘Par’).
phase, indicating that competition between the bacterial and algal
populations for N and P removal may alter the pH. The opposite trend
was observed under laboratory conditions, and higher nutrient re-
movals were observed by the protozoan strain. There was more varia-
tion in the pH in this case, which may be associated with the lower
temperatures under laboratory conditions (Table 1) compared to plant
factory conditions.
Stoichiometric coefficient for nitrate and phosphate ions
The stoichiometric equation for microbial growth through photo-
synthetic (forward reactions) and respiratory (reverse reactions) pro-
cesses in natural waters was described by [42]. In that work, the mi-
crobial chemical formula (C106H263O110N16P1) was given by the
Redfield ratio. The following reaction was considered applicable for the
pH value range of 7 to 9 for algal growth:
106HCO3− + 16NO3− + HPO42 − + 16H2 O + 124H+
⇔ C106 H263 O110 N16 P1 + 138O2.
The stoichiometric reaction clearly indicates that the uptake of
HCO3− increases the pH in the media, as was observed under both plant
factory and laboratory conditions. Photosynthesis drives the pH higher
as long as NO3− is the major N source for microbes, a principle that is
applicable to both WNS and BBM. The actual stoichiometric uptake of
NO3− and PO43− was calculated for the growth phase (Days 4–10) in
WNS and BBM for comparison with the theoretical stoichiometric
21
New BIOTECHNOLOGY 41 (2018) 15–24
uptake expected for microbial growth based on the growth equation
using the measured pH values. The changes in NO3−, PO43−, and pH
were considered between Day 4 and Day 10 (the logarithmic growth
phase). Table 4 summarizes the actual stoichiometric uptake for NO3−
and PO43− under plant factory and laboratory conditions. The results
suggest that P. saepenatans had a higher PO43− uptake rate under both
plant factory and laboratory conditions compared to the microbial
consortium in BBM. This indicates that P. saepenatans required more
PO43− for growth than did the microbial consortium. On the other
hand, the microbial consortium consumed more PO43− than did P.
saepenatans under plant factory conditions in WNS. The opposite pat-
tern of PO43− consumption by the microbial consortium was observed
under laboratory conditions in the same medium. In addition, PO43−
was removed in higher amounts than was the stoichiometric PO43−
required for microbial growth (NO3−:PO43− = 16:1) (Table 4), sug-
gesting PO43− removal through mineral precipitation and further re-
inforcing the MINEQL+ modeling results.
Microbial community composition after nutrient removal
The community composition of the microbial consortium in WNS
and BBM on Day 16 was investigated. DNA sequencing showed that
Vorticella (ciliate protozoan, 58%) and Scenedesmus (green alga, 33%) in
WNS, and Scenedesmus (89%) in the BBM (Fig. 6) were the predominant
microbial eukaryote species, while Brevundimonas (37%) and Sphingo-
monas (4%) in WNS, and Sphingobacterium (13%), Brevundimonas
J.Y. Lee et al. New BIOTECHNOLOGY 41 (2018) 15–24

Fig. 5. Temporal variation in NO3− and PO43− concentrations (mg L−1 as N and P) with pH under laboratory conditions for a microbial consortium (‘Micro’) and Paracercomonas
saepenatans (‘Par’).

Table 4
Stoichiometric coefficient calculated from measured NO3−, PO43−, and pH data for comparison with theoretical stoichiometric coefficients for evaluating the microbial growth and
environmental conditions.

Experimental setup Plant Factory Laboratory Comments

NO3− PO43− NO3− PO43−

Microbial consortium + BBM 16 4.01 No data Paracercomonas had more phosphate uptake than did the microbial consortium in BBM at the
Microbial consortium + BBM 16 4.80 16 3.99 plant factory and laboratory
Paracercomonas + BBM 16 5.13 16 8.68
Paracercomonas + BBM 16 3.53 16 9.64
Microbial consortium + Waste nutrient 16 2.42 16 1.23 The microbial consortium consumed more phosphate than did Paracercomonas in WNS
solution (WNS) medium at the plant factory and less at the laboratory
Microbial consortium + Waste nutrient 16 2.19 16 1.37
solution (WNS)
Paracercomonas + WNS 16 1.87 16 2.49
Paracercomonas + WNS 16 1.44 16 2.08

Note: Paracercomonas indicates Paracercomonas saepenatans

(10%), and Stenotrophomonas (12%) in BBM were the dominant bac-
terial species. Despite the presence and diversity of bacteria, the most
abundant species were Scenedesmus (green alga) and Vorticella sp.
(ciliate protozoan), which have been widely investigated due to their
mixotrophic metabolic activity [43,44]. Microalgae and ciliate proto-
zoans capable of mixotrophic metabolism can assimilate both inorganic
and organic substrates through concurrent respiratory and photo-
synthetic processes [44,45]. Thus, the abundance of Vorticella and/or
Scenedesmus sp. after nutrient removal suggests that these species are
able to out-compete other microbial species and have great potential for
waste nutrient treatment from hydroponic systems. The role of Vorti-
cella and/or Scenedesmus sp. could be further implemented in different
types of hydroponic systems as inoculum for enhancing the rate and
extent of nutrient removal. Enrichment of Vorticella and/or Scenedesmus
sp. could be easily adapted to different plant factory conditions, as
shown by the different experimental conditions in this study.

22
J.Y. Lee et al.
Fig. 6. Microbial community analysis of eukaryote (a and b) and prokaryote (c and d) in WNS and BBM on Day 16.
It is also possible that removal efficiency might be influenced by
other bacterial species due to their presence as observed here. Since the
main focus of this study was to treat hydroponic wastewater by a mi-
crobial mixed culture predominantly of algae and protozoa, the pre-
sence of bacterial species confirmed that this type of wastewater can be
treated by algal and protozoan species together with bacteria.
Furthermore, it is evident that there might be specific roles for different
types of microorganisms (bacteria, protozoa, microalgae and fungi) on
nutrient removal efficiency which requires further investigation.
Potential of photosynthetic microbes for the treatment of waste nutrients in
plant factory systems
The plant factory system examined was designed to provide the
optimal environmental conditions (temperature, light intensity, hu-
midity) for lettuce growth. These same conditions are likely to support
faster growth of photosynthetic microbes. Although the light intensity
in the plant factory was much lower than that under the laboratory
conditions, the rate and extent of microbial growth was similar to that
in the laboratory regimes. These results suggest that other environ-
mental conditions (i.e., relatively constant temperature and humidity,
and the specific red/blue LED light) were favorable for the growth of
microbes under plant factory conditions. In addition, the growth of the
microbial consortium in WNS was similar to, or even greater than, that
in BBM. As a result, the waste nutrient ions produced from the plant
factory were effectively treated by the microbial consortium (or P.
saepenatans) and were reduced to below the regulatory guidelines
within several days. The uninhibited microbial growth of enriched
microorganisms (e.g., Vorticella and/or Scenedesmus sp.) in mixed-cul-
ture conditions in the plant factory and the laboratory demonstrates the
robustness of microbial treatment in hydroponic systems. Furthermore,
P. saepenatans growth in BBM and WNS confirms its ability to survive
and perform well under real-life conditions.
23
New BIOTECHNOLOGY 41 (2018) 15–24
Conclusions
Microbial consortium- or P. saepenatans-mediated WNS treatment
could be a cost-effective and eco-compatible treatment technology for
simultaneous N and P removal from waste nutrient effluents from a
plant factory. The removal efficiency of major waste nutrients from
WNS was approximately 100% of NO3−-N and PO43−-P within 16 days
for plant factory systems. The removal of major and trace nutrients was
mainly due to microbial uptake and/or chemical precipitation.
Vorticella and Scenedesmus in WNS were the dominant species in the
microbial consortium and should be potential targets for future cutting-
edge research on microbial-based water treatment processes. In addi-
tion, the identification of dominant photosynthetic algal and protozoan
species capable of nutrient removal under WNS conditions could pro-
vide significant guidelines to the hydroponic industry.
Disclosure statement
No potential conflicts of interest is reported by the authors.
Acknowledgements
This work was supported by the KIST Open Research Program
(Grant no. 2E25701) and a National Research Council of Science &
Technology (NST) grant from the Korean government (MSIP) (No. CRC-
15-01-KIST).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at https://doi.org/10.1016/j.nbt.2017.11.003.
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