NewBIOTECHNOLOGY41(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

ARTICLEINFO
ABSTRACT
Keywords:
The potential of microbial processes for removal of major nutrients (e.g., N, P) and inorganic cations (e.g., Ca2+,
Algae
Hydroponic system
Mg2+, and Fe2+) from hydroponic systems was investigated. Microbial consortium- and axenic culture-
Nitrogen and phosphorous uptake based experiments were conducted in a waste nutrient solution (WNS). A microbial consortium grown in the
Microbial community WNS and selected microalgae species of PARACERCOMONAS SAEPENATANS were inoculated in two different
Mineral precipitation synthetic media (Bold’s Basal Medium (BBM) and synthetic WNS) in batch systems, and the microbial growth
characteristics and 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 PO 3−) within
16 days. Significant nutrient removal was observed during the growth 4 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 PO 43− 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
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
contains highly concentrated nitrogen (N) (150–500 mg L−1) and
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+, NO −, PO 3−, and SO 2−) compared to
3 4 4
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: m a n j a e k w o n @ k o r e a . a c . k r (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
Availableonline2
1November2017
1871-6784/©2017ElsevierB.V.Allrightsreserved.
J.Y. Lee et AL. NewBIOTECHNOLO
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mental conditions of a plant factory for removal of excess nutrients in
regulations on WNS discharged from hydroponic systems have yet WNS from plant factory effluent has been investigated. It had three
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-

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incubation batch was approxi- mately 90% of the original volume. The
specific objectives: 1) to investigate the growth characteristics of final volume in each incubation batch was further affected by
a microbial consortium and a strain of the flagellate evaporation during bubbling.
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

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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, NO 3−-N and SO 2−
4 ) 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

J.Y. Lee et AL. NewBIOTECHNOLO
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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 −0.0632 0.2518 −0.0090 0.0009 0.0486 −0.0174 0.0079 0.0033 −0.0034
(WNS)
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 −0.0488 0.1087 0.1028 0.0086 0.0285 0.0113 0.0064 0.0077 −0.0004
(WNS)
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 (NO −-N), phosphate (PO 3−-
3 4
P), and sulfate (SO4 2−) 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 DYNAMICS of pH AND nutrient REMOVAL during MICROBIAL growth

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
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
3
as HCO − 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, PO 3P, SO 2−, Ca2+, Mg2+, and Fe2+) (Table S1) that
4̄– 4
10.2) to determine potential relationships of nutrient concentrations,
J.Y. Lee et AL. NewBIOTECHNOLO
GY41(2018)15–24
including micronutrients on each operational day, with pH and 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
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 PO 43−-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
clearly match the growth phase.
phase. In general, the removal trends of NO −-N matched well with the
3
microbial growth phases, suggesting that most of the N-removal me-
Nutrient REMOVAL by MICROBIAL UPTAKE AND CHEMICAL PRECIPITATION
chanism was actually used for microbial growth. The SO 2− and Ca2+
removal trends also matched closely with the microbial 4growth rates.
Meanwhile, the removal rates of PO 3-P, Mg2+, and Fe2+ did not The nutrient removal kinetics and efficiency of the microbial
4
con- sortium and P. SAEPENATANS in the plant factory and under
laboratory

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.
3
2). NO −-N was completely removed within 16 days in the presence of
microbes (Fig. 2a–d), and the low concentration 4 of
ammonium (NH +;
∼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−, PO 3−, SO 2−, Ca2+, Mg2+, and Fe2+ in
4 4 2−
the plant factory and under laboratory conditions. In the case of SO4 ,
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,
PO4 3−-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
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 PO4 3−-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 NH , Ca2+, Mg2+, and PO 3−
4 4
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
accordingly.in thepoor
The laboratory and that
correlation microbes
between PO removed
3− nutrient
and other ionsions
sug-
4
of PO43−. This is likely to be due to differences in the composition of the
medium,
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
Fig. 4. Temporal variation in NO 3− and PO 43− 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

uptake expected for microbial growth based on the growth
populations for N and P removal may alter the pH. The opposite trend
equation using the measured pH values. The changes in NO3−, PO 3−,
was observed under laboratory conditions, and higher nutrient re-
and pH were considered between Day 4 and Day 4 10 (the
movals were observed by the protozoan strain. There was more varia- logarithmic growth phase). Table 4 summarizes the actual
tion in the pH in this case, which may be associated with the lower stoichiometric uptake for NO − and PO 3− under plant factory and 3
temperatures under laboratory conditions (Table 1) compared to plant laboratory
4 conditions. The results suggest that P. SAEPENATANS had a
factory conditions. higher PO 3− uptake rate under both plant4 factory and laboratory
conditions compared to the microbial
Stoichiometric coefficient for NITRATE AND PHOSPHATE ions consortium in BBM. This indicates that P. SAEPENATANS required
more PO43− for growth than did the microbial consortium. On the
The stoichiometric equation for microbial growth through photo- other hand, the microbial consortium consumed more 4 PO 3− than
synthetic (forward reactions) and respiratory (reverse reactions) pro- did P. SAEPENATANS under plant factory conditions in WNS. The
cesses in natural waters was described by [42]. In that work, the mi- opposite pat-
4
tern of PO 3− consumption by the microbial consortium
crobial chemical formula (C H O N P ) was given by the was observed under laboratory conditions in the same medium. In
addition, PO43−
was removed in higher amounts than was the stoichiometric PO43−
Redfi
10 263 110 16 1 required for microbial growth (NO −:PO 3− = 16:1) (Table 4), sug-
6 3 4
eld ratio. The following reaction was considered applicable for the
pH value range of 7 to 9 for algal growth: that is applicable to both WNS and BBM. The actual stoichiometric
uptake of NO − and PO 3− was calculated for the growth phase (Days
106HCO − + − 2− 4–10) in
+
16NO + HPO
C106 H263 O110 N16 P1 + 138O 2.

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 long3as NO − is the major N source for microbes, a principle

3 4
gesting PO 3− removal through mineral precipitation and further re-
inforcing the MINEQL+ modeling results.
MICROBIAL community composition AFTER nutrient REMOVAL
WNS and BBM for comparison with the theoretical stoichiometric
The community
4 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
Fig. 5. Temporal variation in NO3 − and PO4 3− 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 NO −,3PO 3−, 4and pH data for comparison with theoretical stoichiometric coefficients for evaluating the microbial growth and
environmental conditions.

Experimental setup Plant Factory Laboratory Comments

4 3 4
NO3− PO 3− NO − PO 3−
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 [44,45]. Thus, the abundance of VORTICELLA and/or
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
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.
 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

Conclusions
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-
Microbial consortium- or P. SAEPENATANS-mediated WNS treatment
crobial mixed culture predominantly of algae and protozoa, the pre-
could be a cost-effective and eco-compatible treatment technology for
sence of bacterial species confirmed that this type of wastewater can be
simultaneous N and P removal from waste nutrient effluents from
treated by algal and protozoan species together with bacteria.
a plant factory. The removal efficiency of major waste nutrients
Furthermore, it is evident that there might be specific roles for
from WNS was approximately 100% of NO3−-N and 4 PO
3−
-P within 16
different types of microorganisms (bacteria, protozoa, microalgae and
fungi) on nutrient removal efficiency which requires further days for plant factory systems. The removal of major and trace nutrients
investigation. 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
POTENTIAL of photosynthetic microbes for the TREATMENT of WASTE nutrients in
processes. In addi- tion, the identification of dominant photosynthetic
PLANT FACTORY systems
algal and protozoan species capable of nutrient removal under WNS
conditions could pro- vide significant guidelines to the hydroponic
The plant factory system examined was designed to provide the
industry.
optimal environmental conditions (temperature, light intensity, hu-
midity) for lettuce growth. These same conditions are likely to support
Disclosure statement
faster growth of photosynthetic microbes. Although the light intensity
in the plant factory was much lower than that under the
No potential conflicts of interest is reported by the authors.
laboratory conditions, the rate and extent of microbial growth was
similar to that in the laboratory regimes. These results suggest that
Acknowledgements
other environ- mental conditions (i.e., relatively constant temperature
and humidity, and the specific red/blue LED light) were favorable for
This work was supported by the KIST Open Research Program
the growth of microbes under plant factory conditions. In addition, the
(Grant no. 2E25701) and a National Research Council of Science &
growth of the microbial consortium in WNS was similar to, or even
Technology (NST) grant from the Korean government (MSIP) (No.
greater than, that in BBM. As a result, the waste nutrient ions
CRC- 15-01-KIST).
produced from the plant factory were effectively treated by the
microbial consortium (or P. SAEPENATANS) and were reduced to
Appendix A. Supplementary data
below the regulatory guidelines within several days. The
uninhibited microbial growth of enriched microorganisms (e.g.,
Supplementary data associated with this article can be found, in
VORTICELLA and/or Scenedesmus sp.) in mixed-cul- ture conditions in the
the online version, at https://doi.org/10.1016/j.nbt.2017.11.003.
plant factory and the laboratory demonstrates the robustness of
microbial treatment in hydroponic systems. Furthermore,
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