Science of the Total Environment 757 (2021) 144189

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Science of the Total Environment

journal homepage: www.elsevier.com/locate/scitotenv

Green walls to treat kitchen greywater in urban areas: Performance from

a pilot-scale experiment
Nicola Dal Ferro a,⁎, Chiara De Mattia a, Mario Andres Gandini b, Carmelo Maucieri a, Piergiorgio Stevanato a,
Andrea Squartini a, Maurizio Borin a
a
Department of Agronomy, Food, Natural resources, Animals and Environment, University of Padova, Viale dell'Universita' 16, 35020 Legnaro, Padova, Italy
b
Department of Energy and Mechanics, Universidad Autónoma de Occidente, Calle 25 No. 115-85, Cali, Colombia

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• Green wall systems can be used to treat

greywaters in urban environments.
• Kitchen greywater pollutants should be
recycled for non-potable uses.
• Green wall greywater treatment re-
quires improvements in nutrient bal-
ance valuation.
• Relative abundance of bacteria com-
munities is indicator of treatment
effectiveness.

a r t i c l e i n f o a b s t r a c t

Article history: An increase in water use in urban areas is forcing scientists and policy makers to find alternative solutions for
Received 23 August 2020 freshwater management, aimed at attaining integrated water resources management. Here, we tested in a 2-
Received in revised form 28 November 2020 year experiment (June 2017–April 2019) the treatment performance of an innovative wall cascade constructed
Accepted 30 November 2020
wetland (WCCW) system. The aim was to combine the multifunctional benefits of green walls (e.g. aesthetic, sur-
Available online 4 December 2020
face area requirements) with those of constructed wetland systems (e.g. high pollutants removal efficiencies,
Editor: Paulo Pereira water recycling) to treat kitchen greywaters. The WCCW was a terraced system of six phytoremediation lines,
each of which was composed of three plastic tanks (3 × 0.04 m3), filled with lightweight porous media, and veg-
Keywords: etated with different ornamental species, namely Mentha aquatica L., Oenanthe javanica (Blume) DC., and
Constructed wetlands Lysimachia nummularia L. Physicochemical (temperature, pH, electrical conductivity, dissolved oxygen, turbidity)
Green infrastructures and chemical parameters (chemical oxygen demand, biochemical oxygen demand, anionic surfactants, Kjeldahl,
Microbial analysis ammonium and nitric nitrogen, total orthophosphate) were monitored at a frequency of at least 15 days, depend-
NGS sequencing ing on the season and WCCW management. Results showed that the WCCW significantly reduced the main water
Nutrients
pollutants (e.g. organic compounds, nutrients), suggesting its potential application in urban environments for
Urban areas
water recycling in the context of green infrastructures and ecological sanitation. A culture-independent taxo-
nomic assessment of suspended bacterial communities before and after the treatment showed clear
treatment-related shifts, being the functional ecology attributes changed according to changes in greywater

⁎ Corresponding author.
E-mail address: [email protected] (N. Dal Ferro).

https://doi.org/10.1016/j.scitotenv.2020.144189
0048-9697/© 2020 Elsevier B.V. All rights reserved.
N. Dal Ferro, C. De Mattia, M.A. Gandini et al. Science of the Total Environment 757 (2021) 144189

chemical parameters. Future research should attempt to optimize the WCCW system management by regulating
the nutrients balance to avoid macronutrients deficiency, and setting the most suitable water flow dynamics (hy-
draulic retention time, saturation-desaturation cycles) to improve the greywater treatment.
© 2020 Elsevier B.V. All rights reserved.

1. Introduction of 60 physical (e.g. suspended solids), chemical (e.g. pH, COD, BOD,
metals and metalloids) and biological (e.g. Escherichia coli, Salmonella)
Green infrastructures (GIs) are increasingly demanded and broadly parameters, is more specific by far than many others around the
considered environmentally friendly and low-cost solutions that can world (Salgot et al., 2006).
provide multiple ecosystem services and human well-being (Bortolini An efficient green wall system is based on reliable species that
and Zanin, 2018; Tzoulas et al., 2007). Freshwater availability is of a should tolerate the local climate, not be prone to suffering diseases, nu-
major concern in dense urban settlements. In addition, its disposal trient deficiency, or chemical toxicity. Plant selection for green walls is
poses serious environmental and health uncertainties more particularly also closely related to the growing substrate and its volume which influ-
where greywater disposal has a potential of polluting both surface and ences both the size of the plants that can be grown and, to some extent,
underground water sources. In this context, GIs can improve greywater how much water will be available for plants. Finally, wastewater quality
treatment, enhance the hydrological cycle, as well as the aesthetics and under restricted growth conditions might compromise the vegetation
enjoyment of urban areas (Prodanovic et al., 2017; Zanin et al., 2018). development, with consequences in terms of belowground root growth
Moreover, several studies suggest that for any given urban density, apparatus, and aboveground translocation capacity of pollutants as well
with appropriate consideration as regards the proportion and configu- as aesthetics (Manso and Castro-Gomes, 2015; Prodanovic et al., 2019).
ration of green space and tree cover, there is substantial scope for max- By extending CW technology to green walls, we designed and monitored
imizing ecological performance (Panagopoulos et al., 2016; Zhang and an innovative wall cascade constructed wetland system (WCCW). To our
Chui, 2019). Traditional constructed wetlands (CWs), either vertical, knowledge, this is a pioneering study where ornamental species were
horizontal, or hybrid, are green wastewater treatment solutions, tested in a living wall with the aim of: (i) testing its performance in
whose applicability in dense urban areas has been hindered by high treating kitchen greywaters under real-world conditions; (ii) under-
space demand (Foladori et al., 2013; Ghosh and Gopal, 2010; Kivaisi, standing to what extent different ornamental aquatic plant species can
2001). Generally, horizontal flow CWs are sized in Europe with 5 m2 affect the pollutant removal processes.
per population equivalent (PE), while vertical flow CWs are more effi-
cient although they still require 2–4 m2 per PE (Dotro et al., 2017). 2. Material and methods
Green walls, also called vegetated walls, have been proposed as GIs
that can control the temperature (T) in residential and office buildings 2.1. Experimental set-up
of urban areas, improve air quality, increase urban biodiversity, and
add to the amenities of urban spaces (Manso and Castro-Gomes, The WCCW was set up in June 2017 at the “Agripolis” campus of the
2015; Perini et al., 2011). However, growing vegetation in media- University of Padova in Legnaro (8 m a.s.l., 45°21′N 11°58′E, north-
filled and suspended boxes has high management costs, and may eastern Italy). It consisted of a terraced system (2.13 m high) with six
have high water demand, especially in the dry season of the Mediterra- treatment lines and occupied a total surface area of about 16.4 m2
nean area. In this context, green walls have recently been proposed as (Fig. 1A, C). The system functioned as a vertical flow system, with alter-
innovative space-saving systems that can combine the benefits of nation of phases in which the treatment units were completely satu-
greening the buildings with the aquatic vegetation capacity of treating rated (anaerobic conditions) and phases with treatment units partially
wastewater through CW systems (da Cunha et al., 2018). Early studies empty with prevalence of aerobic conditions. The greywater flowed in-
conducted in Italy (Tamiazzo et al., 2015) and in Western India (Masi termittently through plastic pipes at the lower levels by gravity, and after
et al., 2016) monitored the pollutant removal performance of a green a theoretical hydraulic retention time, it was accumulated in a plastic
wall system treating carwash polluted waters –anionic surfactants tank before being collected and transferred to the laboratory for the sub-
(MBAS) ≈ 100 mg l−1– and light greywater from wash basins – sequent analyses. The entering greywater forced the greywater already
chemical oxygen demand (COD) ≈ 60 mg l−1, biochemical oxygen de- present inside a treatment unit to pass through the substrate, and then
mand (BOD5) ≈ 25.1 mg l−1, ammonium nitrate (N-NH4) ≈ 3.5 mg l−1, to rise along a tube to exit and fall in the lower unit (Fig. 1E). Between
suspended solids ≈ 55 mg l−1– respectively, highlighting that effluent two feeding cycles, the water level in the cells decreased due to evapo-
quality was strongly enhanced. More recently, Fowdar et al. (2017) transpiration, determining aerobic conditions inside the tank as in previ-
tested the seasonal treatment performance of living walls that were ous experiments (Tamiazzo et al., 2015). The WCCW was composed of
vegetated with ornamental plants in sand filters. Prodanovic et al. an iron scaffold that supported plans where phytoremediation treat-
(2017) focused on finding the best performing lightweight porous ment lines were placed. A 1-m3 accumulation tank (Fig. 1A) was used
media to treat greywater in green walls at the column scale. These stud- to store the greywater, from which a submersible hydraulic pump trans-
ies showed that only a few attempts have been made to develop green ferred the greywater into six feeding drums, one for each treatment line.
walls for wastewater treatments, whose properties should be tested at Each line was composed of three plastic tanks (0.35 × 0.45 × 0.27 m)
site-specific level. In particular, there is currently little available perfor- (Fig. 1C) that were filled with lightweight inert planting material
mance data regarding the use of these systems for greywater treatment, LECA® (light expanded clay aggregates; total porosity = 0.55 m3 m−3)
despite their ability to simultaneously maintain the living wall and treat to ensure that the only nutrient source for the plants was the greywater.
greywater for reuse purposes (Fowdar et al., 2017). In addition, only Based on the communicating vases principle the incoming greywater
few studies focusing on real-world conditions were experimentally pushed the water already present through the substrate towards the
tested (Fowdar et al., 2017), while the majority of these studies focused exit and, once the overflow was reached, it came out at the lower level.
on laboratory experiments or used synthetic water solutions (Picard The WCCW was planted with different species (Mentha aquatica L. –
et al., 2005; Prodanovic et al., 2020). Finally, greywater reuse legislation hereafter called Mentha; Oenanthe javanica (Blume) DC. – hereafter
is not uniform internationally, highlighting that evaluation of CW sys- called Oenanthe; a mixture of Lysimachia punctata L. and Lysimachia
tem performance is still necessary across different countries. For exam- nummularia L. – hereafter called Lysimachia) (Fig. 1B, D). Lysimachia
ple, the Italian guidelines concerning wastewater reuse, including a list punctata L. did not survive the initial transplant and adaptation phase

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N. Dal Ferro, C. De Mattia, M.A. Gandini et al. Science of the Total Environment 757 (2021) 144189

Fig. 1. Sketch of the WCCW system (A, C) and WCCW system in operation during May 2018 (B, D). A 1-m3 accumulation tank was used to store the kitchen greywater (A). A submersible
hydraulic pump was used to transfer the greywater into the feeding drums, one for each phytoremediation line, each of which had three treatment units in series. Water flow dynamics
within each treatment unit is reported in the inner pane (E).

to the WCCW, so it was not considered in the experiment and from the grease trap system and transferred to the accumulation tank.
substituted by Lysimachia nummularia L. Therefore, each treatment The tank worked as a storage system to fill the drums, and then the veg-
line was vegetated with one plant species (6 plants per plastic tank – etation lines, as well as a sedimentation unit. The trial was divided into
18 plants per line). All chosen plants were rhizomatous perennial spe- four monitoring periods, corresponding to data collected during: 14th
cies that can live in wetland habitats as emergent species. They were June 2017–4th August 2017; 6th October 2017–9th March 2018; 28th
selected for their adaptability to saturated conditions, tolerance to pol- March 2018–14th September 2018; 9th October 2018–14th April 2019.
luted water, size and aesthetic appeal. They are suitable for use in a During the monitoring periods, each line was fed with hydraulic loading
wall CW because they have limited development of the aerial and root rates of between 36.2 and 72.6 l m−2 d−1, corresponding to hydraulic re-
systems compared to other wetland plants. Six rooted plants (10 cm tention times in the range of 2.0–4.1 days (Table 1). Different retention
high) were transplanted into each plastic tank (May 2017), and initially times were scheduled to assess the system's ability to adapt to different
watered (1 week) with tap water that was gradually mixed with greater loads. Rainfall data were also collected using a rain gauge that was placed
amounts of greywater, up to the use of greywater only after two weeks. at the center of the WCCW system.
The experiment began in June 2017 and ended in April 2019. During the Physicochemical water quality parameters (pH; electrical conduc-
two autumn-winter seasons (October–April), the WCCW was covered tivity – EC, μS cm−1; T, °C; dissolved oxygen – DO, mg l−1) were moni-
with a greenhouse plastic film to extend the vegetation growing season. tored regularly throughout the experiment at a frequency that changed
according to the season and WCCW management. The measurements
2.2. Greywater management and monitoring were performed in situ with a portable HQ40D multi-parameter
multi-sensor meter (Hach, Loveland, CO). Water turbidity as nephelo-
Kitchen greywater, used in the experiment, was regularly taken from metric turbidity units (NTU) was also measured on a portable 2100P
the kitchen sink of the canteen of the University of Padova, which collects Turbidimeter (Hach, Loveland, CO). During the summer season June–
the greywater that are produced during food preparation (washing of August 2017, parameters were recorded on a daily basis (Monday to Fri-
vegetables and fruits, cooking waters, etc.) and dishwashing. A total vol- day) in each line, at both the inlet and outlet, and in the intermediate
ume of about 90 m3 is produced yearly, and only subjected to a primary tanks that compose each line. During the monitoring period October
treatment of grease trap and septic tank before being transferred to a con-
ventional wastewater treatment plant. The research was concentrated on
kitchen greywater for several reasons: i) the interest on this particular Table 1
Greywater loading management in the WCCW.
category of wastewater, that can be easily isolated from a domestic sys-
tem of wastewater collection and discharge; ii) their potential adequacy Monitoring period Monitoring season Hydraulic loading Hydraulic retention
to be treated in green walls, due to reduced drawbacks (e.g., lower con- rate (l m−2 d−1) time (d)
tamination, odors) compared to yellow/brown wastewater; iii) the con- 14/06/17–04/08/17 Summer 2017 45.30 3.3
venience, being the experiment conducted on a pilot-scale system in 06/10/17–28/03/18 Winter 2017 36.83 4.0
the University campus; iv) the accessibility to the accumulation basin 28/03/18–14/09/18 Summer 2018 72.57 2.0
09/10/18–14/04/19 Winter 2018 36.19 4.1
(septic tank). One cubic meter of greywater was periodically collected

3
N. Dal Ferro, C. De Mattia, M.A. Gandini et al. Science of the Total Environment 757 (2021) 144189

2017–March 2018, parameters were evaluated every 48–72 h. In the evaluate whether nutrients were sufficient for biomass growth, ensur-
third period, the values were collected twice a week and those of the ing the greywater treatment (Tchobanoglous et al., 1991).
input waters twice a month. Instead, in the winter season 2018–2019 Inflow and outflow greywater parameters as well as data about
parameters were collected weekly. treatment performance were asymmetrical and did not follow a normal
The WCCW treatment performance in removing organic compounds distribution. Therefore, the distribution-free nonparametric Kruskal-
(COD; BOD5), MBAS, nutrients (N-NH4; nitric nitrogen – N-NO3; Kjeldahl Wallis test (Gilbert, 1987) was used to compare: i) the inflow and out-
nitrogen – TKN, total orthophosphate – P-PO4) and turbidity, was quanti- flow water quality parameters; ii) the WCCW treatment performances
fied by monitoring the inflow and outflow concentrations, at an average as affected by the combination of vegetation and seasons/loading man-
frequency of 15 days. Collected water was stored at 5 °C prior to chemical agements. The Siegel and Castellan (1988) test was used for post hoc
analyses in the laboratory. N-NH4, N-NO3 and P-PO4 were determined comparisons of mean ranks of all pairs of groups.
through ion chromatography (Nicoletto et al., 2019) while TKN was de- The relation between physicochemical parameters and treatment
termined following the Kjeldahl method (Latimer, 2012). The COD and performances of pollutants was investigated by performing linear re-
MBAS were measured by a field spectrophotometer (Hach, Loveland, gression analysis and a correlation-based principal component analysis
CO) using either LCK 314 (measurement range 15–150 mg l−1) or LCK (PCA) on 11 variables: pH, EC, DO, T, turbidity, COD, BOD5, MBAS, N-
514 COD (100–2000 mg l−1), and LCK 432 MBAS (measurement range NH4, TKN, P-PO4. Before PCA, the Kaiser-Meyer-Olkin (KMO) test as a
0.1–4.0 mg l−1) cuvette test vials depending on the concentration to be measure of sampling adequacy was performed. The overall KMO was
measured (Dimitrova et al., 2013). The BOD5 was measured by the respi- 0.70, indicating that selected variables were suitable for PCA analysis
rometric BOD OxiTop® (WTW, Xylem Analytics, D) method, based on an (Kaiser, 1974). Rotated orthogonal components with eigenvalues of
accurate pressure measurement (Vähäoja et al., 2005). the correlation matrix >1 were extracted (Kaiser, 1960) and the relative
scores were determined. Statistical analyses were performed with
2.3. Microbial analysis STATISTICA Version 7.0 (Statsoft Inc., USA).

At the end of the 2-year experiment (April 2019), two inflow 3. Results and discussion
greywater samples (IN-1, IN-2), and a bulk of two outflow effluents
grouping the replication lines (OUT-1, OUT-2), were processed by total 3.1. Greywater monitoring of physicochemical and chemical parameters
DNA extraction, amplification with primers targeting seven hypervari-
able regions of the small subunit 16S rRNA operon, library preparation Inflow kitchen greywater was generally characterized by anoxic
and nucleotide sequencing using an IONS5 (Thermo Fischer Scientific, conditions (DO = 2.03 mg l−1) and moderate-low salinity (median
Waltham, MA, USA) unit as described by the manufacturer. EC = 992 μS cm−1), whereas pH was between sub-basic and basic con-
ditions, the median being = 8.7 and percentiles 25%–75% = 7.85–9.46
2.4. Data analysis (Table 2). Other studies reported generally lower pH values for kitchen
greywater, hypothesizing a high dependence of tap water supply on pH
Treatment performance was evaluated as percentage reduction (%P) (Li et al., 2009). In our study, tap water was rather neutral (pH = 6.6 ±
according to different monitoring seasons (Table 1) as follows: 0.6) as also reported in a recent study conducted in the same area
(Zanin et al., 2020). Therefore, it was likely that higher pH values were
due to the high conductivity of kitchen greywater (Noutsopoulos
ðC In −C Out Þ
%P ¼  100; ð1Þ et al., 2018), as well some algal-mediated CO2 extraction that readjusted
C In
carbonates equilibrium. Indeed, the higher pH values were likely due to
the use of salt in food preparation, such as cooking pasta.
where CIn and COut are the inflow and outflow concentrations of each Looking at the greywater dynamics (Fig. 2A), T did not change be-
tested parameter (turbidity, COD, BOD5, TKN, N-NH4, N-NO3, P-PO4). tween inflow and outflow. Greywater came from dish washing and
The COD:BOD5 ratio was estimated to qualify the biodegradability of kitchen sinks and likely had a constant T, however its storage in the ac-
the greywater and the WCCW treatment potential. The higher the ratio cumulation tank –which was not insulated– changed T to that moni-
(COD:BOD5 ≥ 1), the more refractory organic matter it contains (Morel tored in the outflow, which followed the seasonal climatic conditions.
and Diener, 2006). Moreover, the COD:N:P ratio was calculated to During winter, the greenhouse plastic film always maintained T > 2 °C.

Table 2
Inflow and outflow greywater parameters during the two-year experiment. Treatments labelled with different letters in the row were significantly different at p < 0.05.

IN Mentha Oenanthe Lysimachia Italian Jordan

limitsa limitsb
Median (25%–75%) Mentha (25%–75%) Oenanthe 25%–75% Lysimachia 25%–75%

pH 8.74 (7.85–9.46) a 7.81 (7.45–8.30) a 7.93 (7.53–8.60) ab 7.92 (7.56–8.61) b 6.0–9.5 6.0–9.0
EC (μS cm−1) 992.0 (879.5–1064.5) c 1338.0 (1221.8–1842.5) a 1202 (1093.0–1259.0) b 1074 (1022.0–1146.0) c 3000 –
DO (mg l−1) 2.03 (0.87–7.85) b 3.40 (1.15–6.70) a 4.34 (2.74–8.59) ab 4.82 (2.28–7.45) a – >2.0
Turbidity (NTU) 139.0 (87.90–332.50) a 16.10 (6.04–38.13) b 14.25 (5.27–26.00) b 17.10 (8.69–27.75) b 4.3c 10.0
COD (mg l−1) 855.0 (454.5–1102.0) a 98.2 (52.85–200.1) b 82.4 (39.6–139.0) b 66 (39.8–136) b 100 100
BOD5 (mg l−1) 200.0 (70.0–255.0) a 41.5 (20.0–65.0) b 30 (10.0–50.0) b 25.0 (10.0–45.0) b 20 30
MBAS (mg l−1) 12.60 (8.90–19.16) a 2.13 (1.00–4.05) b 2.28 (0.97–3.27) b 2.70 (0.95–3.54) b 0.5d 0.5
TKN (mg l−1) 28.05 (15.83–45.06) a 13.62 (9.87–21.22) b 12.09 (8.44–21.91) b 13.5 (7.61–20.91) b 15e 45e
N-NH4 (mg l−1) 6.89 (2.45–11.26) a 3.18 (2.45–5.51) ab 2.52 (1.91–3.83) b 2.53 (1.92–5.24) b 1.6 –
N-NO3 (mg l−1) 0.01 (<0.005–0.02) c 0.04 (0.01–0.07) b 0.07 (0.02–0.13) ab 0.08 (0.03–0.25) a – 30
P-PO4 (mg l−1) 6.44 (5–7.48) b 8.95 (7.63–10.39) a 6.72 (5.71–9.49) ab 7.11 (5.87–9.86) ab – –
Ptot (mg l−1) 15.7 (13.86–17.68) a 11.9 (8.5–16.43) ab 9.43 (6.52–13.75) b 10.32 (7.44–14.06) ab 2.0 2–5
a
Italian guideline limits for urban wastewater reuse (DM No 182/2003).
b
Jordanian technical base No 893/2006. Irrigation purposes in urban areas (playgrounds, parking areas, etc.)
c
Estimated from legal limits of total suspended solids (TSS) according to Kadlec and Wallace (2008), where NTU/TSS = 0.43.
d
Total surfactants.
e
Total nitrogen.

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N. Dal Ferro, C. De Mattia, M.A. Gandini et al. Science of the Total Environment 757 (2021) 144189

outflow, Mentha showed the highest values throughout the experi-

ment, suggesting higher evapotranspiration capacities that increased
salt concentration in the discharged water, especially during the sum-
mer period. The EC in Mentha was significantly higher (p < 0.05) (me-
dian = 1338 μS cm−1) than that monitored in Oenanthe (median =
1202 μS cm−1) and Lysimachia (1074 μS cm−1) (Table 2). Notably, the
differences increased up to 1426 and 818 μS cm−1 during the 2017
and 2018 summer periods. Anyway, outflow EC values were always
lower that the Italian guidelines limits for wastewater reuse.
Different DO dynamics were found between the first and second half
of the experiment, with initially higher values than the final monitoring
(Fig. 2D), especially in the outflow. As already observed in other studies
comparing small-scale cascade CWs treating greywaters (Kadewa,
2010), a low vegetation cover –as observed here in the first more than
the second year– would have enhanced oxygen exchange between the
atmosphere and the wetland system, whereas a more dense vegetation
cover would have had a higher oxygen demand, provided by a deeper
root growth. Accordingly, DO was slightly higher in Lysimachia (me-
dian = 4.82 mg l−1) > Oenanthe (median = 4.34 mg l−1) > Mentha
(median = 3.40 mg l−1), which followed an opposite trend compared
to vegetation growth. Anyway, a significant (p < 0.05) increase in DO
was observed in outflow (overall median = 3.72 mg l−1) than inflow
greywaters (median = 2.03 mg l−1) (Table 2). The median turbidity
in the inflow greywater was 139 NTU (Table 2), similar to findings by
Ghaitidak and Yadav (2013) who reported values in the 133–311 NTU
range for kitchen sink greywaters, as affected mainly by food residues,
oils and fat, and dishwashing detergents. Turbidity removal dynamics
highlighted values always lower than 62 NTU for all vegetated lines,
suggesting the substrate system capacity to remove suspended solids
by filtration processes. Notably, turbidity removal did not increase
over time when plant roots grew and stabilized the medium (Fig. 2E).
Expanded clay was initially loose and could have favored the washout
of fine particles at the beginning of the experiment, as also found in pre-
vious studies (Prodanovic et al., 2017). The WCCW functioned as a ver-
tical flow system with intermittent flow that likely enhanced particle
sedimentation. It resulted that the effective retention of particles was
mainly due the filtration beds, which corroborated slight variations be-
tween summer and winter (Vymazal, 2019). All vegetated lines removed
substantial suspended solids compared to the inflow (p < 0.05). Among
vegetation treatments, only a slight increase in treatment efficiency was
observed with Oenanthe, which always had values lower than 40 NTU
despite the vegetation not being the most vigorous. Nevertheless, values
were often higher than those set by the Italian limits (Table 2), suggest-
Fig. 2. Inflow and outflow dynamics of monitored physicochemical parameters
(temperature, A; pH, B; electrical conductivity, C; dissolve oxygen, D; turbidity,
ing that the WCCW should be further implemented if the objective was
E). Bars indicate standard errors. greywater reuse. Wetland systems involve the use of living materials,
with biomass production that hinders suspended solids removal below
a background threshold (Kadlec and Wallace, 2008). It is worth noting
The pH (Fig. 2B) ranged between a minimum of 6.5 and a maximum that, the Italian legislation is more specific by far than many others
of 9.0 (both found in the inflow greywater during June–October 2018), a around the world (e.g., Jordanian limits are reported in Table 2) (Salgot
variability that might be due to changes in kitchen management. By con- et al., 2006; Bortolini et al., 2018), however, improvements are required
trast, pH values of outflow were generally smoothed, ranging between on the possible use of WCCW in other international contexts as well as
7.4 and 8.6. Notably, it was slightly higher in the inflow than outflow for experiment on other ornamental plants or plants in general. The re-
during October 2017–March 2018, and vice versa slightly lower inflow lationship between total suspended solids and NTU is highly specific
values were observed later. This difference could be due to some stabili- for each CW, therefore care must be taken in the extrapolation from
zation phenomenon in the WCCW system that could provide a selective one site to another.
anion uptake due to vegetation establishment and growth, which in turn
increased pH (Haynes, 1990; Savvas et al., 2013). A significant relation- 3.2. Chemical parameters
ship was found with a linear regression model between pH and T of out-
flow greywater (pH = 7.67 × T + 0.016; R2 = 0.20; p = 0.001): as water Total organic compounds was represented by median values of
T increased, a decrease in hydrogen ion concentration was likely, in turn COD = 855.0 mg l−1 and BOD5 = 200.0 mg l−1, despite having high var-
providing an increase in pH; this may be due to denitrification processes, iability with percentiles 25%–75% of COD = 454.5–1102.0 mg l−1, and of
which increased the performance at high T, therefore basic pH values BOD5 = 70.0–255.0 mg l−1) (Table 2). Fluctuating input values were
were found (Prinčič et al., 1998). observed throughout the experiment, with maxima in April 2018
On average the content of mineral salts, expressed by EC (Fig. 2C), in (COD = 1561.0 mg l−1, BOD5 = 720.0 mg l−1) and minima between
the incoming greywater was lower than that observed at the outflow, October 2017 (BOD5 = 30.0 mg l−1) and January 2018 (COD =
due to evapotranspiration that increased salts concentration. At the 242.0 mg l−1) (Fig. 3A, B). Also MBAS followed the same dynamics

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N. Dal Ferro, C. De Mattia, M.A. Gandini et al. Science of the Total Environment 757 (2021) 144189

Fig. 3. Inflow and outflow dynamics of monitored COD (A), BOD5 (B) and MBAS (C). Bars indicate standard errors.

(Fig. 3C), being this parameter linearly associated with both COD anoxic conditions (Table 2), in turn suggesting that TKN embedded
(MBAS = 0.011 × COD + 7.472; R2 = 0.42; p = 0.004) and BOD5 most of the total N in the inflow. Nitrogen dynamics fluctuated in the in-
(MBAS = 0.032 × BOD5 + 8.319; R2 = 0.68; p ≤ 0.001). However, flow TKN, which ranged between 12.9 (January 2018) and 63.4 mg l−1
MBAS concentration would not allow outflow greywater reuse, as it (December 2017) (Fig. 4A). A different dynamic was observed for N-
would have for COD and BOD5 (Table 2). The COD:BOD5 ratio had a NH4 (Fig. 4B), as it showed a peak of 29.2 mg l−1 during August 2018,
median value of 3.0, which was slightly higher than findings in pre- while it never exceeded 17 mg l−1 in all other monitoring periods. No-
vious studies that showed a COD:BOD5 ratio of 2.2 (Li et al., 2009; tably, Noutsopoulos et al. (2018) reported that N, particularly in the am-
Noutsopoulos et al., 2018). This difference could be due to several moniacal form, was rather deficient, urine being its major contributor in
factors, such as greywater management that is markedly affected domestic wastewaters. Several authors have therefore suggested that
by the living habits of people (Eriksson, 2002), also in the catering kitchen greywater should be mixed with other greywater streams to
services. Outflow greywater always showed significantly lower balance the COD:N:P ratio to ca. 100:20:1, avoiding the deficiency of
values of organic compounds than the inflow (p < 0.001). The COD macronutrients and trace nutrients (Li et al., 2009; Tchobanoglous
was reduced to median values of 36.3, 32.9 and 30.8 mg l −1 in et al., 1991). Here greywater inflow was unbalanced by an N deficiency
Mentha, Oenanthe and Lysimachia, respectively, without significant in the COD:N ratio (100:3.3), while P-PO4, with a median of 11.9 mg l−1,
differences among vegetated treatment lines (Table 2). Anyway, an suggested satisfactory concentrations. Outflow TKN was significantly
increase in the COD:BOD5 ratio was not observed in the outflow com- lower than inflow (p < 0.001) in all vegetated lines, and usually lower
pared to the inflow, despite slightly higher values in experimental than values for wastewater reuse. In contrast, only Oenanthe and
seasons with lower hydraulic loading rate (Table 1). This suggested Lysimachia significantly removed ammonia compared to the inflow
that some additional retention time might have enhanced the re- (p = 0.002), the results observed with Mentha being intermediate
moval of organic compounds. The low COD:BOD5 ratio also revealed (Table 2). In all cases, N-NH4 in the outflow was higher than the limit
that biological oxidation of organic matter was not the main mecha- of the legislation. Some slight increase was observed for N-NO3
nism behind the reported COD reduction. (Fig. 4C, Table 2) in the outflow than inflow, although with values gen-
In the case of nitrogen (N), the inflow greywater contained organic erally <1 mg l−1.
and ammonia N, with median TKN values of 28.1 mg l−1. The N-NH4 Regarding P-PO4, the outflow was significantly higher than the inflow
had a median of 6.9 mg N l−1, thus representing ca. 25% of TKN. As ex- with Mentha (p < 0.005), while Oenanthe and Lysimachia did not con-
pected, nitrates were always negligible in the inflow greywater due to tribute to significant changes, the median being 6.7 and 7.1 mg l−1,

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N. Dal Ferro, C. De Mattia, M.A. Gandini et al. Science of the Total Environment 757 (2021) 144189

Fig. 4. Inflow and outflow dynamics of monitored TKN (A), N-NH4 (B), N-NO3 (C) and P-PO4 (D). Bars indicate standard errors.

respectively (Table 2). These results were corroborated by P-PO4 concen- Similar findings were also observed for MBAS, but in this case all vege-
tration dynamics, which were slightly higher in the outflow than inflow tated lines reduced the treatment performance in both summer periods
(Fig. 4D). compared to the winter ones. This aspect is particularly relevant with
vigorous vegetation in CWs, and especially when treated wastewater
3.3. Treatment performances is to be reused (Borin et al., 2011; Morari et al., 2015). By contrast, the
water lost through plant transpiration can increase the hydraulic reten-
The combined effect of vegetation and seasonality modified the re- tion time enhancing nutrients reduction (Gagnon et al., 2012), as well
moval treatment performance of organic compounds, with lower values contributing to other ecosystem services provided by green infrastruc-
that were generally observed In Mentha, Oenanthe and Lysimachia dur- tures, e.g. mitigation of extreme rainfall events in urban areas that can
ing summer 2018 compared to the vegetated lines in other periods be channeled into the system (Keath and Brown, 2009).
(Fig. 5). The high summer T likely increased pollutant degradation pro- Regarding nutrients, treatment performance was not significantly
cesses, but contemporarily it maximized the evapotranspiration and the affected by the vegetation species in the different tested seasons
pollutant concentration in greywaters. In summer 2018, this effect was (Fig. 6). For TKN, performances ranged between a median of 33.5% in
particularly relevant for Mentha, being performances significantly Lysimachia, winter 2018, and 73.6% in Mentha, summer 2017. Perfor-
lower (COD = 64.9%; BOD5 = 58.6%) compared to Oenanthe (COD = mances lowered for N-NH4 until a median of 26.3% in Lysimachia, sum-
75.1%; BOD5 = 67.2%) and Lysimachia (COD = 76.7%; BOD5 = 67.3%). mer 2017. Notably, some fluctuation in N-NH4 removal was observed

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N. Dal Ferro, C. De Mattia, M.A. Gandini et al. Science of the Total Environment 757 (2021) 144189

Fig. 5. Boxplots of treatment performances by seasonal periods estimated on monitored COD (A), BOD5 (B), turbidity (C) and MBAS (D). The white squares are median values inside
colored 25th (lower limit)-75th (upper limit) percentile boxes. Bars indicate the non-outlier range (1.5 × height of the box). Boxplots labelled with different lowercase letters were
significantly different at p < 0.05.

Fig. 6. Boxplots of treatment performances by seasonal periods estimated on monitored TKN (A), N-NH4 (B), N-NO3 (C) and P-PO4 (D). The white squares are median values inside colored
25th (lower limit)-75th (upper limit) percentile boxes. Bars indicate the non-outlier range (1.5 × height of the box).

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N. Dal Ferro, C. De Mattia, M.A. Gandini et al. Science of the Total Environment 757 (2021) 144189

comparing the different seasons. It is likely that higher retention times communities was also reduced in the outflow water samples, being
during winter (Table 1) offset the more favorable T for treatment in the number of species lower than at the inflow (Table 3). A total of 75
the summer period (Kim et al., 2016; Prochaska et al., 2007). By con- taxonomical operational units at species rank level were identified. Re-
trast, oxidized nutrients showed different dynamics: for N-NO3, esti- sults showed that Aquaspirillum serpens (Betaproteobacteria,
mated median performances ranged from −380% in Oenanthe, Neisseriaceae) was the most abundant taxon encountered in the analy-
summer 2018, to −7.9% in Mentha, summer 2017; for P-PO4, fluctuat- sis. It was already present in the inflow greywater, but it showed a four-
ing values ranged below and above zero. In the case of N-NO3, the abso- to six-fold increase in relative abundance after treatment (Table S2). It
lute values were always very low, whereas improvements in treatment belongs to a genus typical of freshwater systems and has a wide toler-
performances were required for P-PO4. In this context, the WCCW ance to the variation of salt concentrations, having been isolated both
might suffer from a high P-PO4 content of kitchen greywater, which from distilled water and waters with different inputs of solutes
showed dynamics similar to those reported in other living walls in (Leifson, 1962). Being an autotrophic (chemolitotrophic) aerobic spe-
urban environments treating greywater (Fowdar et al., 2017). A poor cies that thrives on the oxidation of iron and manganese and does not
P-PO4 translocation capacity was due to low vegetation biomass pro- require organic carbon for energy and growth (Aragno and Schlegel,
duction that was selected for vertical systems, and the large expanded 1978), its increase was therefore in line with the treatment practice
clay particles likely had little P sorption capacity. abating BOD5, which in turn affected oxygen solubility.
A general overview of factors influencing greywater treatment was A second case of rather abundant species that showed an increase in
provided by the PCA. Four principal components were extracted with ei- the treated samples –although not so pronounced– was Bacteroides
genvalue >1 (Table S1), totally accounting for 80.9% of the variance. The graminisolvens (Phylum Bacteroidetes). It is a xylanolytic anaerobe that
distribution of variables in the xy-plane (Fig. 7) of the first two principal was originally isolated from a methanogenic reactor treating cattle
components highlighted that the first one was explained turbidity and waste (Nishiyama et al., 2009). Xylan being a hemicellulose, its increase
TKN (on the right side of the graph), while the second one mainly grouped after processes in treatment wetlands could be explained. Likewise, the
MBAS and N-NH4 (on the top of the graph) that were negatively associ- increase of Parabacteroides chartae, a bacterium of the same phylum that
ated with pH and T. These results suggested different dynamics involved can be found in paper mill effluent, was in line with the treatment hab-
in the treatment process regardless the vegetation species, as highlighted itat. A fourth taxon, uniformly present in all inflow and outflow samples,
by plotting the factor scores of Mentha, Oenanthe and Lysimachia: the ef- was Aeromonas sharmana (Gammaproteobacteria, Aeromomdaceae);
fect of low T on N-NH4 and MBAS removal performances likely hid the ef- the genus is facultative anaerobic and ubiquitous in fresh water, while
fect of the longer retention time during winter (Table 1); instead, TKN the species was originally isolated from warm springs (Saha and
removal was associated with the filtration process that also involved Chakrabarti, 2006).
suspended solids (measured by turbidity), being mainly composed of or- The next case, which showed the highest relative abundance in-
ganic material (about 75–80%) according to greywater concentration data crease (about ten-fold) after greywater treatment was Prevotella
(Table 2). Notably, the BOD5 and COD were located centrally, and mainly paludivivens (Bacteroidetes, Prevotellaceae); this gram-negative anaer-
associated with DO in the principal component 3 (Table S1). obic bacterium is an ascertained hemicellulose-decomposer bacterium
originally found in plant residues including roots in irrigated rice-
3.4. Microbial analysis fields (Ueki et al., 2007). Unlike the other members of the genus
Prevotella, this species is not reported as living in animal guts and is
Inflow pre-treated samples had a nearly double content of DNA than associated to paddy plant environments. This occurrence, as that of
outflow indicating that, a high microbial load was conspicuously abated B. graminisolvens, appeared quite compatible with the treatment wet-
by the WCCW treatment (Table 3). The complexity of the resulting land setting.
Greywater treatment strongly reduced Desulfovibrio desulfuricans
(Deltaproteobacteria, Desulfovibrionaceae). This facultative anaerobic
microorganism is the typical representative of the dissimilatory sulfur
cycle reductions as it uses sulfate as electron acceptor in anaerobic res-
piration, ultimately generating H2S and the neurotoxic methylmercury
(Compeau and Bartha, 1985). The presence of this sulfate-reducer in
greywaters was favored by the high BOD5, being a heterotrophic species
that likely obtained energy from organic matter via anaerobic respira-
tion. Its presence and metabolism are the most classical causes of
the unpleasant odor and corrosive action of sewage water, as also expe-
rienced in our inflow greywater. Therefore, its abatement counted
among the main benefits of the greywater treatment process.
Another case of a species which diminished with the greywater
treatment is that of Pseudoxanthomonas spadix (Gammaproteobacteria,
Xanthomonadales), a bacterium which is an indicator of oil-contaminated
environments (Young et al., 2007). The treatment resulted in its complete
disappearance, featuring zero reads in both the outflow processed sam-
ples. Another taxon that was totally removed by the treatment was

Table 3
DNA yield in the extraction from 50 ml of water samples and number of observed species
after the annotation of the sequenced bacterial amplicons.

Sample ID Recovered DNA(ng μl−1) Number of taxa in sequencing output

IN-1 0.300 49
IN-2 0.210 46
Fig. 7. Physicochemical and chemical parameters (labelled by black rings) projected in the
OUT-1 0.100 27
xy-plane determined by the first two principal axes. Scores identifying vegetation
OUT-2 0.130 34
treatments (M, Mentha; O, Oenanthe; L, Lysimachia) were also reported.

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N. Dal Ferro, C. De Mattia, M.A. Gandini et al. Science of the Total Environment 757 (2021) 144189

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