Journal of Water Process Engineering 59 (2024) 105066

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Journal of Water Process Engineering

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

Biochar as a superior material for enhancing the performance and microbial

diversity in biofilters treating greywater
Fatma Öykü Çömez a, Adam Sochacki a, *, Jaroslav Vacula a, Jiří Bárta b, Ljuba Zídková c,
Martin Lexa d, Dana Komínková a
a
Czech University of Life Sciences Prague, Faculty of Environmental Sciences, Department of Applied Ecology, Kamýcká 129, 165 00 Prague 6, Czech Republic
b
University of South Bohemia in České Budějovice, Faculty of Science, Department of Ecosystem Biology, Branišovská 1760, 37005 České Budějovice, Czech Republic
c
DEKONTA, Dřetovice 109, 273 42 Stehelčeves, Czech Republic
d
Czech University of Life Sciences Prague, Faculty of Forestry and Wood Sciences, Department of Wood Processing and Biomaterials, Kamýcká 129, 165 00 Prague 6,
Czech Republic

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

Keywords: This research evaluated the efficiency of greywater treatment in biofilters amended by different additives:
Ecotoxicity biochar (5 % and 10 % v/v), woodchips, crushed bricks, and limestone chippings. The effect of the additives on
Microbial community the removal of selected organic (benzotriazole, caffeine, diclofenac, DEET, methylparaben, and an anionic sur­
Micropollutants
factant) and inorganic (boron, copper, nickel, and zinc) pollutants was tested. The biofilters provided almost
Nature-based solutions
Greywater
complete removal of all organic pollutants, except for benzotriazole (38 %–49 %). This compound was 99 %
removed only in biofilters with biochar. This enhanced removal stemmed from greater sorption capacity of the
biofilters with biochar. The removal of metals was high (>90 %), however, boron removal was negligible. The
treatment reduced the toxicity to negligible or stimulatory levels. Analysis of the prokaryotic microbiome
showed a significantly greater diversity in the biofilters with 5 % biochar and with woodchips. The biochar
amendment offers comparable treatment effect at both doses suggesting the application of the lower ratio in full-
scale systems due to economic reasons. The use of biochar brings very important engineering added values,
which are longevity and the ability to selectively retain organic contaminants, but not the nutrients. This may
qualify the use of biofilters as an attractive pretreatment of greywater for irrigation purposes.

1. Introduction
Greywater (GW) has been proposed as an alternative non-potable
water resource due to its low concentrations of contaminants (patho­
gens and organic content) and high daily household production [1,2],
which can reach up to 85 % of total water consumption [3]. The reuse of
GW in households can decrease the amount of potable water needed for
toilet flushing, car washing, and garden watering [4]. GW originates
from washing machines, the kitchen, and the bathroom (sinks, showers,
and baths), excluding toilet water. Although it is expected to have a
lower contamination level than mixed wastewater, it contains high
concentrations of some organic pollutants (such as pharmaceuticals,
personal care and household products) and inorganic pollutants, which
can be washed from the skin or are used for washing [5,6]. The
composition of GW can vary greatly from source to source depending on
whether it is a household - with handwashing, showering, kitchen
wastewater - or at a public utility building - with mostly handwashing
and dishwashing wastewater (the so-called light GW). These types of
GW usually contain a range of organic pollutants, which are present in
topically applied drugs, cosmetics, and detergents, or are ingredients of
food or beverages whose leftovers may reach GW. One of such con­
taminants is N,N-diethyl-m-toluamide (DEET), which is an active
ingredient in many commercial insect repellents and has been detected
in various types of water worldwide, including groundwater, drinking
water, effluents of wastewater treatment plants, and many ranges of
surface waters [6,7]. DEET together with caffeine (CAF) and several

Abbreviations: B10, biochar (10 % v/v) (columns); B5, biochar (5 % v/v) (columns); Br, bricks (columns); BTA, benzotriazole; C, control (columns); CAF, caffeine;
DCF, diclofenac; DEET, N,N-diethyl-m-toluamide (commonly referred to as DEET); GW, greywater; L, limestone (columns); MPB, methylparaben; S1DS, sodium 1-
decanesulfonate; SGW, synthetic greywater; W, woodchips (columns).
* Corresponding author.
E-mail address: sochacki@fzp.czu.cz (A. Sochacki).

https://doi.org/10.1016/j.jwpe.2024.105066
Received 23 December 2023; Received in revised form 11 February 2024; Accepted 23 February 2024
Available online 28 February 2024
2214-7144/© 2024 Elsevier Ltd. All rights reserved.
F.Ö. Çömez et al.
other substances have been proposed as tracers of contamination of
natural waters with GW [6]. Other GW contaminants are surfactants,
which are present in high concentrations, especially in laundry effluents
- mainly as anionic surfactants, which make up to 30 % of used de­
tergents [8]. Similarly, methylparaben (MPB), a preservative used in
cosmetics, benzotriazole (BTA), a corrosion inhibitor used in detergents,
and diclofenac (DCF), a topically applied anti-inflammatory drug, are
commonly used compounds in various products which can easily enter
GW both in households and public utility buildings [9,10]. Apart from
organic pollutants, copper (Cu), nickel (Ni), and zinc (Zn) are metals
that have been reported at high concentrations in GW due to leaching
from galvanized pipes and tanks, plumbing materials, and fittings and
coatings [11]. Also a non-metal boron (B) has been commonly detected
in GW and has been linked to the usage of detergents in household for
bathing and dishwashing [12]. Results reported by Turner et al. [13]
demonstrate that long-term GW irrigation can lead to high metal con­
centration in groundwater (Cu, Ni, and Zn) and surface waters (Cu and
Zn) as well as causing accumulation of B and Cu in soil. In addition, a
high concentration of Ni is reported as harmful to plants [12]. The reuse
of untreated GW might cause a threat to public health and environ­
mental systems, such as contamination of ground and surface waters due
to the presence of micropollutants and should undergo a proper treat­
ment process [9,14,15].
Nature-based treatment systems such as green roofs, green walls,
vegetated parking lots, and biofilters are an emerging solution to on-site
GW treatment [16–18]. The nature-based systems have been widely
indicated as promising solutions for the treatment of GW [19], but their
specific removal efficiency of organic and inorganic pollutants and
detoxification effect has been scarcely discussed in the literature
[20,21].
Biofilters are commonly used nature-based treatment solutions for
suspended solids, metals, and nutrients, especially from stormwater
runoff in urban areas [22,23]. In addition, they are multipurpose sys­
tems that use physical (sorption) and biological (biotransformation by
microbiome) treatment for the removal of contaminants and, therefore,
can be used for wastewater treatment [24]. The main challenges of
biofilters include operational aspects (such as peak flows) and physical
design aspects (such as filter media type and depth). The irregular flow
of GW due to specificity of activities and working hours within a
household or public building may negatively affect the performance of
biofilters [25]. Additionally, the low load of biodegradable organic,
nutrient deficiency and the presence of surfactants in GW may limit the
growth of biomass [26,27] making the biological removal of organic
compounds a challenging task. Filter media in a biofilter can consist of
natural or synthetic porous media, such as soil, sand, compost, or spe­
cific amendments, which can serve multiple purposes: support the
growth of microbial biofilms [28] and provide the source of organic
carbon and nutrients in the case when wastewater is deficient in these
substances, buffer the pH, and provide sorption capacity. Additionally,
various sorbents like biochar could be used as amendments of biofilters
to increase their sorption capacity especially for organic contaminants
[12]. Different media characteristics based on hydraulic conductivity,
water retention capacity, porosity and capacity to support plant growth
have been studied to enhance biofilter performance [29–31].
Recently, biochar has been proposed as one of the increasingly
studied alternative filter media with especially high removal of organic
compounds and low removal of nutrients, which can be especially
attractive for water reuse for irrigation [32,33]. The removal of various
organic pollutants by biochar has been explored and proposed as a
promising medium for on-site treatment systems [33,34]. Woodchips
were used to improve the efficiency of processes relying on the avail­
ability of organic carbon, especially denitrification [30,35]. Limestone
has been also used to buffer the acidity released in microbial processes
and to provide inorganic carbon for denitrifiers [36] and also to enhance
the removal of Cu, Ni, and Zn [37,38] by the surface reaction with
precipitated Fe, Al and Mn. Crushed bricks were used to increase
2
Journal of Water Process Engineering 59 (2024) 105066
sorption capacity for phosphates [39], metals [40], and potentially DCF
and organic compounds with similar properties [41].
Numerous studies elucidated the role of biofilter media additives in
the treatment of sewage, but only very few publications focused on the
treatment of GW taking into account the simultaneous removal of
inorganic pollutants and the specific organic compounds like surfac­
tants, DCF, BTA, MPB, CAF and DEET. The findings regarding the
treatment of sewage in biofilters cannot be transferred to GW context,
because of specific qualities of GW making its treatment difficult using
biological treatment technologies. In this study, the novelty arises also
from examining the impact of simple media additive on the removal of
multiple pollutants during biofiltration of a household and public fa­
cility relevant GW in relation to specific microbiomes and its ecotoxi­
cological assessment.
This study aims to evaluate concurrent removal efficiency for organic
and inorganic pollutants in biofilters with five different additives (bio­
char 5 % and 10 % by volume, woodchips, crushed bricks, and limestone
chippings).
2. Methodology
2.1. Experimental design and operation
The experiment was designed to simulate the treatment of GW from
households or public utility buildings that are used only during the
working days and do not produce grey water over the weekend. For this
purpose, batch-operated biofilter systems with self-priming and self-
draining device (syphon) could serve as a viable solution. This is
because the system would better adapt to the irregular flows of GW and
the cyclic feeding and draining would enhance oxygenation of the sys­
tem at the beginning of the reaction phase and thus it would enable both
oxic and anoxic process [42,43].
The experimental lab-scale system included 30 columns biofilters
with dimensions of 53 cm height and 7.9 cm in diameter (total volume
2.5 L). The filter media of the column were divided into three layers,
with a total thickness of 44.9 cm (Fig. 1):
- Upper filter layer (thickness 28.6 cm): mixture of sand, compost and
organic soil (5:2:2) creating 85–100 % of the volume and a selected
additive: biochar 5 % (v/v) and biochar 10 % (v/v), woodchips 10 %
(v/v), crushed bricks 10 % (v/v), and limestone chippings 15 % (v/
v). Additionally, a system without an additive was set up as a control.
- Bottom filter layer (thickness 13 cm): coarse sand to avoid clogging
(0–4 mm).
- Drainage layer (thickness 3.4 cm): gravel (4–8 mm) to allow efficient
draining of the columns.
Five types of biofilters were used in the experiment (Table 1),
depending on the type of filtration material in the upper layer of the
filters. Each type was operated in 3 to 5 replicates. The application of
compost and natural soil in the control system was to provide a source of
organic matter for microbial biota because GW is deficient in organic
carbon. Biochar was produced at 600 ◦ C from spruce wood and the
detailed information on the biochar used in this research is given by
Sochacki et al. [33]. A circular geotextile was placed between layers and
onto the top to avoid risks related to interference between layers of the
biofilter, preferential flow development, and surface erosion. Each col­
umn was covered with aluminium foil to avoid sunlight exposure.
The biofilter columns were intermittently fed with synthetic grey­
water (SGW) added to the top of the filtering medium. This arrangement
was preferred to represent possible gravitational draining in biofilters
under field conditions. The biofilters were fed with synthetic greywater
(SGW) to ensure consistency of composition throughout the experiment.
SGW was prepared freshly at the beginning of each cycle. The volume of
a single batch of SGW per column was 400 mL, which translated to the
hydraulic loading of 0.082 m3/m2 per batch. The biofilters were
F.Ö. Çömez et al. Journal of Water Process Engineering 59 (2024) 105066

Fig. 1. Biofilter columns: a) configuration, b) photo of a column.

operated with batch feeding mode with 0.5-h phase of filling, 16-h or 66-
Table 1
h reaction phase, and 7.5-h draining phase (Fig. 2a). The barrels were
Types of biofilters used in the experiment depending on the additive used.
emptied in the morning and loaded with SGW afternoon every weekday.
Symbol Main fraction Additive in the upper layer Therefore, the cycle between batches was 1 day on weekdays and was 3
C Sand: compost: organic soil (5:2:2) No additive – control days at weekends (Fig. 2b).
85–100 % (v/v)a column Prior to the start of the experiment, 30 columns were filled with an
B5 Biochar 5 % (v/v)
activated sludge from a system treating greywater to inoculate the
B10 Biochar 10 % (v/v)
W Woodchips 10 % (v/v) biofilter media. The sludge was retained in the biofilters for 48 h. The
Br Crushed bricks 10 % (v/v) experiment was operated in a greenhouse with a temperature range of
L Limestone chippings 15 % was 20–24 ◦ C.
(v/v) The total duration of the experiment was 125 days and divided into
a
Depending on the proportion of an additive added. three different periods which are denoted as acclimation period, steady-
state period, and microbial-activity inhibition test period (Fig. 2c). After
running the experiment for 100 days, a microbial-activity inhibitor

Fig. 2. Operation of the experimental system: a) operational sequences within 1-d and 3-batches, b) sequence of batches within a cycle, c) timeline of the experiment.

3
F.Ö. Çömez et al.
sodium azide (NaN3) was applied to 1 or 2 selected columns from each
group. This procedure was applied to distinguish the role of biotic and
abiotic processes in the removal of mainly organic pollutants (organic
compounds, anionic surfactants, TOC, and N-NH4) in the biofilters. 10
columns in total were fed by SGW with sodium azide. For this, separate
SGW was prepared with sodium azide at a concentration of 800 mg/L
based on the protocol of [44]. Sodium azide was intentionally not added
in the beginning of the experiment to enable formation of biofilm and
chemical precipitates and adsorbed fraction that could play important
role in the abiotic removal of the contaminants apart from the inherent
properties of the filtering media and the additives. The rest of the col­
umns continued to be irrigated with regularly prepared SGW to compare
the removal process with the inhibited columns.
2.2. Synthetic greywater preparation
Domestic GW composition varies depending on different aspects,
such as commercial products, household habits and preferences, and
seasonal changes [2]. The synthetic greywater (SGW) composition was
formulated based on literature data (S1, Supporting information) and
high-concentration compounds detected in real GW samples from the
campus of Czech University of Life Sciences Prague (data unpublished).
The SGW (70 L) was prepared weekly in tap water by dissolving the
following components: NH4Cl (95.18 mg/L), KH2PO4 (7.16 mg/L),
glycerine (25.5 mg/L), CuSO4.5H2O (0.79 mg/L), ZnSO4.7H2O (0.88
mg/L), NiSO4.6H2O (0.89 mg/L), and H3BO3 (5.72 mg/L). The SGW also
contained six proxy compounds, namely BTA, DEET, DCF, CAF, MPB,
and sodium 1-decanesulfonate (S1DS) (Characteristics given in S2,
Supporting information), purchased from Sigma Aldrich, Czech Re­
public. S1DS was added to the SGW at a concentration of 1 mg/L. DCF
was added in the form of sodium salt DCF and the rest as pure substances
at the concentration of 2.5 mg/L of each compound. The reasons for the
rather high concentrations were, firstly, based on the assumption that
their concentration can occur in milligram-per-litre in household efflu­
ents due to the mode of their application [45]; secondly, to simplify the
analytical procedure by avoiding sample preconcentration step. The
prepared SGW was stored in an opaque plastic barrel in the greenhouse.
The barrel was emptied, cleaned with disinfectant soap, and thoroughly
rinsed for each new influent batch.
2.3. Sampling and analytical methods
Effluent and SGW samples were collected two times per week
(Monday and Thursday) and after each batch in the inhibition period.
The samples were taken by completely draining the columns within 7.5
h. Immediately after collection, all the samples were taken to the labo­
ratory for analyses. The samples were filtered using 0.22 μm polyether-
sulfone syringe filters Rotilabo (Carl Roth) for all the analyses.
The high-performance liquid chromatography (HPLC) coupled with
a diode array detector system Ultimate 3000 (Thermo Scientific, Pra­
golab, Czech Republic) was used for quantification of CAF, BTA, MPB,
DEET, and DCF. LC separations were achieved using C18 HypersilTM
Gold column (250 mm × 4.6 mm; pore size: 5 μm) (Thermo Scientific,
Pragolab, Czech Republic) with a compatible precolumn (Thermo Sci­
entific, Pragolab, Czech Republic). The mobile phase was acetonitrile
(Chromasolv, for HPLC, gradient grade, >99.9 %, Honeywell) and a
buffer: 10 mM ammonium formate/formic acid (pH adjusted to 3.3) in
ultrapure water. The separation was obtained using a gradient mode:
0–1 min 20 % acetonitrile, 13–16 min 90 %, 18–20 min 20 %. Injection
volume was 20 μl for all type of samples. The compounds were quanti­
fied using linear calibration computed using at least 7 calibration levels.
The retention times of the analysed compounds were: 4.9 min for CAF,
6.8 min for BTA, 9.3 min for MPB, 11.6 min for DEET, and 13.8 min for
DCF. The compounds were qualified at the following wavelengths: 220
nm for DEET, 256 nm for MPB and BTA, and 275 nm for DCF and CAF.
The limit of detection (LOD) was 0.01 mg/L, and the limit of
4
Journal of Water Process Engineering 59 (2024) 105066
qualification (LOQ) was 0.05 mg/L and for BTA, DCF and MPB, and for
DEET and CAF the LOD and LOQ were 0.05 mg/L and 0.1 mg/L,
respectively. The LOD values were defined for S/N (signal to noise) at
the level of 3 and the LOQ values at the level of 10. The data was
evaluated by means of Dionex Chromeleon 7.2 software.
For the analysis of S1DS, LCK 332 anionic surfactants cuvette test
(Hach-Lange, USA), which employs methylene blue method (ISO 7875-
1-2-1984), was used. The measurement of the absorbance and quanti­
fication were performed using a DR3900 Spectrophotometer (Hach-
Lange, USA).
Samples were analysed for metals (Cu, Ni, and Zn) and B by an
inductively coupled plasma optical emission spectrometry (ICP-OES,
Agilent 730, Agilent Technologies, USA). All samples were additionally
analysed for total nitrogen (TN), total carbon (TC), total organic carbon
(TOC) and inorganic carbon (IC) using FORMACSHT/TN TOC/TN
ANALYZER HTAccess version 3 (Skalar Analytical B. V., NL.), ammonium
(NH+ 4 ) using Cary 60 UV–Vis spectrometer (Agilent Technologies Inc.,
USA)., and anions (Fl− , Cl− , NO−2 , Br− , NO−3 , PO3− 2−
4 , and SO4 ) using 883
Basic IC Plus Metrohm MagIC NET™. In addition, pH and conductivity
values were measured using IDS pH Electrode SenTix® 940.
The content of elements (shown as normalized percent weight) was
determined by scanning electron microscopy (SEM) and Energy-
dispersive X-ray spectroscopy (EDX) of the samples were performed
using a MIRA 3 electron microscope (Tescan Orsay Holding, Brno, Czech
Republic) with a secondary electron detector operated at 12 kV accel­
eration voltage. The EDX of the samples was conducted using an energy
dispersive spectroscopy system (Bruker XFlash X-ray detector, Karls­
ruhe, Germany, and ESPRIT 2 software).
2.4. Ecotoxicological assessment of effluents
Two model organisms were used for ecotoxicological analyses -
bacterium Aliivibrio fischeri (ČSN EN ISO 11348-2) and seeds of Sinapis
alba. The Aliivibrio fischeri bioluminescence inhibition bioassay is based
on inhibition or stimulation of bacterial bioluminescence due to the
presence of sample at 15 ◦ C. A 0.5 mL sample is mixed with 0.5 mL of
bacterial solution and relative inhibition (Ht) is measured using
luminometer LUMIStox 300, where “t” is 5, 15, and 30 min after expo­
sure to the sample. The control sample is prepared by mixing 2 % so­
lution of NaCl and bacterial solution. The Sinapis alba ecotoxicological
assay is based on 72 h germination capacity of 30 seeds in the presence
of a 10 mL sample in a Petri dish filled with a filter paper.
Positive values of both methods indicated the inhibition of germi­
nation or bioluminescence, and negative values mean their stimulation.
The samples for the ecotoxicity assessment were taken on days 79 and
104 of the experiment to represent the middle (approximately) and the
final day of the steady-state period before the inhibition test. Even
though the steady-state period and the inhibition period ran parallelly
from day 105 to 125, the number of columns unaffected by the inhibitor
was different than before day 105, which could have affected the com­
parison of the ecotoxicity analysis between days 79 and 104.
2.5. Prokaryotic community structure analysis
Total bacterial genomic DNA was extracted to describe the pro­
karyotic microbial community in the biofilters. The samples of the upper
and bottom layer of the filtering material were taken befor the start of
the inhibition period from one replicate of each type of the treatment.
The samples were taken only from the columns that were not fed with
the SGW containing inhibitor. To ensure homogeneity of the sample, the
entire filtration bed was removed from the column to avoid mixing of
the layers. Next, each layer was thoroughly homogenized and divided
into three replicates both from upper and bottom layers (36 samples in
total). The samples were stored at − 25 ◦ C prior to the analysis. The
amount of 0.25 g was used for the DNA extraction using PowerSoil DNA
isolation kit (MO BIO Laboratories, USA). The aliquots of DNA extracts
F.Ö. Çömez et al. Journal of Water Process Engineering 59 (2024) 105066

were sent to SEQme lab (Prague, Czech Republic) for the preparation of
a library and sequencing using MiSeq platform. The Earth Microbiome
Project (EMP) protocol was used for library preparation with modified
universal primers 515FB/806RB [46] for prokaryotic 16S rDNA ampli­
cons. The coverage of prokaryotic primer pair 515FB/806RB was
additionally tested in-silico using ARB Silva database release 128. The
primer pair 515FB/806RB covers almost uniformly all major bacterial
and archaeal phyla. Bacterial 16SrDNA raw pair-end reads (150 bp)
were joined using usearch11 (mergepairs script) to obtain reads of
approx. 250 bp length. Quality filtering of reads was applied as previ­
ously described [46]. We obtained 598,614 bacterial sequences after
quality trimming and filtering. Before picking the zero-radius OTUs
(zOTUs) by unoise3 algorithm which is equal to amplicon sequence
variants (ASVs) all reads were trimmed to equal length of 300 bp, reads
with Ns, homopolymers with >6 bases were discarded. Taxonomy was
assigned to each read by accepting Silva132 taxonomy string of the best
matching Silva 132 sequence using megablast. Raw sequences were
deposited at ENA under the project n. PRJEB61002.
Wilcoxon test were used to assess the differences in pollutant removal
performance between hydraulic retention times. The comparison of the
removal efficiency of organic compounds and metals from filter media
was calculated using the Kruskal-Wallis rank sum test followed by the
Dunn's test with Benjamin-Hochberg correction. In the case of the
parametric test, ANOVA was chosen followed by Tukey HSD. The effect
of inhibition of microorganism populations in the filter medium and its
impact on filtration efficiency before and after the inhibition test was
analysed by the Wilcoxon signed rank exact test or paired t-test. The use
of parametric and non-parametric tests depended on meeting the as­
sumptions for individual analyses.
The microbiome zOTU table was analysed in R 4.2.2 using phyloseq
and microViz R package. For calculating the dissimilatory matrix of
relative proportion of zOTU in different samples, the Bray-Curtis equa­
tion and metric principal-component analysis (PCA) was used.
3. Results and discussion
3.1. Removal of standard contaminants, metals, and boron

2.6. Data analysis

Data describing biofilter performance were divided into three pe­
riods: acclimation period (the first 37 days of the experiment), steady-
Microsoft Excel spreadsheet was used for data management and
state period (from day 37 until the end of the experiment), and inhibi­
removal efficiency calculations. RStudio (v. 4.1.2) was used for statis­
tion period (includes the data from inhibited columns after the day 105).
tical data evaluation. Two sample t-test and the unpaired two-samples

Table 2
The basic parameters of synthetic greywater quality, boron, metals for the steady-state period according to the batch duration (average ± standard deviation (removal
efficiency)).
Compounds (mg/L)

Media Batch pH TOC IC TN N-NO3 N-NH4 Phosphate Boron Copper Nickel Zinc

Influent
7.2 ± 18.44 ± 12.65 ± 30.73 ± 2.46 4.4 ± 21.5 ± 2.7 3.94 ± 1.19 ± 0.13 ± 0.05 0.18 ± 0.02 0.21 ± 0.04
0.32 5.58 2.51 1.3 0.63 0.18

Effluents
C 1d 7.0 ± 17.50 ± 28.06 ± 29.47 ± 4.52 25.13 1.30 ± 1.85 9.12 ± 1.206 ± 0.007 ± 0.002 ± 0.002 ±
0.14 3.53 (5.1 12.81 (4.08 %) ± 2.46 (93.83 %) 2.51 0.151 0.014 0.000 0.003
%) (94.60 %) (98.90 %) (99.14 %)
3d 7.0 ± 18.10 ± 30.95 ± 23.79 ± 2.29 20.57 1.64 ± 2.95 9.60 ± 1.205 ± 0.002 ± 0.003 ± 0.002 ±
0.17 4.36 (1.8 16.40 (22.58 %) ± 3.05 (92.26 %) 2.34 0.136 0.002 0.001 0.004
%) (98.52 %) (98.61 %) (99.02 %)
B10 1d 7.0 ± 10.07 ± 27.64 ± 27.57 ± 4.55 23.74 0.20 ± 0.64 11.10 ± 1.189 ± 0.001 ± 0.002 ± 0.003 ±
0.27 1.51 (45.4 12.75 (10.26 %) ± 5.30 (99.06 %) 3.42 0.127 0.001 0.000 0.007
%) (98.94 %) (98.90 %) (98.60 %)
3d 7.0 ± 10.60 ± 29.04 ± 24.21 ± 2.49 22.11 0.34 ± 1.48 10.83 ± 1.193 ± 0.002 ± 0.002 ± 0.003 ±
0.29 2.37 (42.5 15.81 (21.20 %) ± 1.76 (98.40 %) 3.13 0.146 0.002 0.000 0.004
%) (98.59 %) (98.81 %) (98.60 %)
B5 1d 7.1 ± 11.12 ± 27.44 ± 26.75 ± 3.07 24.29 0.03 ± 0.09 11.38 ± 1.189 ± 0.001 ± 0.003 ± 0.004 ±
0.23 1.55 (39.7 11.98 (12.96 %) ± 2.03 (99.86 %) 3.53 0.143 0.000 0.003 0.007
%) (99.14 %) (98.34 %) (98.01 %)
3d 7.0 ± 11.70 ± 30.72 ± 24.00 ± 1.67 22.35 0.03 ± 0.08 11.52 ± 1.201 ± 0.002 ± 0.002 ± 0.002 ±
0.23 2.02 (36.5 14.86 (21.91 %) ± 1.74 (99.85 %) 3.25 0.134 0.001 0.000 0.003
%) (98.75 %) (98.90 %) (99.30 %)
Br 1d 7.2 ± 16.64 ± 44.58 ± 31.28 ± 5.63 27.39 0.27 ± 0.69 3.30 ± 1.167 ± 0.004 ± 0.003 ± 0.009 ±
0.19 2.78 (9.8 8.38 (− 1.79 %) ± 3.39 (98.72 %) 0.20 0.160 0.007 0.003 0.014
%) (96.53 %) (98.45 %) (96.00 %)
3d 7.2 ± 16.94 ± 47.71 ± 25.71 ± 3.96 23.93 0.14 ± 0.26 3.29 ± 1.197 ± 0.002 ± 0.002 ± 0.008 ±
0.14 3.38 (8.2 10.22 (16.33 %) ± 3.19 (99.34 %) 0.28 0.147 0.002 0.000 0.010
%) (98.58 %) (98.90 %) (96.09 %)
L 1d 7.0 ± 15.51 ± 39.42 ± 31.42 ± 6.40 26.54 0.34 ± 0.35 5.83 ± 1.188 ± 0.007 ± 0.002 ± 0.002 ±
0.13 2.54 (15.9 5.96 (− 2.26 %) ± 3.23 (98.40 %) 0.61 0.146 0.015 0.000 0.004
%) (94.47 %) (98.90 %) (99.12 %)
3d 7.1 ± 16.04 ± 44.20 ± 24.82 ± 2.53 21.75 0.51 ± 0.70 5.67 ± 1.139 ± 0.011 ± 0.002 ± 0.015 ±
0.18 3.26 (13.0 7.86 (19.22 %) ± 2.20 (97.57 %) 0.53 0.135 0.018 0.000 0.016
%) (91.55 %) (98.90 %) (93.07 %)
W 1d 6.8 ± 18.05 ± 37.73 ± 24.64 ± 2.34 9.42 ± 9.67 ± 4.24 4.07 ± 1.192 ± 0.008 ± 0.003 ± 0.002 ±
0.28 4.20 (2.1 18.15 (19.81 %) 3.52 (54.27 %) 1.16 0.147 0.018 0.003 0.003
%) (94.01 %) (98.34 %) (99.18 %)
3d 6.8 ± 18.14 ± 37.31 ± 23.25 ± 2.17 9.52 ± 8.09 ± 4.03 4.11 ± 1.151 ± 0.004 ± 0.004 ± 0.002 ±
0.25 4.87 (1.6 21.25 (24.35 %) 3.36 (61.75 %) 1.19 0.141 0.005 0.004 0.004
%) (96.92 %) (97.60 %) (99.05 %)

5
F.Ö. Çömez et al.
The division between the acclimation and the steady-state periods was
arbitrarily assumed based on the behaviour of BTA in the effluents
(Section 3.1). The summary of the concentration of basic greywater
quality parameters for influents (SGW) and effluents with the removal
efficiency is presented in Table 2. The average TOC (total organic car­
bon) concentrations in effluents, except for B10 and B5, were close to
influent values. Only, B10 and B5 exhibited moderate removal of TOC,
which was in the range of 36–45 %. The improved removal of TOC in the
biochar columns was assumed to be associated with increased adsorp­
tion capacity, which can be explained by its high surface area and hy­
drophobic properties due to high pyrolysis temperature (as evaluated for
this specific batch of biochar by [33]). However, the inhibition test
indicated a major role for biodegradation in the removal of TOC (S3,
Supporting information). The removal of IC (inorganic carbon) was
observed to be negative during the experiment and the highest release
was measured for Br biofilters. Additionally, longer batch duration
increased the release of IC, which could be associated with denitrifica­
tion process [65]. TN (total nitrogen) removal efficiency was lower than
reported in the literature in similar systems [47]. On the other hand, it is
possible to observe that the longer batch duration increased the TN
removal efficiency in all types of biofilters probably as a result of
enhanced activity of denitrifiers. TN removal was very low or negligible
during the 1-day batch in the C (4 %), Br (− 2 %), and L (− 2 %) biofilters,
while 23 %, 16 %, and 19 % removal was achieved during the 3 days
batch, respectively. In B10, B5, and W biofilters, the TN removal was
>10 % during the 1 day batch and >20 % during the 3-day batch. The
reason might be due to the higher proportion of nitrifiers in biochar
columns that implies more efficient nitrification (Section 3.4). The
removal of N-NH4 was >90 % in all biofilters except biofilters with
woodchips (58 %). The removal of phosphate was not observed in any
biofilters except for Br (16.29 %) biofilters, which provided low
removal. This behaviour of Br biofilters was probably due to the rich
surface in iron (Fe), calcium (Ca), and aluminium (Al) (S4, Supporting
information) [48].
The performance of the biofilters in removing inorganics was high,
except for boron. During the steady period, removal of Cu, Ni, and Zn
were efficient (>90 %) in all biofilters. Biofilters with biochar additive
demonstrated stable removal efficiency whereas occasional break­
through of metals which were observed during the steady-state period
may have happened due to desorption or dissolution of metal binding
phases like iron and manganese oxides. Nevertheless, boron removal in
all types of biofilters studied was negligible. Boron was released from the
medium materials until day 51 (− 10 % removal efficiency on average of
all biofilters). Then, the removal efficiency showed an increasing trend
(S5, Supporting information), and <20 % removal efficiency was
observed. On day 100, L biofilters had 15 % boron removal efficiency
followed by W (11 %), B5, B10 and Br (10 %), and C (8 %). By this
means, additive media predictably showed a positive impact on the
removal of boron. The removal of B remained below 20 % until the end
of the experiment. No statistically significant difference (p > 0.05) was
found between the inhibition and the steady period for boron removal in
all media configuration of biofilters, suggesting negligible effect of biotic
processes on the removal of this element.
Metals were almost completely removed (>90 %), with no signifi­
cant differences between the tested types of biofilters (p > 0.05).
Although no statistically significant differences were found between the
steady-state and inhibition test periods, slightly lower removal effi­
ciency after the inhibition test could suggest that metal removal was due
to binding to the filter medium with a minor role of interactions with the
active microbiota. Such high metal removal in biofilters has been re­
ported before by Blecken et al. [49] for Cu (95.2 % ± 5.7) and Zn (97.4
% ± 1.7) and by Hasan et al. [50] for Cu and Zn (>95 %) in biochar-
based biofilters have been reported to be efficient in the removal of [50].
6
Journal of Water Process Engineering 59 (2024) 105066
3.2. Removal of organic pollutants
A summary of average concentration for influent and effluent values,
as well as removal efficiency of the compounds according to the bio­
filters, are given in S6 (Supporting information) for the steady period.
The results from the acclimation and steady-state period showed that the
removal of organic compounds was >90 % in the biofilters for each
media type, except for BTA. BTA removal was achieved on average > 95
% only in B5 and B10 biofilters while the other biofilters had <60 % on
average during the acclimation period. Similarly, during the steady
period, a high average removal of BTA continued in B10 and B5 bio­
filters with an average of 98 % and 99 %, respectively however, the BTA
removal was lower than 30 % in the other biofilters (Fig. 3ab), showing a
decreasing trend. As it was already stated above, the experiment was
divided into three main phases: acclimation, steady-state, and inhibition
periods. The effluent concentrations of BTA were very low at the
beginning of the experiment but showing growing tendency until day 34
(apart from B5 and B10 biofilters, they stayed low during the whole
experiment) and increased to a level within a range of 1.20–1.70 mg/L,
indicating moderate removal, and remained at this level until the end of
the experiment. The time profile of BTA was used as an indication for the
selection of a steady period (Fig. 4).
High removal performance of biofilters with biochar additive were
probably due to its high adsorption capacity which was supported with
the results from the inhibition of biofilters. A higher removal of BTA
could be otherwise achieved in biofilters with free-draining operation
mode (constantly unsaturated bed fed intermittently), which increases
the oxygenation of the system. For example, Felis et al. [9] obtained over
80 % BTA removal via unsaturated intermittently fed treatment wetland
system with indicating no positive affect of vegetation on the removal.
Kowalska et al. [51] classified BTA as a compound undergoing partial
removal only if an adapted activated sludge is used, otherwise its
biodegradation is negligible for the removal. Using membrane bio­
reactors, they achieved on average 97 % BTA removal however, the
main process for elimination was referred to biotransformation more
than biodegradation or sorption; and reported that some of the trans­
formation products might be more toxic to aquatic ecosystem than
parent compound [51]. Therefore, BTA removal through low cost bio­
filter media might need further studies focusing on the transformation
products of BTA. After the inhibition of biofilters for 25 days, the
removal of BTA through biofilters ceased and the release of the com­
pound was observed in the effluent apart from B5 and B10 biofilters. The
biofilters with biochar additives continued the elimination of BTA with
99 % efficiency. The preserved efficiency in biochar biofilters can be
attributed to the high sorption capacity of biochar. However, the release
in biofilters C, W, Br, and L after the implementation of inhibitor in­
dicates that low removal during the steady period was probably due to
biodegradation. These results are following the findings of Alotaibi et al.
[52], showing that the limited removal of BTA from a sterile control
column proved that sodium azide had inhibited the activity of BTA-
degrading microorganisms. It should be highlighted that the sorption
capacity of the applied biochar was determined to be 42.2 mg/g in a
dynamic with DCF, MPB and S1DS as co-solutes [33]. Taking into ac­
count the overall load of BTA it can be estimated that the capacity of B5
and B10 columns in the present experiment were saturated only in 2.7 %
and 5.3 %, respectively. This would imply that the longevity of the
biochar for BTA at the influent concentration of 2.5 mg/L in biofilters
would be approx. 12 years, which is more than the design operational
period of biofilters. However, specific longevity tests should be per­
formed in pilot-scale systems treating real greywater. So far, the data
regarding this aspect are missing.
S1DS was removed with >75 % efficiency in all biofilters during both
the acclimation and steady periods, mostly due to its high biodegrada­
tion which is supported later by the results of the inhibition test. The
removal efficiency showed a slightly decreasing trend from the begin­
ning to the end of the experiment. In control biofilters, 86 % removal on
F.Ö. Çömez et al. Journal of Water Process Engineering 59 (2024) 105066

Fig. 3. Removal efficiency of BTA in biofilters during the steady-state period in 1-d batch (A) and 3-d batch (B). The whiskers are presented as min/max quartiles
with data from the steady period. The average is represented with a triangle. The darker colour of the box plots represents the first quartile, and the lighter colour
represents the third quartile.

Fig. 4. Time profile of BTA concentration in the effluent.

7
F.Ö. Çömez et al.
average was obtained during the acclimation period, then it dropped to
80 % removal on average during the steady period. Removal perfor­
mance of biofilters with additives did not show any statistically signif­
icant difference (p > 0.05) as compared with the (C) group biofilters
during the steady-state period, indicating that additive inclusion did not
change the removal performance of S1DS. During the steady period, the
removal of S1DS in all biofilters did not drop below 75 % efficiency.
These results were slightly better than the removal efficiency obtained
through vegetated biofilters (<75 %) reported by Boano et al. [53] and
the removal efficiency obtained through granite sand filters (<75 %)
reported by Tripathi et al. [54]. The inhibition of biotic degradation
processes for S1DS (S3, Supporting information) indicates that their
removal is highly dependent on the biodegradation process and that
adsorption might play a minor role, especially in a long-term process.
However, adsorption might play a moderate role for the removal process
in B10 and B5 biofilters which may also suggest limited capacity of this
sorbent for S1DS. So far, the longevity of biochar cannot be determined
and, moreover, it is still unknown whether the compounds adsorbed
onto biochar can be available for microbial biota, which would enable
bio-regeneration of biochar [55].
DEET removal was at high level (>95 %) in all biofilters during both
the acclimation and the steady periods. While initial results of the
removal performance for C, B10, B5, Br, and L biofilters were in high
levels (>95 %), acclimation took relatively longer for W biofilters to
achieve >95 % removal performance (after day 20). In general, effluent
concentrations of DEET initially exhibited an increasing trend within
several days of the experiment, followed by a steep decline to values
below or at LOQ, which remained until the end of the experiment (S7,
Supporting information). This behaviour of DEET could indicate adap­
tation of microbial biota to the transformation of this compound. During
the steady-state period, statistically significant differences were found
between the removal performance of control biofilters and both Br and L
biofilters (p < 0.001), which might be due to decreasing removal per­
formance of Br and L after day 86 (S7, Supporting information). In the
inhibition test, the removal performance of biofilters for DEET was
partially inhibited, apart from B5 and B10 columns. The removal of
DEET was not significantly affected (p > 0.05) in B10 and B5 biofilters
between the steady-state and inhibition periods, whereas the impact
significance level was <0.001 in C, W, and L biofilters and <0.05 in Br
biofilters. At the end of the inhibition test period (day 125), the removal
efficiency of biofilters were 61 % in Br and L, 53 % in C, and 50 % in W.
On average, the largest decrease in the removal efficiency was observed
for W biofilters by 28 %. While removal performance of biofilters
showed a decline during the inhibition test period, the high removal in
biochar additive filters suggests adsorption as the removal mechanism of
DEET. Supporting this, continuing removal in the other biofilters
(despite the decrease) indicates existence of adsorption. Greenstein et al.
[24] similarly concluded that DEET removal is dependent on sorption
instead of biotransformation during their pilot scale biofilter design with
virgin anthracite-sand and previously used biological activated carbon-
sand dual media filters.
The removal of DCF was efficient (>90 %) in all biofilter types (S8,
Supporting information). A slightly decreasing trend of removal was
observed only for the biofilter with limestone additive, but removal ef­
ficiency remained over 90 %. Even though the removal efficiency of DCF
in B5 and B10 biofilters did not change between the steady-state period
and the inhibition test (remained >90 %), it was shown that some
fraction of DCF was removed by biotic process, suggesting probable
depleting sorption capacity of biochar. For the other types of biofilters,
biotic process played a predominant role. However, it was reported that
brick media enhanced with iron oxides increased the adsorption ca­
pacity for removing DCF from aqueous solution [41].
Despite the use of high influent concentrations, the biofilters pro­
vided high removal (>99 %) for MPB and CAF from the beginning of the
experiment. The average MPB and CAF concentrations were 2.4 and 2.5
mg/L, respectively in the influent during the experiment and no
8
Journal of Water Process Engineering 59 (2024) 105066
degradation trend was observed in the storage barrel. In the effluents,
MPB and CAF concentration were 0.025 ± 0.001 mg/L for all biofilters
during the steady period. The high MPB and CAF removal efficiency of
biofilters can be explained by the high degradability of these substances
[56,57]. However, the increased removal efficiency from a biologically
active environment to a biologically non-active environment may sug­
gest that the removal of these substances mostly depended on sorption
(S3, Supporting information). Similarly, sorption has been reported as a
responsible mechanism for the removal of CAF through biochar filters
[34] and slow-sand filters [58]. This observation might, however, not be
valid considering two factors: their high mobility in soils assumed based
on logDow coefficient (S2, Supporting information); and incomplete in­
hibition of the biodegradation processes, reflected by the partially active
removal of N-NH4. It can be hypothesized that both MPB and CAF were
readily biodegraded even at a reduced capacity of biofilters for
biodegradation. This high potential for biodegradation of CAF and MPB
suggests that these substances can probably also be removed under less
favourable conditions considering temperature, short batch duration,
and presence of inhibitors (e.g., chlorine compounds). Since there was
not a major difference in removal efficiencies between control biofilters
and biofilters with additives, the impact of additives stayed unclear.
Statistical analysis indicated no significant differences (p > 0.05) in
the effect of batch duration on the removal of CAF, MPB, DEET, DCF and
S1DS (S9, Supporting information). The exception was observed for BTA
in the biofilters filled with Br, in which the longer batch duration slightly
improved the BTA removal (p < 0.01) (Fig. 3ab). The improved removal
of BTA, which was noted to be slowly biodegradable in this experiment,
probably stems from the extended batch duration that can be crucial for
biodegradation processes.
3.3. Ecotoxicological assessment
The results of the ecotoxicity tests are shown in Fig. 5. The SGW itself
(influent) exhibited low toxicity to Aliivibrio fisheri (luminescence inhi­
bition 15.3 %) and it was found to stimulate Sinapis alba (germination
inhibition − 29.3 %).
In the samples taken on day 79, there was no significant difference in
toxicity to Aliivibrio fisheri between samples from the control column and
all tested columns, and also among all tested columns with their various
additives. The samples showed slight inhibition ranged between 7.4 %
and 21.5 %. The samples also had a positive effect on Sinapis alba except
for the B10 sample, which showed slight inhibition (15.2 %). The
stimulation ranged from − 12.6 % (Br biofilters) to relatively high
stimulation effects of − 33.7 % (B5 biofilters) and − 41.3 % (L biofilters).
As the used certified method of ecotoxicology has error up to 20–25 %,
the differences among the additives are negligible.
In the case of the samples taken on day 104 of all columns had no
significant effect on Aliivibrio fisheri and had significant positive effect on
Sinapis alba. Relative inhibition of Aliivibrio fisheri after 30 min ranged
from 6.5 % to − 11.2 %. The relative inhibition to Sinapis alba ranged
from − 40.7 % (Br biofilters) and the highest values of stimulation was
observed for W (− 101.5 %) biofilters. No negative effect of added tested
materials was observed for Aliivibrio fisheri or Sinapis alba.
The assessment of ecotoxicological effect of treated GW indicated
that the detoxification efficiency of the treatment increased with the age
of the biofilters, as near to the end of the experiment period, there were
no negative effect on tested organisms, even stimulation effect on Sinapis
alba was observed. The stimulation effect indicates that treated GW due
to the presence of residual nutrients, may be used for irrigation and
fertilization in gardens and in agriculture depending on the types of
crops and legislation. The differences in stimulation effect among the
additives can be contributed to their chemical composition, as wood­
chips contain more organic matters and nutrients, than crushed bricks
and biochar.
The observed stimulative effect on the plant germination indicates
that GW could be a safe source of water for irrigation, but further
F.Ö. Çömez et al.
Fig. 5. Results of ecotoxicology analysis using two indicator organisms: a) Aliivibrio fischeri, b) Sinapis alba (the data for the synthetic greywater are for day 104).
investigation should be considered about the effect on soil respiration
and the transfer of the contaminants to plants, among other issues.
3.4. Microbiome community analysis
The analysis of the prokaryotic community was performed to eluci­
date the role of the additives and the layering of the biofilters on the
diversity and structure of the microbiome.
The data shown in Fig. 6ab shows that B5 columns provided the
greatest diversity of the microbiome based on both the Shannon and
Chao indices. Interestingly, the diversity in the B10 columns was lower
than in B5 counterparts and also in W columns. The W columns provided
slightly lower diversity than B5 columns. The superiority of B5 and W
columns could have been related to various aspects like higher avail­
ability of organic carbon and nutrient retention in the case of biochar
[59]. The diversity of the prokaryotic communities was much greater in
the upper layers of the filters (S10, Supporting information), probably
because of higher concentrations of nutrients, greater aeration of the
filtration bed and the presence of compost (in all the columns) and
various additives and.
Prokaryotic communities were analysed at phyla level. Fig. 7 shows
the composition in both layers was dominated by Proteobacteria and
Actinobacteriota. These results are in line with those obtained by
Rodríguez-Martínez et al. [60]. In the bottom filter layer, there is
considerably higher proportion of Firmicutes (fermentation bacteria).
The upper filter layer supported greater proportion of Myxococcota
(predatory and cellulose degrading bacteria). The proportion of bacte­
rial nitrifiers (Nitrospirota) was greater in the bottom layer, especially in
biofilters with biochar additive. The biofilters with biochar additive,
also, have higher proportion of Crenarchaeota (mainly ammonia oxi­
dizers) and Desulfobacterota (anaerobic sulphate-reducing bacteria) in
the bottom layer. The former indicates higher nitrification potential and
more efficient ammonia removal in the presence of biochar [61]. The
composition of microbiome at genus level is given in S11 (Supporting
information) for genera other than nitrifying organisms. Noteworthy,
biochar was observed to be responsible for greater abundance of
9
Journal of Water Process Engineering 59 (2024) 105066
particular genus of ammonia-oxidizing organisms, which are Nitro­
sarcheum (S12, Supporting information). Specifically, it is a group of
mesophilic, ammonia-oxidizing archaea that play a crucial role in the
nitrogen cycle, particularly in the process of nitrification [62]. This
process is essential in maintaining nitrogen balance in ecosystems and is
therefore crucial in various applications, including wastewater treat­
ment, agriculture, and remediation. The study of Nitrosarchaeum and
other ammonia-oxidizing archaea has provided valuable insights into
the nitrogen cycle and its role in ecosystems [63], and it has implications
for understanding how these microorganisms can be harnessed for
practical purposes, like improving nitrogen removal in sewage treat­
ment plants or enhancing agricultural practices. The abundance of
Nitrosomonas bacteria, which commonly found ammonia oxidizing or­
ganisms in wastewater treatment systems was at a comparable level in
all the treatments, with slightly higher abundance in the W biofilters.
The factors affecting the dominance of ammonia-oxidizing bacteria or
ammonia-oxidizing archaea in wastewater treatment systems have not
been clearly indicated despite many studies. It seems that the important
factors involved in niche partitioning is the ammonia concentration,
temperature, levels of organic matter and metals [64]. However, the role
of biochar in wastewater treatment on shaping the abundance of archaea
was not so far discussed and thus needs further research.
4. Conclusions and outlook
Biochar was found to be a superior amendment in biofilters treating
SGW, because it improved their performance and promoted the biodi­
versity of the bacterial community compared to the other filter additives
studied. The high adsorption capacity of biochar improved the perfor­
mance of biofilters to obtain efficient removal of non-readily biode­
gradable compounds, such as BTA. There was no significant difference
between the removal performance of the biofilters B5 and B10, showing
that 5 % of biochar to the total media volume is already sufficient for an
efficient removal. Compost, sand, and organic soil mixture are materials
that are traditionally used as biofilter media, however, could be amen­
ded with media with high adsorption capacity, such as biochar, which
F.Ö. Çömez et al.
Fig. 6. Alpha diversity indices of microbiome in the studied biofilters: a) Chao1 index, b) Shannon index.
could increase the removal efficiency of organic pollutants in greywater.
The use of biochar brings very important engineering added values,
which are longevity and the ability to selectively retain organic con­
taminants, but not the nutrients. This may qualify the use of biofilters as
an attractive pretreatment of greywater for irrigation purposes. The
relatively high unit cost of biochar does not seem to be an issue that
could hinder its implementation (S13, Supporting information). This is
caused by the small proportion of biochar that could be implemented in
the biofilters if the present design is considered. The unit cost of
implementing biochar as an additive in public utility buildings would be
10
Journal of Water Process Engineering 59 (2024) 105066
lower than in households because of the lower volume of the produced
greywater. Further discussion of the implementation costs are given in
S13 (Supporting information).
CRediT authorship contribution statement
Fatma Öykü Çömez: Writing – original draft, Methodology, Data
curation, Conceptualization. Adam Sochacki: Writing – review &
editing, Supervision, Methodology, Investigation. Jaroslav Vacula:
Software, Investigation, Formal analysis. Jiří Bárta: Writing – original
F.Ö. Çömez et al.
Fig. 7. The relative abundance of prokaryotic microbiome composition (phylum level, silva ARB v. 132 in the upper and bottom layers of each type biofilters).
draft, Methodology, Investigation. Ljuba Zídková: Software, Investi­
gation, Formal analysis. Martin Lexa: Investigation, Data curation.
Dana Komínková: Writing – review & editing, Supervision,
Conceptualization.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
Data will be made available on request.
Acknowledgements
This work was supported by project SWAMP - Responsible water
management in built-up areas in relation to the surrounding landscape,
No. CZ.02.1.01/0.0/0.0/16_026/0008403, Ministry of Education,
Youth and Sports of the Czech Republic. Adam Sochacki acknowledges
Czech Science Foundation grant (project No. 19-12184Y). The authors
acknowledge Environmental English Ltd. for proofreading.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.jwpe.2024.105066.
11
Journal of Water Process Engineering 59 (2024) 105066
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