Bioresource Technology Reports 24 (2023) 101615

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Bioresource Technology Reports

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Use of biochar and Moringa oleifera in greywater treatment to remove heavy

metals and contaminants of emerging concern
Ivan Kozyatnyk a, *, Mary Njenga b, c
a
Department of Health, Medicine and Caring Sciences, Unit of Clinical Medicine, Occupational and Environmental Medicine, Linköping University, 581 83 Linköping,
Sweden
b
World Agroforestry (ICRAF), P.O. Box 30677-00100, United Nations Avenue, Gigiri, Nairobi, Kenya
c
Wangari Maathai Institute for Peace and Environmental Studies, University of Nairobi, P.O. Box 2905-0065, Nairobi, Kenya

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

Keywords: This study investigated the combination of biochar derived from carbonized local feedstock and Moringa oleifera
Adsorption seed extract-based flocculant, a novel combination in the context of greywater treatment missed in the previous
Flocculation studies, with a focus on the removal of organic contaminants (caffeine, chloramphenicol, trimethoprim, carba­
Sustainable solutions
mazepine, diclofenac, and bisphenol A) and heavy metals (Cr(VI), As(III), Co(II), Ni(II), Cu(II), Zn(II), and Pb
Water contamination
Developing countries
(II)). The research focused on the optimization of dosage, application procedure, and relevance to greywater
(presence of humic acids, clay, and surfactants).
The order of application was found to impact organic compound removal, with the initial application of the
flocculant, followed by biochar, being the more effective approach. The combined use of biochar and flocculant
positively impacted the removal of heavy metals but impeded the removal of organic pollutants. The presence of
humic acids, clay, and surfactants affected the flocculation process, which impeded the removal of heavy metals
and organic pollutants.

1. Introduction
Water contamination and the scarcity of safe drinking water have
been exacerbated by societal problems such as population growth, the
degradation of water catchment areas, climate change, urban growth,
and industrial development. Most developing countries have inadequate
infrastructure for effective wastewater treatment due to perceptions that
these types of investments are exorbitant despite the subsequent benefits
in human health. The conventional wastewater treatment methods like
sedimentation and flotation, secondary biological treatments like acti­
vated sludge, trickling filters, and rotating biological contactors, as well
as tertiary treatments such as membrane filtration, advanced oxidation
processes are often perceived as expensive in many developing countries
(Bassi et al., 2022). Low-cost greywater treatment can be a solution to
these issues. Greywater describes wastewater from baths, sinks and
washing machines, and accounts for about 60 % of the outflow from
residential homes (Khanam and Patidar, 2022). This type of wastewater
contains far less pathogens and 90 % less nitrogen than toilet water, so it
does not require the same treatment process as sewage water. In light of
the continuously growing demand for freshwater, treated greywater
could be extensively used for irrigation purposes, which would decrease
the strain on freshwater for primary uses (Khanam and Patidar, 2022).
At least 280 organic micropollutants can be found in greywater;
these contaminants include pharmaceutical and personal care products,
pesticides, agrochemicals, surfactants, and artificial sweeteners, among
others, most of which are difficult to handle (Dwumfour-Asare et al.,
2018). Emerging pollutants are increasingly being recognized as a latent
threat to human health. In many developing countries, there is limited
regulation regarding water protection from landfill sources, which often
includes ineffective enforcement mechanisms. Currently, the water
scarcity present in developing countries forces people to use drinking
water that is polluted with greywater infiltrate. Therefore, an important
current issue is developing technologies to treat this type of water for
drinking or household purposes.
One of the most broadly used methods for removing contaminants of
emerging concern (CECs) from water is adsorption on carbon adsorbents
such as activated carbons (ACs) (Leite et al., 2018). ACs have various
structural and morphological properties that enhance adsorption po­
tential, such as large specific surface area, high porosity, and reactive
surface chemistry (Tan et al., 2017). For this reason, ACs are effective for

* Corresponding author.
E-mail addresses: ivan.kozyatnyk@liu.se (I. Kozyatnyk), M.Njenga@cifor-icraf.org (M. Njenga).

https://doi.org/10.1016/j.biteb.2023.101615
Received 30 July 2023; Received in revised form 5 September 2023; Accepted 6 September 2023
Available online 9 September 2023
2589-014X/© 2023 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
I. Kozyatnyk and M. Njenga
removing pharmaceuticals (Mestre et al., 2019), natural organic matter
(Kozyatnyk et al., 2014), heavy metals (Cao et al., 2019), and nutrients
from different types of wastewater (Riley et al., 2018). However, the
high production cost and energy consumption of these materials (León
et al., 2020), along with a non-renewable source of raw materials (Zhao
et al., 2023), limit the extent to which commercial ACs are used in
developing countries for water treatment. The adsorption performance
of activated carbons is influenced by their porosity, surface area, and
functional groups, but they can struggle with low molecular weight
compounds, quickly saturate with high pollutant loads, and often need
energy-intensive regeneration or replacement (Kozyatnyk et al., 2020).
The carbonization of local, low-cost feedstocks (oil palm and coconut
shells, sugar cane bagasse, coffee husk, firewood) has attracted much
attention as an alternative method for producing carbonaceous mate­
rials, e.g., biochars, that are suitable for the treatment of greywater and
contaminated drinking water in low- and middle-income countries
(Dalahmeh et al., 2016). Biochar is a stable carbon material obtained by
heating biomass at elevated temperatures (500–800 ◦ C) with little or no
oxygen (Ahmed et al., 2016). Monitoring several key parameters can
have a significant effect on the ability of biochar to adsorb organic
compounds, which involves pH, surface functional groups, organic
matter residuals, surface area and pore volume, and the degree of
carbonization (Oh and Seo, 2016). It has been reported that biochar can
efficiently adsorb both organic and inorganic pollutants (Ramola et al.,
2014), but may show limited ability to selectively adsorb contaminants
which are present at high concentrations due to low porosity (Ahmed
et al., 2016). Several modification methods, including surface oxidation,
impregnation of metal oxides, and functionalization, are used to
improve the environmental remediation performance of biochars.
However, most of these methods require complicated equipment and
reagents which may not be readily available in developing regions.
Therefore, the present study focuses on resources and techniques that
should be available to most developing countries.
Moringa oleifera (MO) is a small tree from sub-Himalayan regions of
north-west India that is indigenous to many parts of Asia, Africa, South
America, and the Pacific and Caribbean Islands. In these regions, MO
leaves, flowers, seeds and roots are consumed as food. The antimicrobial
and flocculant properties of dried MO seeds are well documented in the
literature (Matthew et al., 2022; van den Berg and Kuipers, 2022). The
active component of a powder prepared from dried, crushed MO seeds is
a soluble protein which acts as a natural cationic polyelectrolyte that
causes flocculation (Nouhi et al., 2019). In addition, there are reports
that MO seeds can be used to remove dyes and detergents from aqueous
solutions (Beltrán-Heredia et al., 2009). Despite these clear advantages,
the use of MO seeds in the water treatment process can increase dis­
solved organic carbon (DOC) levels. This influences the odour, colour,
taste, and microbiological stability of water (Beltrán-Heredia et al.,
2012; Okuda et al., 2001), ultimately making it undesirable. To address
these disadvantages MO-induced flocculation can be followed by
filtration processes to remove the flocs and associated DOC. By using MO
seed extracts rather than the whole seed, one can reduce the amount of
organic matter introduced to the water (Nouhi et al., 2019). The
extraction process can be optimized to get maximum coagulating pro­
teins while minimizing the introduction of extraneous organic com­
pounds. Adjusting the MO dosage can also balance the benefits of
contaminant removal against the disadvantages of increased DOC. One
of the main advantages of using MO as a flocculant is that it is readily
available, low-cost, and easy to use. In the context of wastewater
treatment, MO seed flocculant has been shown to be effective in
removing a wide range of contaminants, including heavy metals (Shan
et al., 2017), organic pollutants (Rosmawanie et al., 2018), and patho­
gens (Bancessi et al., 2020).
The primary goal of this work was to investigate the combined po­
tential of using MO seed protein, a natural coagulant and flocculant, and
biochar, an adsorbent material, to remove organic CECs and heavy
metals from spiked tap water and artificial greywater. The study focused
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on optimizing the dosage and application parameters, as well as eluci­
dating which mechanisms are responsible for the removal of contami­
nants by these materials. Furthermore, the research assessed the
potential advantages and limitations of this combined treatment
approach.
2. Material and methods
2.1. Raw materials
The biochar samples utilized in this study were a by-product from
cooking with Eucalyptus and Markhamia lutea firewood in a gasifier stove
with a frame temperature of 740 ◦ C (Gitau et al., 2019). The samples
were collected from Siaya County, Nyabeda sublocation (0.13◦ N;
34.40◦ E), in Western Kenya. These biochars are typical by-products of
pyrolysis when firewood is used in a gasifier stove, making them easily
accessible and relevant for real-world applications in the region. The
biochar was subsequently ground and sieved to obtain a powder fraction
with a particle size of <0.15 mm for use in the experiments.
The MO seeds were sourced from Kenya, and the active substance
was extracted following a the protocol presented by (Nouhi et al., 2019)
with several modifications. The MO seed powder was de-oiled with a
hexane solution (Fisher Scientific, USA), after which the active ingre­
dient was extracted from a solution comprising 2.5 g of MO seed powder
and 50 mL of 0.1 M NaCl (Sigma-Aldrich, USA). The mixture was stirred
at 40 rpm for 1 h using a multirotator PTR-60 (Grant Instruments, UK),
after which the extract was separated from the seed powder by centri­
fugation at 4000 rpm for 20 min using a Rotina 380R centrifuge (Het­
tich, Germany). The concentration of MO proteins in the extract was 2.5
± 0.5 mg mL− 1.
2.2. Water treatment experimental design
The study aimed to investigate the removal of six CECs (caffeine,
chloramphenicol, trimethoprim, carbamazepine, diclofenac, and
bisphenol A, all at a concentration of 0.5 mg L− 1) and several heavy
metals (Cr(VI), As(III), Co(II), Ni(II), Cu(II), Zn(II), and Pb(II), all at the
concentration of 0.1 mg L− 1) from spiked tap water and artificial grey­
water using biochar and MO seed water extract. The artificial greywater
was prepared using humic acids (Sigma-Aldrich, Switzerland), kaolin
clay (Sigma-Aldrich, USA), and surfactants sodium dodecyl sulphate
(SDS) (Sigma-Aldrich, USA), each at a concentration of 50 mg L− 1. The
removal process was carried out in a JLT4 flocculator (Velp Scientifica,
Italy).
The CECs were selected based on distinct hydrophilic-hydrophobic
properties, which significantly impact adsorption on carbon porous
materials. The distribution coefficient (KD), which accounts for the ionic
form of a compound at a specific pH, has been suggested as a suitable
parameter for evaluating these properties (Kozyatnyk et al., 2021; Li
et al., 2018).
In the experiments, different amounts of biochar (100, 200, 500 and
1000 mg L− 1) were combined with 0.5 L of spiked water and stirred in
the flocculator at 60 rpm for 2 h. Next, varying volumes of MO seed
extract, e.g., 0.5, 1.0, 2.0, and 3.0 mL L− 1, which correspond to 1.3, 2.5,
5.0, and 7.5 mg L− 1, MO seed proteins respectively, were added, and the
mixture was stirred at 120 rpm for 5 min, followed by stirring at 20 rpm
for 30 min. The treated water was allowed to settle for 30 min before
being filtered through a 0.45 μm regenerated cellulose membrane filter
(VVR, China) using a syringe.
The influences of distinct greywater components (humic acids,
kaolin clay, and SDS) on treatment efficiency were assessed through
separate experiments. The study was conducted in four stages to eval­
uate the effectiveness of biochar and MO seed extracts in removing
contaminants from spiked tap water and artificial greywater:
1. Determination of the optimal treatment order.
I. Kozyatnyk and M. Njenga
Fig. 1. Morphological characteristics of the surface of wood biochar produced in a gasifier stove. a - pore volume distribution and nitrogen adsorption-desorption
isotherm; b – FTIR spectrum; c – C1s XPS spectrum; d – O1s XPS spectrum.
2. Selection of the appropriate dosages for biochar and MO seed extract.
3. Investigation of the individual effects of humic acids, kaolin clay, and
SDS on the treatment process, as well as their combined effects, in
artificial greywater.
4. Evaluation of increased MO seed extract dosages in scenarios where
flocculation was found to be problematic.
2.3. Determination of biochar structure and surface functionalities
The surface chemistry of the utilized biochar was analyzed using X-
ray Photoelectron Spectroscopy (XPS) and Diffuse Reflectance Infrared
Fourier Transform Spectroscopy (DRIFTS). The XPS spectra were ac­
quired with a Kratos Axis Ultra DLD electron spectrometer, for more
details please see (Latham et al., 2021).
The DRIFTS spectra were acquired with a Bruker IFS 66 v/S FT-IR
spectrometer (vacuum bench) coupled to DTGS and MCT detectors
(Bruker Daltronics, USA). The biochar powder was mixed with KBr at a
ratio of 1:12 and placed in sample holders. The analysis chamber was the
pumped down to <0.7 kPa, and spectra between 4000 and 400 cm− 1 at a
resolution of 2 cm− 1 were obtained.
An ASAP2020 automated nitrogen sorption/desorption instrument
(Micromeritics, USA) was used to analyze the porous properties of the
biochar samples. In the analysis, 0.1 g of dried biochar was degassed
under nitrogen flow at 300 ◦ C for 6 h. The multipoint BET method was
used to calculate the total specific surface area. Micropore surface area,
external surface area, and micropore volume were calculated using the t-
plot method. The pore volumes were determined using the
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Barrett–Joyner–Halenda (BJH) method.
2.4. Evaluation of contaminants in water samples
The CECs concentrations in water samples were determined using an
Agilent 1260 Chromatography System (Agilent, Germany) fitted with a
Purospher STAR RP-18 endcapped 5 μm C18, 150 × 2.0 mm column
with an analogous 4.0 × 4.0 mm pre-column (Merck, Germany). The
column temperature was kept constant at 30 ◦ C. The analyses were
carried out using acetonitrile and a 0.1 mol L− 1 ammonium acetate
buffer solution (Sigma-Aldrich, the Netherlands) at a flow rate of 0.5 mL
min− 1. The injection volume was 20 μL, and caffeine, trimethoprim,
chloramphenicol, carbamazepine, and diclofenac were detected based
on UV absorption at 283 nm. Bisphenol A was detected with a fluores­
cence detector at an excitation wavelength of 280 nm and emission of
340 nm.
The samples subjected to heavy metal analysis were acidified to 1 %
with Suprapur HNO3 (Merck, Germany). The heavy metal concentra­
tions were determined using an ICP MS iCAP RQ (Thermo Fisher Sci­
entific, Germany) instrument fitted with an ESI 4DX autosampler
(Thermo Fisher Scientific, Germany), a Teflon micro-nebulizer, and a
cooled (+4 ◦ C) quartz cyclonic spray chamber for sample introduction.
A quartz micro injector tube was used in combination with Pt cones.
Details of the ICP-MS operating conditions are presented in the Ap­
pendix (Table A1). The instrument was regularly optimized using a
multi-element standard solution (10 ng mL− 1). The calibration curves
for heavy metals were generated using 1000 mg L− 1 of mono-elemental
I. Kozyatnyk and M. Njenga
standard solutions (Inorganic Ventures, USA) diluted to appropriate
concentration levels in 1 % Suprapur HNO3.
Total MO protein content in water samples was determined using the
Bradford method. The Bradford protein assay was performed using a
Bradford assay solution (TCI Europe, Belgium). A standard curve was
prepared using serial dilutions of the in-house prepared MO seed protein
solution (see Appendix) spanning the concentration range of 5 to 50 mg
L− 1. Water samples were filtered through a 0.45 μm regenerated cellu­
lose membrane filter (VWR, China) to remove any suspended particles.
For the assay, 15 μL of the protein standard or water sample was added
to the wells of a 96-well microplate, followed by 250 μL of the Bradford
assay solution. The plate was incubated at room temperature for 5 min,
after which absorbance was measured at a wavelength of 590 nm using
the ClariStar Plus multimode microplate reader (BMG Labtech,
Germany).
All of the collected samples were sent to a commercial laboratory for
DOC analysis and a comprehensive description of initial tap water
characteristics (ALS, Sweden). DOC was analyzed according to a method
based on CSN EN 1484, CSN EN 16192, and SM 5310. Prior to analysis,
all of the samples were acidified by adding 1 % (v/v) of 2 M HCl; this was
followed by filtration through a 0.45 μm regenerated cellulose mem­
brane filter (VWR, Belgium). The detection limit was 2 mg L− 1 and the
determinations had an average relative standard deviation of 4.5 %.
Initial tap water characteristics are presented in the Appendix
(Table A2).
pH and total dissolved solids (TDS) were measured using a HQ2100
meter (HACH, USA). The IntelliCAL PHC101 pH electrode (HACH, USA)
was calibrated using pH 4.00, 7.00, and 10.00 buffer solutions. The
CDC401 conductivity cell (HACH, USA), which was calibrated with a
conductivity standard solution (1413 μS cm-1), was used for TDS mea­
surements. The pH or TDS of each water sample was measured by
immersing the corresponding electrode in the sample and recording the
stabilized reading. The TDS value was automatically calculated and
displayed on the meter based on the measured conductivity and tem­
perature of the sample.
A DR1900 benchtop spectrophotometer (HACH, China) was used to
analyze the colour and turbidity of water samples. The spectropho­
tometer was calibrated using Pt-Co colour standards (0–500 units) ac­
cording to the manufacturer’s guidelines. Water samples were filtered
through a 0.45 μm regenerated cellulose membrane filter (VWR, China)
to remove suspended particles and then introduced into a cuvette with a
2.5 cm path length. The absorbance of each sample was measured at a
wavelength of 455 nm, and the colour was calculated and reported in Pt-
Co units.
The spectrophotometer was calibrated using turbidity standards (20,
100, and 800 nephelometric turbidity units (NTUs)) according to the
manufacturer’s instructions. In the analyses, water samples were gently
mixed, and an aliquot was transferred to a clean cuvette. The cuvette
was then placed into the spectrophotometer, and the turbidity value was
recorded at a wavelength of 600 nm.
3. Results and discussion
3.1. Morphological characterisation of the surface of wood biochar
produced in a gasifier stove
3.1.1. Biochar porosity
The nitrogen adsorption-desorption isotherm presented in Fig. 1a
provides valuable information about the porosity and surface area of the
investigated wood biochar. The presented nitrogen adsorption-
desorption isotherm (Fig. 1a) is a reversible-type isotherm (a combina­
tion of types Ib and II) that is concave with respect to the p/p0 axis at
lower relative pressures, with this trend reversing at relative pressures
close to 1. This is typical for the physisorption of nitrogen at 77 K on
microporous solids, which have relatively small external surfaces with
various pore size distributions, i.e., ranging from wider micropores to
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narrow mesopores (≤2.5 nm). The obtained hysteresis loop appeared to
be type H4, which is often the case for micro-mesoporous carbons
(Thommes et al., 2015). These two isotherms, when considered
together, suggest that the wood biochar has a broad range of pore sizes,
including micropores and possibly narrow mesopores.
The nitrogen adsorption-desorption isotherm of biochar (shown in
Fig. 1a) is an important tool for characterizing the porosity and surface
area of a material. However, the nitrogen adsorption-desorption iso­
therms of some biochars include an open hysteresis feature, which is
incompatible with standard IUPAC classifications or calculation models.
This can be caused by non-equilibrium conditions, such as slow
adsorption or desorption rates, or by pore deformation as a result of low-
pressure nitrogen adsorption. Low-pressure nitrogen adsorption can
cause pore deformation in biochar if the adsorbent structure is non-rigid.
During gas adsorption, pores of varying width are exposed to extreme
stress. This local pressure results in pore deformation, i.e. as a result of
swelling or contraction, if the stress exceeds the rigidity of the carbon
matrix (Maziarka et al., 2021).
The porosity of biochar has a significant impact on adsorption po­
tential since a large amount of pores will increase the surface area of the
biochar. The wood biochar tested in the present study exhibits a broad
range of pore sizes, including micropores and possibly narrow meso­
pores. The surface area of the tested biochar (91.5 m2 g− 1) of the biochar
is not exceptionally large, and rather typical (<150 m2 g− 1) for direct
carbonized wood biochars (Liu et al., 2015); this result is likely due to
excess air in the gasified stove (Sun et al., 2022).
3.1.2. Biochar surface functionality
XPS and FTIR analyses were employed to examine the surface
characteristics of the tested biochar. The XPS spectra revealed that the
surface of the biochar is predominantly comprised of carbon (88.9 at.%)
and oxygen (7.9 at.%). Several inorganic elements, namely, K (1.9 at.%),
F (0.6 at.%), Ca (0.4 at.%), P (0.2 at.%), and S (0.1 at.%), represent the
ash part of the biochar. XPS data from biochar samples are often used to
estimate the surface oxygen:carbon ratio, which serves as an indicator of
the level of environmental oxidation that has occurred in the biochar
sample. The results of curve-fitting to the C 1s and O 1s peaks are
illustrated in Fig. 1c. Analyses of the biochar surface also revealed broad
peaks in the 291 eV region, which can be attributed to the π–π* shift
arising from excitation of π orbitals during the photoemission process.
The main peaks, present at ~285 eV, appear symmetrical; although the
π–π* shift was observed, it does not indicate an extended graphite-like π
orbital system in this case. The C 1s spectral window also includes the K
2p peaks, which – as mentioned above – represent the ash part of the
biochar. For instance, a 2p3/2–2p1/2 doublet with an intensity ratio of
~2:1 can be observed, which is expected based on the reference data for
K. The O 1s peaks in Fig. 1d were fitted using two components, one at
~532 eV (O = C bonds) and the other at ~533.2 eV (O–C bonds). The
lower intensity of the O–C component relative to the O– – C component
is consistent with the relative proportions of these components in the C
1s spectra (Singh et al., 2017).
These findings correlate well with the FTIR assignment of the studied
biochar (Fig. 1b) The presence of C–C/C– – C/C–H bonds correlates
well with the FTIR spectrum, which displays a 1600 cm− 1 band that
represents the formation of aromatic C– – C bonds during biomass
carbonization (Kruse and Zevaco, 2018). The FTIR spectrum obtained
for the studied wood biochar displays well-resolved ν(C– – C) bands at
1590–1600 cm− 1 and out-of-plane C–H deformation bands in the
950–700 cm− 1 region. Biochars prepared at high temperatures, around
700 ◦ C, typically exhibit fewer carboxylic groups and a shift of the ar­
omatic ν(C– – C) band to a lower energy level. Additionally, biochars
produced at high temperatures exhibit fewer carbonate features.
The spectral region between 950 and 700 cm− 1 encompasses con­
tributions from aromatic out-of-plane C–H deformation bands
(δ(C–H)oop), as reported by (Whitman et al., 2013). However, the
spectral features in this region are complex due to potential interference
I. Kozyatnyk and M. Njenga
Fig. 2. Removal efficiency of heavy metals and organic compounds under three
experimental scenarios: 1) Moringa oleifera seed extract-based flocculant fol­
lowed by biochar; 2) Biochar followed by Moringa oleifera seed extract-based
flocculant; and 3) Simultaneous application of both.
from inorganic phases. The spectrum obtained for the biochar samples
exhibits significant peaks in the 1120–1050 cm− 1 range, which repre­
sent C–O stretching. In the case of aromatic rings, C–
– C bond stretching
occurs in alkene within the 842–720 cm− 1 range, with peak intensity
increasing with the temperature used to produce biochar (Antonangelo
et al., 2019; Reza et al., 2020). The sample appears to be well‑carbon­
ized, which suggests that the carbonization temperature exceeded
700 ◦ C (Singh et al., 2017). The XPS and FTIR analyses of biochar sur­
face characteristics revealed that the sample was predominantly
comprised of carbon and oxygen, with the presence of certain inorganic
elements. These characteristics have significant implications for the
adsorption of organic contaminants and heavy metals during greywater
treatment, which was investigated in more detail in subsequent
experiments.
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Bioresource Technology Reports 24 (2023) 101615
3.2. Removal of organic compounds and heavy metals by a flocculant
based on Moringa oleifera seed extract and biochar
3.2.1. Order of application of the flocculant based on Moringa oleifera seed
extract and biochar
One of the questions the study sought to answer was whether the
order of application of the flocculant based on MO seed extract and
biochar would noticeably impact the removal of both heavy metals and
organic compounds from water. There were three experimental sce­
narios of applications: 1) applying the MO seed extract-based flocculant
first, followed by biochar; 2) applying biochar first, followed by the MO
seed extract-based flocculant; and 3) applying both simultaneously. The
results revealed (Fig. 2) that the order of application did not significantly
affect the removal of heavy metals. However, in the case of organic
compounds, it was better to apply the biochar first, followed by the
flocculant. For instance, the removal of trimethoprim increased from 12
% (flocculant first, then biochar) to 31 % when the biochar was applied
first, and then followed by the MO seed extract-based flocculant.
This can be explained by the fact that adding biochar to the waste­
water first enables effective adsorption of contaminants to the biochar
surface. In other words, an initial adsorption step serves to reduce the
concentration of organic compounds in the water, making the subse­
quent flocculation process more efficient (Huang et al., 2020). Addi­
tionally, the presence of biochar in the water can promote the formation
of flocs by providing a suitable substrate for flocculant attachment; the
presence of biochar also increases the overall particle size distribution in
the water, which can enhance the settling rate of flocs.
However, the results also revealed that the combination of biochar
and MO seed extract-based flocculant (in any order) was not effective in
removing arsenic and chromium ions from water. A possible explanation
for this result is that arsenic and chromium ions are present in water as
negatively-charged anions, while both biochar and the MO-based floc­
culant include a negatively-charged, oxygen-containing group. This
similarity in the surface charge of the flocculant and the contaminants
can lead to repulsion, which hinders the ability of the flocculant to
remove these anions from water.
Fig. 3. Dosages of the Moringa oleifera seed extract-
based flocculant and biochar for removing organic
compounds and heavy metals from greywater. The
tested biochar dosages were 100, 200, 500 and 1000
mg L− 1, whereas the tested Moringa oleifera seed
extract-based flocculant were 1.3, 2.5, 5.0, and 7.5
mg L− 1. When biochar and the Moringa oleifera-based
flocculant were tested simultaneously, a constant
flocculant dosage of 5.0 mg L− 1 was used, while
biochar dosages of 100, 200, 500 and 1000 mg L− 1
were tested.
I. Kozyatnyk and M. Njenga
Fig. 4. Influence of kaolin, surfactant and humic compounds on the removal of organic compounds and heavy metals by a Moringa oleifera based flocculant and
biochar. M – Moringa oleifera seed extract (5.0 mg L− 1), B – biochar (1000 mg L− 1), K – kaolin, S – SDS surfactant, H – humic acids, KSH – mixture of kaolin, SDS and
humic acids.
3.2.2. Dosage of the Moringa oleifera seed extract-based flocculant and
biochar for the removal of organic compounds and heavy metals from
greywater
The next experiment in this study involved determining the dosages
of biochar and MO seed extract-based flocculant for the effective
removal of heavy metals and organic pollutants from spiked tap water
(Fig. 3). The results clearly showed that the removal efficiencies of both
heavy metals and organic pollutants improve as the biochar dosage in­
creases. The removal of heavy metals, such as Co2+, Ni2+, Cu2+, and
Zn2+, increased from 5 to 48 % at a biochar dosage of 100 mg L− 1 to
60–90 % at a dosage of 1000 mg L− 1. Similarly, the removal of organic
compounds increased from 3 to 30 % at a biochar dosage of 100 mg L− 1
to 42–99 % at a dosage of 1000 mg L− 1. This result can be attributed to
the surface area and porosity of biochar, i.e., as the amount of biochar in
solution increases, so does the available surface area for adsorption,
which will increase the removal of contaminants (Tan et al., 2017).
In contrast to the clear observed trend between biochar dosage and
removal efficiency, the relationship between MO-based flocculant
dosage and pollutant removal efficiency was not as straightforward.
According to Fig. 3, the highest dosage (7.5 mg L− 1) of the MO-based
extract does not necessarily result in the most efficient removal of
organic pollutants and heavy metals. In fact, it appears that comparable
removal efficiencies can be achieved with lower dosages of the floccu­
lant. It is seen in Fig. 3, a 5.0 mg L− 1 dosage of the MO-based flocculant
had similar, or possibly even higher, removal efficiency for Cu, Zn, Pb,
trimethoprim, diclofenac, and bisphenol as the 7.5 mg L− 1 dosage. This
result can be attributed to several factors, including an optimal balance
between the positively-charged polyelectrolytes from the MO extract
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Bioresource Technology Reports 24 (2023) 101615
and the negatively-charged pollutants, as well as the potential for adding
excessive amounts of flocculant (Blanco et al., 2005; Yukselen and
Gregory, 2004), which may lead to the formation of smaller, and more
stable, flocs that take long to settle and remove.
The combination of biochar and the MO-based flocculant increased
the removal efficiency of certain heavy metals, such as zinc and lead,
relative to the sole use of either compound. The increase in the removal
efficiency of zinc and lead can be attributed to the complementary na­
ture of the two tested water treatment agents. Biochar can adsorb a
significant amount of the metal ions present in greywater, while the MO-
based extract can promote the formation of larger flocs that encapsulate
the remaining ions. Consequently, the combined use of these two agents
enables more effective heavy metal removal than using either alone. It is
interesting to note that the removal of Co, Ni, and Cu was basically the
same when biochar was used alone or in combination with the MO-based
flocculant. As discussed above, the MO seed extract-based flocculant
showed insignificant removal of anionic metals, such as chromium and
arsenic; this could be explained by the fact that arsenic and chromium
anions often exist in water as soluble species, e.g., arsenate (AsO₄3− ) and
chromate (CrO₄2− ) ions, respectively. The high solubility of these an­
ions, as well as potential interactions with other anionic species, e.g.,
CECs, can hinder the ability of flocculants to effectively capture and
remove these contaminants through the coagulation-flocculation pro­
cess. In contrast, insoluble or poorly soluble metal species are more
amenable to flocculation.
The effectiveness of biochar and the MO-based flocculant in
removing organic pollutants, either used alone or in tandem, is
described in the following section. The combination of biochar and the
I. Kozyatnyk and M. Njenga
Fig. 5. Influences of kaolin, surfactant, and humic compounds on the removal of organic compounds and heavy metals by a Moringa oleifera seed extract-based
flocculant and biochar. M – Moringa oleifera seed extract-based flocculant (5.0 or 25 mg L− 1), B – biochar (1000 mg L− 1), H – humic acids, KSH – mixture of
kaolin, SDS, and humic acids.
MO-based flocculant was found to be less efficient in eliminating organic
pollutants when compared to the sole use of biochar. This was particu­
larly noticeable in the case of carbamazepine and bisphenol, with the
removal rates at a biochar dosage of 1000 mg L− 1 decreasing by 15 %
and 40 % when the MO-based flocculant was also added to solution. This
could represent a situation in which organic pollutants and proteins in
the MO extract are competing for active sites on the surface of the bio­
char. As there are a finite amount of active sites on the surface of the
biochar, this competition could cause most of the active sites to become
occupied in a short amount of time, which would decrease the overall
removal efficiency. However, it is worth noting that the addition of MO-
based flocculant can help to effectively remove powdered biochar from
the water. Biochar particles tend to be negatively-charged, which causes
the particles to repel each other and result in poor settling (Tan et al.,
2020). The addition of positively-charged MO-based flocculant can
neutralize the negative charges on the surface of biochar particles,
which will lead to the formation of large particles (flocs) that can readily
settle. This process also aids in the removal of residual MO-associated
protein from the water, which will further enhance the overall water
treatment efficiency.
3.2.3. Influence of kaolin, surfactant and humic compounds on the removal
of organic compounds and heavy metals by the Moringa-based flocculant
and biochar
A major goal of the presented research was to determine how
effective the combination of biochar and an MO-based flocculant is at
removing certain contaminants from household greywater. Therefore,
we simulated greywater by including critical components such as humic
acids, clay (kaolin) and surfactants (sodium dodecyl sulphate). Then, we
tested how effective biochar (1000 mg L− 1) and an MO-based flocculant
(5.0 mg L− 1), both alone and in combination, are at removing contam­
inants from the simulated greywater (see Fig. 4).
The results demonstrated that the presence of humic acids, clay, and
surfactants in greywater can negatively impact the effectiveness of the
tested treatment approach. For instance, the MO-based agent did not
flocculate when humic acids were present in the water. More specif­
ically, the presence of humic acids inhibited the removal of colour and
7
Bioresource Technology Reports 24 (2023) 101615
heavy metals via flocculation of the MO seed extract-based agent. The
mechanism underlying this inhibition is likely the complexation of
heavy metals with humates (Kozyatnyk et al., 2015). Humates are large,
negatively-charged molecules that can form stable complexes with
heavy metals. These complexes show a noticeable degree of solubility,
which means that they do not readily precipitate out of solution; thus, it
can be challenging to remove heavy metals from greywater.
We observed that the presence of kaolinite in the greywater matrix
negatively impacted the removal of organic pollutants via both
adsorption to biochar and MO flocculation. This finding can be attrib­
uted to the high adsorption capacity of clay minerals, which may
compete with biochar in the adsorption of organic contaminants.
Furthermore, fine kaolinite particles can increase the turbidity of the
greywater, which can hinder the flocculation process and reduce overall
treatment efficiency.
The presence of SDS, a surfactant, in the artificial greywater also
impeded the flocculation process. More specifically, the presence of SDS
decreased the removal efficiencies of both heavy metals and organic
pollutants by 10–50 % when biochar and the MO seed extract-based
flocculant were used simultaneously. Previous research has shown
that the surface properties of surfactants can interfere with the adsorp­
tion of contaminants onto biochar, e.g., by blocking active sites (Que
et al., 2018), as well as hinder coagulation and flocculation processes.
Moreover, Shah et al. (2016) suggested that surfactant molecules can
form micelles that encapsulate contaminants which ultimately prevents
interactions with materials that have been added to wastewater for the
purpose of removing contaminants.
Previous studies (Arnoldsson et al., 2008; Desta and Bote, 2021;
Taiwo et al., 2020) have reported that the optimal dosage of MO-asso­
ciated proteins for wastewater treatment is between 0.05 and 0.5 mg
L− 1. However, the results presented in Fig. 4 suggest that this dosage of
biochar and the MO seed extract-based flocculant may not be sufficient
for initiating flocculation in greywater that contains humates, clay, and
surfactants (Oteng-Peprah et al., 2018). Therefore, we tested if a far
higher dosage than 5 mg L− 1 of the MO seed extract-based flocculant
could improve flocculation in a solutions containing humates or mixture
of all artificial greywater components (humates, clay, and SDS) where
I. Kozyatnyk and M. Njenga
Fig. 6. Influence of kaolin, surfactant, and humic compounds on DOC, colour, pH, protein content, turbidity, and TDS after treatment with Moringa oleifera seed
extract-based flocculant (5.0 or 25 mg L− 1) and biochar (1000 mg L− 1).
we did not observe flocculation. We then tested an MO seed extract-
based flocculant dose of 25 mg L− 1 to determine whether such a high
dose could improve the removal efficiency of heavy metals and organic
pollutants from water (Fig. 5).
The higher dose of MO seed extract-based flocculant exhibited the
most substantial enhancement in heavy metal and organic pollutant
removal (Fig. 5). The results demonstrated increased removal efficiency
for both heavy metals and organic pollutants, with a marked improve­
ment in the removal of chromium and arsenic. The increase in chromium
and arsenic removal, from no removal to 50 % efficiency, is particularly
noteworthy as these contaminants are challenging to eliminate using
conventional flocculants. In the case of copper, zinc, and lead, nearly
100 % removal was achieved by using biochar alone (Figs. 3 and 4).
The addition of the MO-based flocculant demonstrated similar levels
of heavy metal removal with reduced amounts of biochar. The impact of
simultaneous MO-based flocculant and biochar treatment on organic
substance removal was less straightforward. In the case of trimethoprim
and diclofenac, the addition of the MO-based flocculant marginally
increased the water purification performance, i.e., by 10–15 %. In
contrast, the addition of the MO-based flocculant decreased the removal
of bisphenol and carbamazepine by 10 %, or resulted in no removal at
all. As demonstrated earlier, biochar can play an important role in the
removal of organic compounds from greywater. Thus, the dosage of
additional flocculants must be carefully considered, as a five-fold in­
crease in the dose of the MO-based flocculant could have led to in­
teractions with the biochar active adsorption centres, ultimately
8
Bioresource Technology Reports 24 (2023) 101615
resulted in the saturation of these adsorption sites and limiting the ca­
pacity of biochar to remove organic compounds from solution.
The results presented in Fig. 6 demonstrate that a high dosage of MO
seed extract-based flocculant significantly improved water quality pa­
rameters. As a result of treatment, both colour and turbidity noticeably
decreased, reaching 72 PCU and 56 NTU, respectively. It is worth
mentioning that we did not observe a change in pH in our experiments,
which was at a slightly alkaline level, around 8, throughout the exper­
iments. This alkalinity was likely caused by the presence of residual
surfactants in the water and the ash part of biochar. The observed in­
crease in TDS was most probably due to additional NaCl, which is used
to extract the active substances of MO and remains in the water after
flocculation. Residual MO-associated proteins may contribute to the
heightened DOC levels in water treated with both biochar and MO seed
extract-based flocculant. However, our observations do not indicate a
direct correlation between DOC and residual protein concentrations. A
higher dose of flocculant enables the flocculation threshold to be ach­
ieved, which results in a more complete flocculation process. More
efficient flocculation will increase protein precipitation, and conse­
quently reduce protein concentrations in the treated water. The DOC
levels measured in the water treated using a combination of biochar and
MO seed extract-based flocculant suggest that the dosage of the MO-
based flocculant may need to be adjusted based on the type and level of
organic matter in the greywater.
I. Kozyatnyk and M. Njenga
4. Conclusions
This study explored the potential of combining biochar and an MO-
based flocculant for treating household greywater. These materials were
selected as they represent a low-cost, sustainable solution for water
treatment. The order of application impacted the removal of organic
compounds; it was more effective to apply the flocculant before the
biochar. The combination of biochar and the MO-based flocculant
improved the removal efficiency of heavy metals but was less effective
for organic pollutant removal due to competition for active sites. The
study revealed that while humic acids, clay, and surfactants in house­
hold greywater challenged the flocculation process using biochar and
MO extract, inhibiting pollutant removal, increasing MO seed protein
dosages enhanced water quality and contaminant removal. The results
suggest that the dosage of MO-based flocculant needs to be adjusted
depending on the organic matter characteristics of greywater.
To overcome the identified bottlenecks, integrating the biochar-MO
approach with other treatment methods might be the key. For instance,
subsequent advanced oxidation processes or microbial degradation can
be investigated for enhanced organic pollutant removal. Extending the
research to pilot-scale studies can provide insights into the practical
challenges and benefits of this approach, setting the stage for potential
full-scale applications.
CRediT authorship contribution statement
Ivan Kozyatnyk - Conceptualization; Data curation; Formal anal­
ysis; Funding acquisition; Investigation; Methodology; Project admin­
istration; Visualization; Writing - original draft; Writing - review &
editing
Mary Njenga - Conceptualization; Data curation; Resources; Writing
- original draft; Writing - review & editing
All authors read and approved the manuscript.
Declaration of competing interest
The authors declare the following financial interests/personal re­
lationships which may be considered as potential competing interests:
Ivan Kozyatnyk reports financial support was provided by Swedish
Research Council Formas.
Data availability
Data will be made available on request.
Acknowledgement
This work was supported by the Swedish Research Council for Sus­
tainable Development, Formas [grant 2019-00458]. The authors thank
Prof. Kajsa Uvdal (FTIR) and Prof. Emma Björk (BET) at Department of
Physics, Chemistry and Biology, Linköping University; as well as Dr.
Andrey Shchukarev (XPS) at Chemical Biological Centre, Umeå Uni­
versity, for their valued help with instrumental analyses of our samples.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.biteb.2023.101615.
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