Use of biochar and Moringa oleifera in greywater treatment to remove heavy metals and contaminants of emerging concern

This study investigated the combination of biochar derived from carbonized local feedstock and Moringa oleifera seed extract-based flocculant, a novel combination in the context of greywater treatment missed in the previous studies, with a focus on the removal of organic contaminants (caffeine, chloramphenicol, trimethoprim, carba-mazepine, diclofenac, and bisphenol A) and heavy metals (Cr(VI), As(III), Co(II), Ni(II), Cu(II), Zn(II), and Pb (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.


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 activated 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 potential, such as large specific surface area, high porosity, and reactive surface chemistry (Tan et al., 2017).For this reason, ACs are effective for 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 materials, 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 dissolved 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 proteins while minimizing the introduction of extraneous organic compounds.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 pathogens (Bancessi et al., 2020).
The primary goal of this work was to investigate the combined potential 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 on optimizing the dosage and application parameters, as well as elucidating which mechanisms are responsible for the removal of contaminants by these materials.Furthermore, the research assessed the potential advantages and limitations of this combined treatment approach.

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 ingredient 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 centrifugation at 4000 rpm for 20 min using a Rotina 380R centrifuge (Hettich, Germany).The concentration of MO proteins in the extract was 2.5 ± 0.5 mg mL − 1 .

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 greywater 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 (K D ), 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 evaluate 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.
2. Selection of the appropriate dosages for biochar and MO seed extract.

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.

Determination of biochar structure and surface functionalities
The surface chemistry of the utilized biochar was analyzed using Xray Photoelectron Spectroscopy (XPS) and Diffuse Reflectance Infrared Fourier Transform Spectroscopy (DRIFTS).The XPS spectra were acquired 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 tplot method.The pore volumes were determined using the Barrett-Joyner-Halenda (BJH) method.

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 fluorescence 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 HNO 3 (Merck, Germany).The heavy metal concentrations were determined using an ICP MS iCAP RQ (Thermo Fisher Scientific, 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 Appendix (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 standard solutions (Inorganic Ventures, USA) diluted to appropriate concentration levels in 1 % Suprapur HNO 3 .
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 cellulose 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 membrane 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 measurements.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 temperature of the sample.
A DR1900 benchtop spectrophotometer (HACH, China) was used to analyze the colour and turbidity of water samples.The spectrophotometer was calibrated using Pt-Co colour standards (0-500 units) according 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.

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 adsorptiondesorption isotherm (Fig. 1a) is a reversible-type isotherm (a combination of types Ib and II) that is concave with respect to the p/p 0 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 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 isotherms 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 lowpressure 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 potential 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 mesopores.The surface area of the tested biochar (91.5 m 2 g − 1 ) of the biochar is not exceptionally large, and rather typical (<150 m 2 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).

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, whichas mentioned aboverepresent the ash part of the biochar.For instance, a 2p 3/2 -2p 1/2 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 aromatic 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 contributions 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 from inorganic phases.The spectrum obtained for the biochar samples exhibits significant peaks in the 1120-1050 cm − 1 range, which represent 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-carbonized, which suggests that the carbonization temperature exceeded 700 • C (Singh et al., 2017).The XPS and FTIR analyses of biochar surface 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.

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 scenarios 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 wastewater 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 subsequent flocculation process more efficient (Huang et al., 2020).Additionally, 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 flocculant 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 extractbased 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.

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 increases.The removal of heavy metals, such as Co 2+ , Ni 2+ , Cu 2+ , and Zn 2+ , 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 flocculant.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 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 nature of the two tested water treatment agents.Biochar can adsorb a significant amount of the metal ions present in greywater, while the MObased 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 (AsO₄ 3− ) and chromate (CrO₄ 2− ) ions, respectively.The high solubility of these anions, 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 process.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 MO-based flocculant was found to be less efficient in eliminating organic pollutants when compared to the sole use of biochar.This was particularly 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 biochar.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 MObased 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.

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 contaminants 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 specifically, the presence of humic acids inhibited the removal of colour and 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 attributed 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 adsorption 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-associated 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 we did not observe flocculation.We then tested an MO seed extractbased 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 improvement 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 increase in the dose of the MO-based flocculant could have led to interactions with the biochar active adsorption centres, ultimately resulted in the saturation of these adsorption sites and limiting the capacity 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 parameters.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 experiments.This alkalinity was likely caused by the presence of residual surfactants in the water and the ash part of biochar.The observed increase 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 achieved, which results in a more complete flocculation process.More efficient flocculation will increase protein precipitation, and consequently 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 MObased flocculant may need to be adjusted based on the type and level of organic matter in the greywater.

Conclusions
This study explored the potential of combining biochar and an MObased 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 household 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.

Fig. 1 .
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.