Characterization of a novel polyfunctional “metalloglyco-protein/polypeptide-organochlorine” bioflocculant containing saturated nitro compounds, sulfones, polysulfides, phosphorus chlorine compounds, magnesium oxide, and metal chlorides, produced from Pseudomonas aeruginosa strain F29, isolated from porcine faeces in Nigeria

Characterization of a novel polyfunctional “metalloglyco-protein/polypeptide-organochlorine” bioflocculant containing saturated nitro compounds, sulfones, polysulfides, phosphorus chlorine compounds, magnesium oxide, and metal chlorides, produced from Pseudomonas aeruginosa strain F29, isolated from porcine faeces in Nigeria | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Characterization of a novel polyfunctional “metalloglyco-protein/polypeptide-organochlorine” bioflocculant containing saturated nitro compounds, sulfones, polysulfides, phosphorus chlorine compounds, magnesium oxide, and metal chlorides, produced from Pseudomonas aeruginosa strain F29, isolated from porcine faeces in Nigeria Ikechukwu K. M. Okorie, Adeniyi A. Ogunjobi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7453669/v2 This work is licensed under a CC BY 4.0 License Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Abstract Antibiotic resistance has reached universal proportions, and the discovery of effective alternatives to the common antibiotics currently used, could aid in solving this problem. The aim of this study was to characterise a bioflocculant produced from Pseudomonas aeruginosa strain F29, accession number OQ734844, that exhibited effective antibacterial activity against two antibiotic resistant bacteria, viz, Staphylococcus aureus SO183, and an identified strain of Pseudomonas aeruginosa , in another study. FTIR detected saturated nitro compounds, sulfones, polysulfides, phosphorus-chlorine bonds, magnesium oxide bonds and metal-chloride bonds. FTIR also detected the following functional groups: carboxyl, amide/peptide, aromatic alcohol, alkene, and halo. SEM showed a clumped and flaky bioflocculant surface, while EDX detected chlorine (56.00%), carbon (20.50%), sodium (12.50%), oxygen (4.00%), phosphorus (3.00%), sulfur (2.43%) magnesium (1.06%), potassium (0.32%) and nitrogen (0.30%). HPLC and MS detected varied peaks of glucose, galactose, inositol and mannose, D-ribose, arabinose, rhamnose and xylose. The phenol sulfuric acid method calculated the concentration of these sugars as 0.0059 g/L. The bioflocculant is a polymeric compound composed mainly of carbohydrates, proteins/polypeptides and organochlorines, possibly a novel “metalloglyco-protein/polypeptide organochlorine” bioflocculant. The presence of the metals: sodium, potassium and magnesium; the non-metals: phosphorus, sulfur and nitrogen; and multiple moieties, likely contributed to the antibacterial activity of the bioflocculant produced from Pseudomonas aeruginosa strain F29. From available documentation, this is the first report of a polyfunctional “metalloglyco-protein/polypeptide organochlorine” bioflocculant, that naturally contains saturated nitro compounds, sulfones, polysulfides, phosphorus chlorine compounds, magnesium oxide and metal chlorides; and of a bioflocculant produced from porcine faeces in Africa. Biopolymers Chemical Biology Biological Chemistry Drug Discovery, Design, & Development characterisation novel “metalloglyco-protein/polypeptide organochlorine” bioflocculant polyfunctional Pseudomonas aeruginosa strain F29 antibiotic resistance Figures Figure 1 Figure 2 Figure 3 Figure 4 INTRODUCTION Antibiotic resistance has reached universal proportions, and the discovery of effective alternatives to the common antibiotics currently used, could aid in solving this problem. Antibiotic resistance occurs when bacteria are no longer susceptible to the usual drugs designed to kill them (bactericidal antibiotics) or to retard their growth (bacteriostatic antibiotics). Antibiotic resistance is the main subdivision of the larger group of antimicrobial resistance (AMR), and is defined as the development, by bacteria, of resistance to specific drugs invented to kill them, or to repress their growth (WHO, 2021). Alternative antibacterial agents are therefore being sought. It has been reported that bioflocculants possess bioactivities against bacteria and certain other groups of microorganisms (Abu Tawila et al ., 2018). Bioflocculants are biological flocculants and are therefore, more generally defined as flocculants of organic origins and not solely microbial (Yang et al ., 2024). Bioflocculants, can be obtained from microorganisms, animals and vegetation (Kurniawan et al ., 2022). With respect to microorganisms, bioflocculants are extracellular polymeric substances that are produced as secondary metabolites and cause flocculation in a given medium (Dih et al ., 2019). These microbial bioflocculants are a result of cytolysis and the release of cellular products by different groups of microbes (Alias et al ., 2022). Bioflocculants aggregate particles and then remove these particles, from the liquid medium in which they were formerly suspended. On the whole, flocculants have the capacity to clump small-sized substances suspended in a medium; these clumped substances are termed flocs (Wang et al ., 2022), and sediment over time. The specific mechanism of action, by which bioflocculants operate, is still unknown. A bioflocculant may trigger the construction of a bridge between particles of the medium and its own molecules; or may kick-start a neutralisation of charges present in the medium (Lai et al ., 2018). Other hypotheses exist, nevertheless, the precise mechanism of bioflocculant antibacterial bioactivity, may be deduced from the inherent capacity of bioflocculants to form flocs, that later sediment. A bioflocculant may therefore, initiate the clumping together of the cell wall components of the bacterium being acted upon, and this could more likely, produce tears in the bacterial cell membrane, with varying degrees of fragmentation. The compromise in bacterial cell membrane integrity that ensues, if progressive and widespread enough, could trigger an influx of noxious substances from the surroundings, that could lead to a destabilisation of the internal milieu of the bacterial cell, and result in bacterial cell lysis and death. Bioflocculants are made up of natural compounds such as cellulose, nucleic acids, glycoproteins and polysaccharides, that render them readily decomposable and less injurious to the environment, therefore, secondary environmental contamination is obviated (Alias et al ., 2022). Microbial bioflocculants are polymeric substances produced during the microbial growth phase (Tsilo et al ., 2022). Plant-derived bioflocculants include mucilage (Das et al ., 2021), alginate, starch (Salehizadeh et al ., 2018) and cellulose ((Fauzani et al ., 2021). There are, however, different kinds of bacterial species, that also produce cellulose (here known as bacterial or microbial cellulose), that provides mechanical support (Choi et al ., 2022), and possesses similar bioflocculant characteristics as plant cellulose. Animal-derived bioflocculants include gelatin and chitosan (Badawi et al ., 2023). Bioflocculants composed mainly of proteins, are easily affected by thermal fluctuations because proteins naturally lose their spatial conformations when exposed to external forces such as thermal energy (Bukhari et al ., 2020). The nucleic acids (deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)), present in bioflocculants, constitute part of the larger pool of environmental nucleic acids; indeed, their presence in the environment is being utilised, now more than previously, in “molecular biomonitoring” (Littlefair et al ., 2022). Glycoprotein bioflocculants are characterised by the presence of oxygen, nitrogen and carbon in their framework; these elements are thought to boost their inherent bioflocculating capacities (Tsilo et al ., 2022). Bioflocculants largely composed of polysaccharides, retain their activities at high temperatures (Tsilo et al ., 2021). It has been documented that the flocculating capacity of any flocculant is directly proportional to the stretch of monomeric subunits it possesses; therefore, flocculants with higher molar masses are said to possess a commensurate degree of stretch and can even house unbound moieties, that can link flocs, to bring about better floc formation (Michaels, 1954; Gao et al . 2006). Apart from their use as antibiotics, bioflocculants can be effective against algae and viruses (Abu Tawila et al ., 2018). Bioflocculants that are primarily composed of polysaccharides, can substantially protect cells against free radical injury (Giri et al ., 2019). Bioflocculants can also be used as stabilisers, thickeners and emulgents, in the food industry and in drug manufacturing (Zhong et al ., 2018). Some exopolysaccharides can demonstrate flocculating properties such as the exopolysaccharide, EPS SM9913, produced from the blue-water, cryophilic bacterium, Pseudoalteromonas sp. SM9913 (Li et al ., 2008). Exopolysaccharides that double as bioflocculants can therefore be utilised as flocculants for wastewater management (Li et al ., 2008), in addition to other uses that stem from the properties they possess as exopolysaccharides. In a bioflocculant, the hydroxyl, carboxyl and amide functional moieties promote the flocculation process; they provide sites of attachment for the bivalent positively-charged ions that principally partake in flocculation (Tang et al ., 2014; Vimala, 2019). Certain microorganisms can assimilate metals into the bioflocculants they produce, and these metal components affect the intrinsic chemical composition and morphology, and the mechanisms of actions of the bioflocculant (Zoaka et al ., 2025). The aim of this study was to characterise a bioflocculant that was produced from Pseudomonas aeruginosa strain F29, accession number OQ734844, that exhibited effective antibacterial activity against two antibiotic resistant bacteria, viz, Staphylococcus aureus SO183, and an identified strain of Pseudomonas aeruginosa , in another study that was part of a Master’s project (Okorie, 2023). This characterisation was necessary in order to identify the physical nature and the chemical composition of the bioflocculant. The bacterial DNA sequence was deposited in the National Centre for Biotechnology Information (NCBI) GenBank database under its accession number. FTIR, SEM coupled to EDX, HPLC coupled to MS and the phenol‒sulfuric acid method, were employed as analytic tools. MATERIALS AND METHODS An aliquot of the bioflocculant was transferred into a sterile bottle and subjected to some analytical procedures to determine its elemental composition, functional groups and physical characteristics. The analytical tools used, were Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) coupled to energy dispersive X-ray spectroscopy (EDX), high-performance liquid chromatography (HPLC) coupled to mass spectrometry (MS) and the phenol‒sulfuric acid method. Fourier transform infrared (FTIR) spectroscopy The functional groups present in the bioflocculant were characterised using a Perkin Elmer Spectrum 100 Series 3000 MX spectrometer. A set of instructions for FTIR, from the NanoScience Technology Centre (NTSC) (2008), was used as a reference point. The bioflocculant sample was thinned between two potassium bromide (KBr) plates for FTIR analysis, and then was placed inside the sample holder of the FTIR device that was wiped clean with acetone. The addition of relevant computer commands, enabled the recording of the generated infrared spectra. Next, the data from these spectra was analysed using the spectroscopic software Win-IR Pro Version 3.0, and saved. The interpretation of the infrared (IR) spectra was performed using Infrared and Raman Characteristic Group Frequencies from Socrates, (2004); IR Spectra Table and Charts from Sigma Aldrich Company (Aldrich, 2019) and FTIR results analyses from Aksnes and Aksnes (1963). Bunaciu et al . (2014), Smith (2017), Nandiyanto et al . (2019), Ji et al . (2020) and Agyapong et al . (2023). Scanning electron microscopy (SEM) and energy dispersive X-ray analysis/spectroscopy (EDS/EDX) (dup: abstract ?) The surface topography of the bioflocculant was revealed using a JEOL JSM-7600F scanning electron microscope (SEM) (Tokyo, Japan), while its elemental composition was determined using an in-built energy dispersive X-ray (EDX) spectrometer. The protocol laid out by Core Facilities (Facilities, 2021) for preparing an SEM sample was used as a reference. Primary fixation of a measured volume of the bioflocculant sample was performed using formaldehyde and glutaraldehyde, while secondary fixation was performed with osmium tetroxide. This step was followed by dehydration with ethanol and sample drying. Mounting of the sample, and firm fixation of the sample, on a specimen stub, were performed next, before placement of the sample in the specimen slot, was performed. Sputter coating of the sample with platinum was performed before sectioning of the sample ensued. The bioflocculant surface was observed by SEM. Analysis of the chemical composition of the bioflocculants was performed via an in-built EDX. The supercharged electron beam from the SEM, hit the bioflocculant sample and caused the release of X-rays from the bioflocculant surface atoms (Thambiratnam et al ., 2020). The X-rays from this interaction, were pooled by the X-ray detector in the EDX, interpreted as elemental type and concentration results, and displayed as an EDX graph (Thambiratnam et al ., 2020). High-performance liquid chromatography (HPLC) and mass spectrometry (MS) (dup: abstract ?) The carbohydrate sugars present in the bioflocculant were analysed using an Agilent 1100 series HPLC system (Agilent Technologies, California, USA) equipped with a diode array detector (DAD) and a reversed-phase C-18 Zorbax Eclipse extradense bundle (XDB-C18) column (250 × 4.6 mm, 5 µm particle size, 300 Å pore size). An Agilent 1100 Series LC/MSD benchtop mass spectrometer connected to the HPLC device was used to determine the mass‒charge ratio of the carbohydrate sugars. A set of HPLC procedures outlined by Anumula (1994), Guzzetta (2001), and Sharma et al . (2020), were used. The Agilent 1100 Series High Value System User’s Guide from Agilent Technologies (1999) was also used. The HPLC solvent conduits were degassed by HPLC-grade isopropanol purging prior to the commencement of the analytical technique. Solvent A (HPLC-grade water combined with 0.1% v/v formic acid) and solvent B (HPLC-grade acetonitrile and 0.1% v/v trifluoroacetic acid), composed the HPLC reversed-phase solvent elution setup (Guzzetta, 2001). Multiple injections of the same samples were enabled through an injector protocol accessed in the HPLC device (Huber, 2010) to separate the monosaccharide moieties. The HPLC procedure involved sorting non-volatile compounds from the analyte to forestall matrix interference (Ho et al ., 2003). The product of the HPLC analysis was channelled into an Agilent 1100 Series LC/MSD (liquid chromatography/mass selective detector) benchtop mass spectrometer. The ionisation of the sample kick-started the analysis; a disaggregation of the sample ions ensued in the electromagnetic field created by the spectrometer on the basis of the differences in ionic mass/charge (m/z) ratios (Reusch, 2013). The disaggregated ions were identified and quantified as they made contact with both ultraviolet and mass selective detectors of the device; this information was combined into an array of graphical results (Reusch, 2013). Consequently, both qualitative and quantitative characterisation of the bioflocculant sample, could be performed via mass spectrometry (Ho et al ., 2003). A total ion chromatogram was the final graphical display of the results of both HPLC and MS. The phenol‒sulfuric acid method The phenol‒sulfuric acid method was used to measure the concentration of the total sugar content of the bioflocculant (expressed in milligrams/litre); this method employed glucose as the standard (Chaplin and Kennedy, 1986). The amount of carbohydrate was determined as described. A quantity of the bioflocculant, 0.1 mL, was mixed with 2 mL of distilled water. To this mixture, 0.1 mL of 6% phenol and 5 mL of 95% sulfuric acid (v/v) were quickly added. This composite mixture was shaken and left to stand for 10 minutes. The optical density was then recorded at 490 nm, as an average of three separate readings. Two millilitres of distilled water mixed with 0.1 mL of 6% phenol and 5 mL of 95% sulfuric acid (v/v) were used as the blank control, and the absorbance was measured. The amount of bioflocculant sugars was determined using a standard glucose curve that had to be prepared. This process was conducted according to the method outlined by DuBois et al . (1956) with some modifications. Glucose (0.1 g) was dissolved in 100 mL of distilled water to make a stock solution. From this stock solution, 10 mL was removed and added to 90 mL of distilled water to make 100 mL of diluted stock solution. The following volumes of the diluted stock solution were poured into five different sterile bottles: 0.1 mL, 0.2 mL, 0.4 mL, 0.8 mL and 1.0 mL. Next, a series of different dilutions of the glucose solution was achieved by making up each volume of stock solution to 3 mL, through the addition of the needed volume of distilled water. For each of these concentrations, 0.1 mL of 6% phenol and 5 mL of 95% (v/v) sulfuric acid were quickly mixed, and the composite mixtures were allowed to stand for 10 minutes. The optical density (OD) at 490 nm was subsequently measured. A standard glucose curve was then plotted with the optical densities of the different glucose dilutions at 490 nm on the y-axis and the corresponding glucose concentration in mg/L on the x-axis. A linear graph of the optical densities of the bioflocculants was also constructed from the best-fit line. From the standard glucose curve, the following mathematical equation was obtained: y = mx + c where y represented the optical density (absorbance) at a wavelength of 490 nm; m represented the mass; x represented the concentration of the bioflocculant sugars; and c represented the mathematical (velocity) constant (Jain et al ., 2017). Substituting values, the exact concentration of bioflocculant sugars was calculated from the following formula: OD bioflocculant = 0.3492 x + 1.2234 where OD bioflocculant represented the average optical density of the bioflocculant sugars RESULTS The bioflocculant produced from Pseudomonas aeruginosa strain F29 was characterised using Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy coupled to energy dispersive X-ray analysis (EDX), high-performance liquid chromatography (HPLC) coupled to mass spectrometry, and the phenol‒sulfuric acid method. These investigations revealed the typical elemental compositions, the functional groups and the physical characteristics of the bioflocculant. Fourier transform infrared (FTIR) spectroscopy The FTIR result of the bioflocculant from Pseudomonas aeruginosa strain F29, at 4000 − 400 cm − 1 frequency range, is shown on Fig. 1 . The functional groups of the bioflocculant from Pseudomonas aeruginosa strain F29, were determined by interpreting the peaks in both the functional group and the fingerprint regions of the FTIR spectrum. The FTIR showed a strong absorption peak at 3241.10 cm − 1 (broad), that correlated with O-H stretching of the hydroxyl group of a carboxylic acid/carboxylate (COOH) or a concentrated aromatic alcohol (-OH). This broad, strong, FTIR absorption peak at 3241.10 cm − 1 also fell within the 3200–3300 cm − 1 range that correlated with N-H moieties that participated in hydrogen bonding. The absorption at 1635.05 cm − 1 was medium and was possibly a result of vibrations from C = C alkene bond stretching in disubstituted (cis) compounds. This absorption at 1635.05 cm − 1 also indicated the presence of carbonyl (C = O) groups in sugars or/ and amide/peptide bonds. Furthermore, the absorption at 1635.05 cm − 1 fell within the 1500–1660 cm − 1 range that is typical for vibrations from saturated nitro compounds. The strong absorption peak at 599.55 cm − 1 was attributed to C-I, C-Br or C-Cl stretching (typically 600–700 cm − 1 ) in a halo compound and was also due of the bending of carbonyl (C = O) groups in an amide/peptide bond. Furthermore, this strong FTIR absorption peak at 599.55 cm − 1 , fell within the 390–610 cm − 1 range for P—Cl bonds; 550–670 cm − 1 range typical for stretching vibrations from magnesium oxide bonds; and the 590–600 cm − 1 range for sulfones. The absorption peak at 474.39 cm − 1 was from the S‒S stretch correlated with polysulfides, while the FTIR of 418.53 cm − 1 was from the vibrations of a metal-chloride (M-Cl) bond. Scanning electron microscopy (SEM) and energy dispersive X-ray analysis/spectroscopy (EDS/EDX) Plate 1 shows an SEM image of the bioflocculant from Pseudomonas aeruginosa strain F29, while Fig. 2 shows the EDS/EDX result. The SEM image showed a high-resolution three-dimensional image of the scanned surface of a narrow stretch of the surface of the bioflocculant. This surface looked clumped and flaky in topography. The X-ray energy emitted by the SEM electron beam was captured and read by an in-built analyser (EDX.) The elemental composition of the bioflocculant was recorded as a percentage of its total weight, and the following results were obtained: chlorine (56.00%), carbon (20.50%), sodium (12.50%), oxygen (4.00%), phosphorus (3.00%), magnesium sulfur (2.43%), magnesium (1.06%), potassium (0.32%) and nitrogen (0.30%). The amount of chlorine was slightly greater than half the total weight of the bioflocculant. The presence of carbon and oxygen suggested the presence of carbonyl and carboxylic bonds, as found in sugars and carbonyl bonds present in proteins and peptides. High-performance liquid chromatography (HPLC) and mass spectrometry (MS) Figure 3 shows the total ion chromatograph of the HPLC and MS performed on the bioflocculant. The monosaccharide composition of the bioflocculant was revealed by HPLC, and the mass was determined by measuring the ionised atoms of the bioflocculant. The different masses that corresponded to different retention times were compared to a standard for identification of the monosaccharide. There were many peaks for a single simple sugar compound because of the different injection rates used in the HPLC and the existence of stereoisomers. Additionally, the total number of peaks for each type of simple sugar moiety were numbered. The total ion chromatogram showed that the bioflocculant from Pseudomonas aeruginosa strain F29 consisted of monosaccharides; namely, glucose, galactose, inositol and mannose, D-ribose, arabinose, rhamnose and xylose. There was a total of sixteen recorded peaks, with glucose possessing four peaks (1, 2, 9 and 11); galactose with three peaks (5, 14 and 15); inositol with two peaks (3 and 16); rhamnose with two peaks (4 and 12); and mannose with two peaks (6 and 10). Arabinose, xylose and ribose had one peak each (7, 8 and 13). The presence of many peaks for a single simple sugar was due to the different rates of injection used in the HPLC process and the possible presence of stereoisomers. Glucose and galactose are stereoisomers. The phenol‒sulfuric acid method This analytic tool was used to determine the concentration of carbohydrate sugars in the bioflocculant from a standard glucose curve. The concentration of sugars in the bioflocculant was 0.0059 g/L. Plate 1: Scanning electron microscopy (SEM) micrograph of the bioflocculant produced from Pseudomonas aeruginosa strain F29 Key: F29 Bioflocculant from Pseudomonas aeruginosa strain F29; WD: Working distance; Mag: Magnification; Hv: High voltage; HFW: Horizontal field switch; MM: millimetres KV: Kilovolts; µm: Micrometres; PA: Pascals Key: y-axis- absorbance (2-log%T); x-axis-wavelength measured in cm − 1 Key- C: carbon; O: oxygen; P: phosphorus; Cl: chlorine; S: sulfur; Na: sodium; Mg: magnesium; K: potassium; N: nitrogen; KeV (x-axis): energy of the X-rays; y-axis: peak intensity (counts); Wt (%): percentage weight; F29: Pseudomonas aeruginosa strain F29 Time (s) Figure 3: Total ion chromatogram of the bioflocculant from Pseudomonas aeruginosa F29 Key: RT: retention time; s: seconds; glu: glucose; xyl: xylose; man: mannose; rha: rhamnose: rib: D-ribose; gal: galactose; ara; arabinose; ino; inositol DISCUSSION The physical characteristics, the composition and relative distribution of the chemical elements, and the functional groups of the bioflocculant that was produced from Pseudomonas aeruginosa strain F29, were determined, using Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM) coupled to energy dispersive X-ray spectroscopy (EDS or EDX), high-performance liquid chromatography (HPLC) coupled to mass spectrometry (MS), and the phenol-sulfuric acid method. Fourier transform infrared (FTIR) spectroscopy of the bioflocculant from Pseudomonas aeruginosa strain F29, revealed the presence of certain functional groups corresponding to the given absorption peaks. The capacity of a bioflocculant to exert its coagulative-flocculative functions, the ambit of its resistance to denaturation, and its amenability to different spatial configurations, depend on the type of the functional group it possesses (Rao et al ., 2013). In fact, Okaiyeto et al . (2015) reported that the functional groups in bioflocculants, provide dedicated areas for the chemical interactivity of positively charged ions and colloids suspended in a liquid medium. The presence of a carboxylic acid/carboxylate compound and/or a concentrated aromatic alcohol, in the bioflocculant from Pseudomonas aeruginosa strain F29, was alluded to from the strong absorption peak at 3241.10 cm − 1 ; this peak was generated from the stretching of O-H (hydroxyl) functional groups. This finding was in partial agreement with the research by Okaiyeto et al . (2013) on a purified bioflocculant, who reported that the peak at 3412 cm − 1 , was attributed to a hydroxyl group. This strong absorption peak at 3241.10 cm − 1 was also attributed to the presence of amino groups, and was within the protein amide A band of 3500–3300cm − 1 (Ji et al ., 2020). The medium absorption peak at 1635.05 cm − 1 in the bioflocculant from Pseudomonas aeruginosa strain F29, was attributed to C = C alkene bending in compounds, whose two alkyl groups are oriented on the same side of the alkene (cis isomer). In a study by Hurairah et al . (2023) on coagulation-flocculation, the seed peel extract from Archidendra jiringa , that was found to be effective as an aid in the elution of lead from man-made residual water, was noted to possibly contain alkene compounds (or aromatic compounds), as indicated by the weak FTIR bands at 3019 cm − 1 and 3001 cm − 1 . In the spectrum of the bioflocculant from Pseudomonas aeruginosa strain F29, the absorption at 1635.05 cm − 1 , also indicated the presence of carbonyl (C = O) groups, typically within the range of 1900–1600 cm − 1 (Smith, 2017), that revealed the presence of carbohydrate sugars (Roberts and Caserio, 1977) in the bioflocculant. Together with the second functional group commonly found in carbohydrates, and detected at 3412 cm − 1 , viz, the hydroxyl group, the presence of carbohydrates in the bioflocculant from Pseudomonas aeruginosa strain F29, was confirmed. In another light, the carbonyl (C = O) groups indicated by the medium absorption at 1635.05 cm − 1 , also fell within the amide I band, documented as typical for peptides or protein macromolecules, while the strong absorption peak, observed at 599.55 cm − 1 , fell into another typical range for peptides or proteins, the amide VI band, that comprises “out-of-plane” bending of carbonyl (C = O) groups in amide/peptide bonds (Krimm and Bandekar, 1986; Bandekar, 1992; Li, 2016). Additionally, Li (2016), documented that the amide band I is the most readily detected, out of the nine amide bands typically characteristic for a protein macromolecule, and thus can be used to study its composition and surroundings. Yatsyna et al . (2016) stated, in their research, that amide IV, V and VI bands, can be accurately used to identify the shape of the amide functional groups, and the framework of bound atoms present in a protein macromolecule. In addition, Yatsyna et al . (2016) reported that, amide IV, V and VI bands, also reveal that, amide bonds, as may occur in peptides whose building blocks are arranged in rings, possess amino acid molecules oriented on the same side of the amide/peptide bond (“cis-conformation”). In addition, amide A, I, II and III bands, were documented, by Ji et al . (2020), as the amide bands used to ascertain the construction of a protein macromolecule. The presence of a protein (or polypeptide) component in the bioflocculant from Pseudomonas aeruginosa strain F29, was therefore, reasonably established, by the amide I, VI and A bands that were detected in it. Additionally, Wang et al (2021) reported that amidation boosted the flocculation of superfine pyrite. Therefore, the inherent amide bonds present in the bioflocculant from Pseudomonas aeruginosa strain F29, were more than likely, partly responsible for its flocculation bioactivity. The absorption at 1635.05 cm − 1 fell within the 1660–1500 cm − 1 range that is typical for vibrations from saturated nitro compounds. From an extensive search of documented studies, it would appear that there is no information on the presence of nitro compounds in either conventional flocculants or bioflocculants. While research such as that by Smith (2020) may have suggested the inherent potential explosivity of the bioflocculant from Pseudomonas aeruginosa strain F29, the study by Noriega et al . (2022) on the vast array of the activities of nitro compounds, suggested that the bioflocculant from Pseudomonas aeruginosa strain F29, may exhibit antimicrobial, antiparasitic and antitumour bioactivities in addition to certain other activities. Nonetheless, Noriega et al . (2022) also stated that the sum total of the bioactivities of the nitro moiety, whether advantageous or harmful, is completely contingent on its chemical reduction (by receiving as many as 6 electrons) to the amide compound. As stated previously, the amide condensation reaction has been observed to enhance flocculation (Wang et al ., 2021). This amide bond, characterizes the amide/peptide moiety. Furthermore, Kurniawan et al . (2022) reviewed research that called attention to certain mechanisms by which moieties such as the amide moiety, subserve the coagulation-flocculation brought about by the Moringa plant. Therefore, it is inferable that the nitro moiety may display flocculation via the amide compounds obtained from the nitro-reduction pathway it undergoes, and that the net flocculation exhibited by a nitro compound-containing polymeric flocculant, such as the bioflocculant from Pseudomonas aeruginosa strain F29, would be partly due to this nitro moiety. Yet again, research such as by Kovacic and Somanathan (2014) and Gupta and Ronen (2024) have reported some nitro compounds as harmful contaminants in the environment and in wastewaters, in need of remediation. Ma et al . (2020) observed that environmental contaminants such as chromium and nitrobenzene, were flocculated out of the experimental aqueous mixtures of these contaminants that they had created, via activated biomass. Consequently, it would be worthwhile to observe if the flocculation triggered by a nitro compound-containing flocculant such as the bioflocculant from Pseudomonas aeruginosa strain F29, is effective in remediating toxic nitro compounds, from wastewater. If effective, it may be quite instructive to determine how this flocculation compares with effective, non-nitro compound-containing flocculants in common use. The strong FTIR absorption peak at 599.55 cm − 1 was also within the 670–550 cm − 1 range, typical for stretching vibrations from magnesium oxide bonds (Agyapong et al ., 2023). Although studies such as by Liu et al . (2023) and by Das et al ., (2023), reported on the flocculation property of magnesium oxide, the study by Sofi et al . (2021) that reported on some other properties of magnesium oxide, may give better insight into its presence in the bioflocculant produced from Pseudomonas aeruginosa strain F29. It may be that, as reported by Sofi et al . (2021), for pigments and zero-resistance materials, magnesium oxide enhanced and improved the properties and performance of the bioflocculant produced from Pseudomonas aeruginosa strain F29. These enhancements and improvements would no doubt, have boosted the overall properties of the bioflocculant produced from Pseudomonas aeruginosa strain F29, and not only bioflocculation. The strong FTIR absorption peak at 599.55 cm − 1 , fell within the 600–590 cm − 1 range for sulfones (Socrates, 2004). There seems to be an apparent lack of documentation on the existence of naturally-occurring sulfonated bioflocculants. However, lignosulfonates, that are bye products in the pulp and paper-making industry, have been observed to possess flocculation properties (Anukam et al ., 2021; Chan et al ., 2021). As such, lignosulfonates are strictly-speaking, man-made compounds. Tang et al . (2020), functionalized a bioflocculant, chitosan, with a sulfone. This synthetic, chemically modified, sulfone-containing bioflocculant, CS-g-P(AM-AMPS), was observed to effectively remediate heavy metals from wastewater, by the binding of the sulfone moiety to the heavy metal ions, to form a stable ring structure, in addition to heavy metal-binding to other moieties, and simultaneous sedimentation (Tang et al ., 2020). The presence of sulfones in the bioflocculant produced from Pseudomonas aeruginosa strain F29, was therefore, very likely, contributory to its overall flocculation capacity. The strong absorption peak at 599.55 cm − 1 was additionally, attributed to the presence of halogen compounds (carbon-iodide, carbon-bromide or carbon-chloride bonds) (Aldrich, 2019), in the bioflocculant from Pseudomonas aeruginosa strain F29. Maliehe et al . (2016), also reported the presence of halo compounds with carbon-chloride bonds at the 599.14 cm − 1 absorption peak, and carbon-iodide bonds at the 509.5 cm − 1 absorption peak in their research on the bioflocculant TMT 1 . Bodîrlău et al . (2009) reported the stretching from a carbon-chloride (C-Cl) bond at 700–600 cm − 1 , in their research on amalgams made from two categories of wood, Tripathy and De (2006) described polyamines as polymeric compounds that possess flocculating activity and are formed by a condensation reaction between halogenated compounds and amines. Therefore, halo compounds can be said to possess inherent coagulating-bioflocculating properties and their presence in the bioflocculant produced from Pseudomonas aeruginosa strain F29, may be partly responsible for its flocculation capacity. The strong FTIR absorption peak at 599.55 cm − 1 was within the 610–390 cm − 1 range for P-Cl bonds (Socrates, 2004). However, ascertaining the exact type of phosphorus-chlorine compounds in the bioflocculant from Pseudomonas aeruginosa strain F29, fell outside the scope of this study. Roshchin and Molodkina (1977) documented that phosphorus chlorides particularly the oxychloride, trichloride and pentachloride, are noxious substances that are considered occupational dangers. However, Kudzin et al . (2021) used gaseous forms of phosphorus trichloride to formulate a novel antimicrobial compound. Additionally, both phosphorus trichloride and phosphorus pentachloride, have been reported as chlorinating substances (Dillon et al ., 1976; Xiao and Han, 2019). Zhang et al . (2018) reported that crystalline ferric chloride is a moderately strong electron pair-acceptor that is used as both catalytic and chlorinating substances, in the manufacture of carbon-based compounds. Nonetheless, research such as by Ettaloui et al . (2021) has reported on the coagulation-flocculation property of ferric chloride. Therefore, it may be that the phosphorus chlorides present in the bioflocculant from Pseudomonas aeruginosa strain F29, also possess coagulation-flocculation properties, that synergistically contribute to its total flocculation capacity. Although a phosphorus-chlorine (P-Cl) bond on FTIR, can also emanate from a phosphoryl chloride, the absence of the phosphoryl moiety in the bioflocculant from Pseudomonas aeruginosa strain F29 bond, negated the possibility of the presence of a phosphoryl chloride. The FTIR of 418.53 cm − 1 was from the vibrations of a metal-chloride (M-Cl) bond (Socrates, 2004), in the bioflocculant from Pseudomonas aeruginosa strain F29. Three metals are naturally present in the bioflocculant, viz sodium potassium, and magnesium, and any, or all of them, could have formed the corresponding chloride salt. The study by Zhao and Zhang (2017) reported on the use of the chlorides of sodium, potassium and magnesium as aids to maximize the medium for the production of a bioflocculant from Bacillus subtilis . However, the use of sodium chloride, potassium chloride and magnesium chloride either as flocculants or flocculant aids have been well documented (Pérez et al ., 2021; Zhou et al ., 2022; Ozkan and Yekeler, 2004). Therefore, any of these chlorides, if present in the bioflocculant from Pseudomonas aeruginosa strain F29 would likely have contributed to its flocculation property. The bioflocculant from Pseudomonas aeruginosa strain F29 also contained polysulfides. This was alluded to, by the peak at 474.39 cm − 1 , that was attributed to S‒S disulfide bond stretching. Asmare et al . (2021) used the coagulation-flocculation induced by a mixture of calcium polysulfide and ferrous sulphate, for remediating waste water from a leather-making industry. They discovered that although the use of a high concentration of calcium polysulfide (4% v/v), mixed with ferrous sulphate, did not eliminate the chemical oxygen demand entirely, it still resulted in a relatively high reduction (86.13%) in waste, and achieved all other indices of proper wastewater treatment. This finding highlights the relative effectiveness of polysulfide compounds in the coagulation-flocculation process. High-performance liquid chromatography (HPLC) and mass spectrometry (MS) analyses, revealed different monosaccharides, indicating that the bioflocculant produced from Pseudomonas aeruginosa strain F29 possessed a heteropolysaccharide component. Polysaccharides have been observed to possess bioflocculating properties (Salehizadeh et al ., 2018). In a research study conducted by Ma et al . (2022), the heteropolysaccharide bioflocculant BP50-2, produced from discarded banana skin, demonstrated effective agglomerating activity. Therefore, the polysaccharide component of the bioflocculant produced from Pseudomonas aeruginosa strain F29 would more likely have contributed to the net coagulation-flocculation capacity of the bioflocculant. Scanning electron microscopy of the bioflocculant produced from Pseudomonas aeruginosa strain F29, revealed clumped and flaky surfaces. Xiong et al . (2010) reported that the nature of the surface of a bioflocculant holds immense significance with regard to its flocculating capacity. The topographic layout of a bioflocculant, thus determines its potency or lack thereof (Tsilo et al ., 2022). EDX of the bioflocculant produced from Pseudomonas aeruginosa strain F29, revealed a relative abundance of chlorine (56.00%) and carbon (20.50%). Sodium (12.50%) and oxygen (4.00%) were next in abundance. Phosphorus (3.00%) and sulfur (2.43%) were also present in the bioflocculant produced from Pseudomonas aeruginosa strain F29, with the least abundant elements being magnesium (1.06%), potassium (0.32%) and nitrogen (0.30%). The carbon and oxygen compositions hinted at the presence of polysaccharides (Awuchi and Amagwula, 2021) and proteins (Biswas, 2020) in the bioflocculant produced from Pseudomonas aeruginosa strain F29. The bioflocculant in the research conducted by Tsilo et al . (2022), had a much higher oxygen (43.76%) and phosphorus (14.44%) content, and a much lower chlorine content (0.31%), when compared with the same elements in the bioflocculant produced from Pseudomonas aeruginosa strain F29. The potassium content (0.34%) was almost the same as that present in the bioflocculant produced from Pseudomonas aeruginosa strain F29 (0.32%). Calcium was absent, in the bioflocculant produced from Pseudomonas aeruginosa strain F29, but was relatively abundant (20.35%) in the bioflocculant studied by Tsilo et al . (2022). These differences in bioflocculant composition were probably due to species differences and the type and ratio of the constituents of the bacterial medium that was used in bioflocculant production. Some metal cations have been observed to aid in floc-formation (Kuriyama et al ., 1991; Tawila et al ., 2019). Sodium alginate, a compound found in nature, was observed to possess good flocculating activity during sewage treatment, in research conducted by Tian et al . (2020), while sodium polyacrylate was used to aggregate tiny hematite particles and thereby cause them to drift away from the intermixed quartz particles (Cheng et al ., 2022). Ozkan and Yekeler (2004), in their research to determine the floc-forming properties of strontium sulphate (“celestite”), by utilising two classes of flocculants, discovered that magnesium 2 + ions from magnesium chloride, worked best on the strontium sulphate, than did calcium 2+ (from calcium chloride) and aluminium 3+ (from aluminium chloride). Also, in research conducted by Hejazi (2013), potassium compounds were combined with sodium dodecyl sulphate, to trigger floc formation in a mixture that contained megestrol acetate particles. The non-metal, chlorine, was utilised as an aid in flocculation, in addition to its use as a deodoriser and a disinfectant, in water purification experiments (Weston, 1924). Ou et al . (2016), in an experiment conducted, observed that the chlorine-containing compound, sodium chloride (common salt) displayed good floc-forming activity against small-grained particles of industrial kaolinite, and this activity was directly proportional to the quantity per unit volume, of both flocculant and industrial kaolinite. Therefore, it may be deduced that the presence of the metals, sodium, potassium, magnesium, and the non-metal, chlorine, in the bioflocculant produced from Pseudomonas aeruginosa strain F29, would have contributed, to some significant degree, to its bioflocculating activity. The non-metal elements, phosphorus, sulfur and nitrogen have been stated as constituents of certain carbohydrates (Gangasani et al ., 2022), and the bioflocculant produced from Pseudomonas aeruginosa strain F29, possessed these elements in varying proportions. Nevertheless, these elements- phosphorus, nitrogen and sulfur - have been described by research such as by Kakade et al . (2022), as lesser constituents of carbohydrates, and occur in those carbohydrates that have been modified. On the other hand, phosphorus, sulfur and nitrogen have also been documented as constituents of proteins (Biswas, 2020). Although, nitrogen was described by Biswas (2020) as a main constituent of a protein macromolecule, with phosphorus and sulfur present in relatively less proportions, the bioflocculant from Pseudomonas aeruginosa strain F29 possessed relatively more abundance of phosphorus (3.00%) and sulfur (2.43%), than nitrogen (0.30%). In this manner, the protein component of the bioflocculant from Pseudomonas aeruginosa strain F29 could more likely be described as atypical. The existence of a protein/proteins were alluded to, from the occurrence of phosphorus, nitrogen, sulfur, and amide/peptide bonds within amide I, VI and A bands (Li, 2016; Yatsyna et al ., 2016; Ji et al ., 2020; Kakade et al ., 2022), in the bioflocculant from Pseudomonas aeruginosa strain F29. However, the more specific differentiation between a protein and that of a polypeptide, in the bioflocculant, could not be exactly ascertained. This was because the use of a more diagnostic method, such as x-ray crystallography, that can provide a stereographic view of a protein (Alberts et al ., 2002), fell outside the scope of this study. Additionally, the discovery that more than 75% of the bioflocculant from Pseudomonas aeruginosa strain F29, consisted of carbon and chlorine (with chlorine accounting for more than half of the total amount of elements present), strongly indicated that the bioflocculant, was largely an organochlorine compound (organochloride). The sodium, potassium and magnesium ions, accounted for the total metal composition of the bioflocculant from Pseudomonas aeruginosa strain F29. This metal composition was 13.32%, with sodium ion accounting for 90% of all metals present. Therefore, with the confirmed presence of metals, carbohydrate sugars, proteins and organochlorines, in the bioflocculant from Pseudomonas aeruginosa strain F29, it was deduced that this bioflocculant was a metal-containing polymeric compound composed mainly of carbohydrates, proteins/polypeptides and organochlorines; possibly a “ metalloglyco-protein/polypeptide-organochlorine” bioflocculant. Literature is rife with documentation on polyfunctional compounds. However, it was particularly intriguing and puzzling to discover multiple moieties associated with a variety of well-documented bioactivities, and chemical compounds with diverse applications, packed into one macromolecule namely, the bioflocculant from Pseudomonas aeruginosa strain F29. It would appear that this bioflocculant was produced by Pseudomonas aeruginosa strain F29, to execute a wide range of functions. The incorporation, into the bioflocculant, of multiple functional groups that provided dedicated reactive zones that determined its chemical reactiveness, characteristics and biological functions (Ertl et al ., 2020), was probably therefore, a response of Pseudomonas aeruginosa strain F29, to varied stimuli it had received in its environment. Further studies on this polyfunctional bioflocculant from Pseudomonas aeruginosa strain F29, could provide valuable insight into its range of chemical properties, bioactivities and industrial applications. An extensive search of documented research, failed to reveal any report of either metal-containing “glycoprotein/polypeptide-organochlorines” (“metallo- glycoprotein/polypeptide-organochlorines”), or “glycoprotein/polypeptide-organochlorines”. There seemed to be an absence of documentation on the discovery of these categories of glycoconjugates. There was a report by Fujimori et al . (2012) on Pseudomonas species producing the vaporescent organochloride compound, chloromethane. However, an extensive search of documented studies did not reveal any report of an organochloride bioflocculant, a “glyco-protein/polypeptide-organochlorine” bioflocculant, or a “metallo- glycoprotein/polypeptide-organochlorine” from Pseudomonas species or any other microorganism. Additionally, there was also an apparent lack of documentation on the natural occurrence of saturated nitro compounds, sulfones, polysulfides, phosphorus chlorine compounds, magnesium oxide and metal chlorides, in a bioflocculant. From available documentation, this is the first report of a polyfunctional “metalloglyco-protein/polypeptide organochlorine” bioflocculant that naturally contains saturated nitro compounds, sulfones, polysulfides. phosphorus chlorine compounds, magnesium oxide and metal chlorides; and of a bioflocculant produced from porcine faeces in Africa. CONCLUSION A bioflocculant produced from Pseudomonas aeruginosa strain F29 (accession number OQ734844) was characterised using investigative procedures. These procedures revealed that the bioflocculant was possibly a novel glycoconjugate, a “metallo-glycoprotein/polypeptide-organochloride”, that contained metals and naturally-occurring saturated nitro compounds, sulfones, polysulfides, phosphorus chlorine compounds, magnesium oxide and metal chlorides. From available documentation, this is the first report of a polyfunctional “metallo-glycoprotein/polypeptide-organochlorine” bioflocculant that naturally contains saturated nitro compounds, sulfones, polysulfides, phosphorus chlorine compounds, magnesium oxide, and metal chlorides; and of a bioflocculant produced from porcine faeces in Africa. Further research on this bioflocculant should prove helpful in establishing the ambit of its properties, activities and applications. AUTHORS CONTRIBUTION I, Ikechukwu Kenneth M. Okorie (corresponding author), carried out all the work in this study as my Masters (MSc.) research project. Professor Adeniyi A. Ogunjobi supervised my Master’s research study. Declarations SOURCE OF FUNDING This study was self-funded by me, Ikechukwu. Kenneth M. Okorie. No external funding was received. COMPETING INTEREST Both authors declare no financial or nonfinancial competing interest. CONFLICTING INTEREST Both authors declare no conflicting interest. ACKNOWLEDGEMENTS The corresponding author would like to thank Professor Adeniyi A. Ogunjobi for supervising this study. Many thanks also go to Professor Tonye G. Okorie for editing and proofreading the article and to Dr. Augustine Akpoka for his useful suggestions. Mr. Gabriel O. Bamidele helped greatly with the laboratory work. References Abu Tawila, Z. M., Ismail, S., Dadrasnia, A. and Usman, M. M. 2018. 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Journal of the Science of Food and Agriculture 102. 9: 3752–3761. https://doi.org/10.1002/jsfa.11723 Additional Declarations The authors declare no competing interests. Cite Share Download PDF Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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F29\u003c/p\u003e","description":"","filename":"Plate1BioflocculantSEM.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7453669/v2/465797055e1c4b12346b0a46.jpg"},{"id":99790018,"identity":"855d16aa-3e22-47e4-96d2-b7cd477527fb","added_by":"auto","created_at":"2026-01-08 12:52:34","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":510484,"visible":true,"origin":"","legend":"\u003cp\u003eFigure 1: FTIR spectrum of bioflocculant from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain F29\u003c/p\u003e","description":"","filename":"Figure1BioflocculantFTIR.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7453669/v2/bf8452271c52ec1b153cf3ad.jpg"},{"id":99790553,"identity":"d0335a5e-2ce4-4cba-a6f1-e6f412f478f3","added_by":"auto","created_at":"2026-01-08 12:58:19","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":818500,"visible":true,"origin":"","legend":"\u003cp\u003eFigure 2: Energy dispersive X-ray analysis (EDX) spectrum of the bioflocculant from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain F29\u003c/p\u003e","description":"","filename":"Figure2BioflocculantEDX.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7453669/v2/0e1e3c7735fdf813d81da50e.jpg"},{"id":99483359,"identity":"1291b3d7-4fb5-4889-abdd-13ab15cf4a6e","added_by":"auto","created_at":"2026-01-05 01:31:07","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":911644,"visible":true,"origin":"","legend":"\u003cp\u003eFigure 3: Total ion chromatogram of the bioflocculant from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e F29\u003c/p\u003e","description":"","filename":"Figure3BioflocculantTIC.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7453669/v2/26d84e917f084598fe53b82c.jpg"},{"id":100356259,"identity":"2911eccd-e2e8-4585-b599-1c6613a2f93e","added_by":"auto","created_at":"2026-01-16 06:58:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4315855,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7453669/v2/e680ee54-4f4f-43a7-97e6-b835a8fdd98e.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"Characterization of a novel polyfunctional “metalloglyco-protein/polypeptide-organochlorine” bioflocculant containing saturated nitro compounds, sulfones, polysulfides, phosphorus chlorine compounds, magnesium oxide, and metal chlorides, produced from Pseudomonas aeruginosa strain F29, isolated from porcine faeces in Nigeria","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eAntibiotic resistance has reached universal proportions, and the discovery of effective alternatives to the common antibiotics currently used, could aid in solving this problem. Antibiotic resistance occurs when bacteria are no longer susceptible to the usual drugs designed to kill them (bactericidal antibiotics) or to retard their growth (bacteriostatic antibiotics). Antibiotic resistance is the main subdivision of the larger group of antimicrobial resistance (AMR), and is defined as the development, by bacteria, of resistance to specific drugs invented to kill them, or to repress their growth (WHO, 2021). Alternative antibacterial agents are therefore being sought. It has been reported that bioflocculants possess bioactivities against bacteria and certain other groups of microorganisms (Abu Tawila \u003cem\u003eet al\u003c/em\u003e., 2018).\u003c/p\u003e \u003cp\u003eBioflocculants are biological flocculants and are therefore, more generally defined as flocculants of organic origins and not solely microbial (Yang \u003cem\u003eet al\u003c/em\u003e., 2024). Bioflocculants, can be obtained from microorganisms, animals and vegetation (Kurniawan \u003cem\u003eet al\u003c/em\u003e., 2022). With respect to microorganisms, bioflocculants are extracellular polymeric substances that are produced as secondary metabolites and cause flocculation in a given medium (Dih \u003cem\u003eet al\u003c/em\u003e., 2019). These microbial bioflocculants are a result of cytolysis and the release of cellular products by different groups of microbes (Alias \u003cem\u003eet al\u003c/em\u003e., 2022). Bioflocculants aggregate particles and then remove these particles, from the liquid medium in which they were formerly suspended. On the whole, flocculants have the capacity to clump small-sized substances suspended in a medium; these clumped substances are termed flocs (Wang \u003cem\u003eet al\u003c/em\u003e., 2022), and sediment over time. The specific mechanism of action, by which bioflocculants operate, is still unknown. A bioflocculant may trigger the construction of a bridge between particles of the medium and its own molecules; or may kick-start a neutralisation of charges present in the medium (Lai \u003cem\u003eet al\u003c/em\u003e., 2018). Other hypotheses exist, nevertheless, the precise mechanism of bioflocculant antibacterial bioactivity, may be deduced from the inherent capacity of bioflocculants to form flocs, that later sediment. A bioflocculant may therefore, initiate the clumping together of the cell wall components of the bacterium being acted upon, and this could more likely, produce tears in the bacterial cell membrane, with varying degrees of fragmentation. The compromise in bacterial cell membrane integrity that ensues, if progressive and widespread enough, could trigger an influx of noxious substances from the surroundings, that could lead to a destabilisation of the internal milieu of the bacterial cell, and result in bacterial cell lysis and death.\u003c/p\u003e \u003cp\u003eBioflocculants are made up of natural compounds such as cellulose, nucleic acids, glycoproteins and polysaccharides, that render them readily decomposable and less injurious to the environment, therefore, secondary environmental contamination is obviated (Alias \u003cem\u003eet al\u003c/em\u003e., 2022). Microbial bioflocculants are polymeric substances produced during the microbial growth phase (Tsilo \u003cem\u003eet al\u003c/em\u003e., 2022). Plant-derived bioflocculants include mucilage (Das \u003cem\u003eet al\u003c/em\u003e., 2021), alginate, starch (Salehizadeh \u003cem\u003eet al\u003c/em\u003e., 2018) and cellulose ((Fauzani \u003cem\u003eet al\u003c/em\u003e., 2021). There are, however, different kinds of bacterial species, that also produce cellulose (here known as bacterial or microbial cellulose), that provides mechanical support (Choi \u003cem\u003eet al\u003c/em\u003e., 2022), and possesses similar bioflocculant characteristics as plant cellulose. Animal-derived bioflocculants include gelatin and chitosan (Badawi \u003cem\u003eet al\u003c/em\u003e., 2023).\u003c/p\u003e \u003cp\u003eBioflocculants composed mainly of proteins, are easily affected by thermal fluctuations because proteins naturally lose their spatial conformations when exposed to external forces such as thermal energy (Bukhari \u003cem\u003eet al\u003c/em\u003e., 2020). The nucleic acids (deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)), present in bioflocculants, constitute part of the larger pool of environmental nucleic acids; indeed, their presence in the environment is being utilised, now more than previously, in \u0026ldquo;molecular biomonitoring\u0026rdquo; (Littlefair \u003cem\u003eet al\u003c/em\u003e., 2022). Glycoprotein bioflocculants are characterised by the presence of oxygen, nitrogen and carbon in their framework; these elements are thought to boost their inherent bioflocculating capacities (Tsilo \u003cem\u003eet al\u003c/em\u003e., 2022). Bioflocculants largely composed of polysaccharides, retain their activities at high temperatures (Tsilo \u003cem\u003eet al\u003c/em\u003e., 2021). It has been documented that the flocculating capacity of any flocculant is directly proportional to the stretch of monomeric subunits it possesses; therefore, flocculants with higher molar masses are said to possess a commensurate degree of stretch and can even house unbound moieties, that can link flocs, to bring about better floc formation (Michaels, 1954; Gao \u003cem\u003eet al\u003c/em\u003e. 2006). Apart from their use as antibiotics, bioflocculants can be effective against algae and viruses (Abu Tawila \u003cem\u003eet al\u003c/em\u003e., 2018). Bioflocculants that are primarily composed of polysaccharides, can substantially protect cells against free radical injury (Giri \u003cem\u003eet al\u003c/em\u003e., 2019). Bioflocculants can also be used as stabilisers, thickeners and emulgents, in the food industry and in drug manufacturing (Zhong \u003cem\u003eet al\u003c/em\u003e., 2018). Some exopolysaccharides can demonstrate flocculating properties such as the exopolysaccharide, EPS SM9913, produced from the blue-water, cryophilic bacterium, \u003cem\u003ePseudoalteromonas\u003c/em\u003e sp. SM9913 (Li \u003cem\u003eet al\u003c/em\u003e., 2008). Exopolysaccharides that double as bioflocculants can therefore be utilised as flocculants for wastewater management (Li \u003cem\u003eet al\u003c/em\u003e., 2008), in addition to other uses that stem from the properties they possess as exopolysaccharides.\u003c/p\u003e \u003cp\u003eIn a bioflocculant, the hydroxyl, carboxyl and amide functional moieties promote the flocculation process; they provide sites of attachment for the bivalent positively-charged ions that principally partake in flocculation (Tang \u003cem\u003eet al\u003c/em\u003e., 2014; Vimala, 2019). Certain microorganisms can assimilate metals into the bioflocculants they produce, and these metal components affect the intrinsic chemical composition and morphology, and the mechanisms of actions of the bioflocculant (Zoaka \u003cem\u003eet al\u003c/em\u003e., 2025).\u003c/p\u003e \u003cp\u003eThe aim of this study was to characterise a bioflocculant that was produced from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain F29, accession number OQ734844, that exhibited effective antibacterial activity against two antibiotic resistant bacteria, viz, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e SO183, and an identified strain of \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, in another study that was part of a Master\u0026rsquo;s project (Okorie, 2023). This characterisation was necessary in order to identify the physical nature and the chemical composition of the bioflocculant. The bacterial DNA sequence was deposited in the National Centre for Biotechnology Information (NCBI) GenBank database under its accession number. FTIR, SEM coupled to EDX, HPLC coupled to MS and the phenol‒sulfuric acid method, were employed as analytic tools.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003eAn aliquot of the bioflocculant was transferred into a sterile bottle and subjected to some analytical procedures to determine its elemental composition, functional groups and physical characteristics. The analytical tools used, were Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) coupled to energy dispersive X-ray spectroscopy (EDX), high-performance liquid chromatography (HPLC) coupled to mass spectrometry (MS) and the phenol‒sulfuric acid method.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eFourier transform infrared (FTIR) spectroscopy\u003c/h2\u003e \u003cp\u003eThe functional groups present in the bioflocculant were characterised using a Perkin Elmer Spectrum 100 Series 3000 MX spectrometer. A set of instructions for FTIR, from the NanoScience Technology Centre (NTSC) (2008), was used as a reference point. The bioflocculant sample was thinned between two potassium bromide (KBr) plates for FTIR analysis, and then was placed inside the sample holder of the FTIR device that was wiped clean with acetone. The addition of relevant computer commands, enabled the recording of the generated infrared spectra. Next, the data from these spectra was analysed using the spectroscopic software Win-IR Pro Version 3.0, and saved. The interpretation of the infrared (IR) spectra was performed using Infrared and Raman Characteristic Group Frequencies from Socrates, (2004); IR Spectra Table and Charts from Sigma Aldrich Company (Aldrich, 2019) and FTIR results analyses from Aksnes and Aksnes (1963). Bunaciu \u003cem\u003eet al\u003c/em\u003e. (2014), Smith (2017), Nandiyanto \u003cem\u003eet al\u003c/em\u003e. (2019), Ji \u003cem\u003eet al\u003c/em\u003e. (2020) and Agyapong \u003cem\u003eet al\u003c/em\u003e. (2023).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eScanning electron microscopy (SEM) and energy dispersive X-ray analysis/spectroscopy (EDS/EDX) (dup: abstract ?)\u003c/h3\u003e\n\u003cp\u003eThe surface topography of the bioflocculant was revealed using a JEOL JSM-7600F scanning electron microscope (SEM) (Tokyo, Japan), while its elemental composition was determined using an in-built energy dispersive X-ray (EDX) spectrometer.\u003c/p\u003e \u003cp\u003eThe protocol laid out by Core Facilities (Facilities, 2021) for preparing an SEM sample was used as a reference. Primary fixation of a measured volume of the bioflocculant sample was performed using formaldehyde and glutaraldehyde, while secondary fixation was performed with osmium tetroxide. This step was followed by dehydration with ethanol and sample drying. Mounting of the sample, and firm fixation of the sample, on a specimen stub, were performed next, before placement of the sample in the specimen slot, was performed. Sputter coating of the sample with platinum was performed before sectioning of the sample ensued. The bioflocculant surface was observed by SEM.\u003c/p\u003e \u003cp\u003eAnalysis of the chemical composition of the bioflocculants was performed via an in-built EDX. The supercharged electron beam from the SEM, hit the bioflocculant sample and caused the release of X-rays from the bioflocculant surface atoms (Thambiratnam \u003cem\u003eet al\u003c/em\u003e., 2020). The X-rays from this interaction, were pooled by the X-ray detector in the EDX, interpreted as elemental type and concentration results, and displayed as an EDX graph (Thambiratnam \u003cem\u003eet al\u003c/em\u003e., 2020).\u003c/p\u003e\n\u003ch3\u003eHigh-performance liquid chromatography (HPLC) and mass spectrometry (MS) (dup: abstract ?)\u003c/h3\u003e\n\u003cp\u003eThe carbohydrate sugars present in the bioflocculant were analysed using an Agilent 1100 series HPLC system (Agilent Technologies, California, USA) equipped with a diode array detector (DAD) and a reversed-phase C-18 Zorbax Eclipse extradense bundle (XDB-C18) column (250 \u0026times; 4.6 mm, 5 \u0026micro;m particle size, 300 \u0026Aring; pore size). An Agilent 1100 Series LC/MSD benchtop mass spectrometer connected to the HPLC device was used to determine the mass‒charge ratio of the carbohydrate sugars.\u003c/p\u003e \u003cp\u003eA set of HPLC procedures outlined by Anumula (1994), Guzzetta (2001), and Sharma \u003cem\u003eet al\u003c/em\u003e. (2020), were used. The Agilent 1100 Series High Value System User\u0026rsquo;s Guide from Agilent Technologies (1999) was also used. The HPLC solvent conduits were degassed by HPLC-grade isopropanol purging prior to the commencement of the analytical technique. Solvent A (HPLC-grade water combined with 0.1% v/v formic acid) and solvent B (HPLC-grade acetonitrile and 0.1% v/v trifluoroacetic acid), composed the HPLC reversed-phase solvent elution setup (Guzzetta, 2001). Multiple injections of the same samples were enabled through an injector protocol accessed in the HPLC device (Huber, 2010) to separate the monosaccharide moieties. The HPLC procedure involved sorting non-volatile compounds from the analyte to forestall matrix interference (Ho \u003cem\u003eet al\u003c/em\u003e., 2003).\u003c/p\u003e \u003cp\u003eThe product of the HPLC analysis was channelled into an Agilent 1100 Series LC/MSD (liquid chromatography/mass selective detector) benchtop mass spectrometer. The ionisation of the sample kick-started the analysis; a disaggregation of the sample ions ensued in the electromagnetic field created by the spectrometer on the basis of the differences in ionic mass/charge (m/z) ratios (Reusch, 2013). The disaggregated ions were identified and quantified as they made contact with both ultraviolet and mass selective detectors of the device; this information was combined into an array of graphical results (Reusch, 2013). Consequently, both qualitative and quantitative characterisation of the bioflocculant sample, could be performed via mass spectrometry (Ho \u003cem\u003eet al\u003c/em\u003e., 2003). A total ion chromatogram was the final graphical display of the results of both HPLC and MS.\u003c/p\u003e\n\u003ch3\u003eThe phenol‒sulfuric acid method\u003c/h3\u003e\n\u003cp\u003eThe phenol‒sulfuric acid method was used to measure the concentration of the total sugar content of the bioflocculant (expressed in milligrams/litre); this method employed glucose as the standard (Chaplin and Kennedy, 1986). The amount of carbohydrate was determined as described. A quantity of the bioflocculant, 0.1 mL, was mixed with 2 mL of distilled water. To this mixture, 0.1 mL of 6% phenol and 5 mL of 95% sulfuric acid (v/v) were quickly added. This composite mixture was shaken and left to stand for 10 minutes. The optical density was then recorded at 490 nm, as an average of three separate readings. Two millilitres of distilled water mixed with 0.1 mL of 6% phenol and 5 mL of 95% sulfuric acid (v/v) were used as the blank control, and the absorbance was measured. The amount of bioflocculant sugars was determined using a standard glucose curve that had to be prepared. This process was conducted according to the method outlined by DuBois \u003cem\u003eet al\u003c/em\u003e. (1956) with some modifications. Glucose (0.1 g) was dissolved in 100 mL of distilled water to make a stock solution. From this stock solution, 10 mL was removed and added to 90 mL of distilled water to make 100 mL of diluted stock solution. The following volumes of the diluted stock solution were poured into five different sterile bottles: 0.1 mL, 0.2 mL, 0.4 mL, 0.8 mL and 1.0 mL. Next, a series of different dilutions of the glucose solution was achieved by making up each volume of stock solution to 3 mL, through the addition of the needed volume of distilled water. For each of these concentrations, 0.1 mL of 6% phenol and 5 mL of 95% (v/v) sulfuric acid were quickly mixed, and the composite mixtures were allowed to stand for 10 minutes. The optical density (OD) at 490 nm was subsequently measured. A standard glucose curve was then plotted with the optical densities of the different glucose dilutions at 490 nm on the y-axis and the corresponding glucose concentration in mg/L on the x-axis. A linear graph of the optical densities of the bioflocculants was also constructed from the best-fit line.\u003c/p\u003e \u003cp\u003eFrom the standard glucose curve, the following mathematical equation was obtained:\u003c/p\u003e \u003cp\u003e \u003cem\u003ey\u003c/em\u003e\u0026thinsp;=\u0026thinsp;\u003cem\u003emx\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003ec\u003c/em\u003e\u003c/p\u003e \u003cp\u003ewhere \u003cem\u003ey\u003c/em\u003e represented the optical density (absorbance) at a wavelength of 490 nm; \u003cem\u003em\u003c/em\u003e represented the mass; \u003cem\u003ex\u003c/em\u003e represented the concentration of the bioflocculant sugars; and \u003cem\u003ec\u003c/em\u003e represented the mathematical (velocity) constant (Jain \u003cem\u003eet al\u003c/em\u003e., 2017).\u003c/p\u003e \u003cp\u003eSubstituting values, the exact concentration of bioflocculant sugars was calculated from the following formula:\u003c/p\u003e \u003cp\u003eOD \u003csub\u003ebioflocculant\u003c/sub\u003e = 0.3492\u003cem\u003ex\u003c/em\u003e\u0026thinsp;+\u0026thinsp;1.2234\u003c/p\u003e \u003cp\u003ewhere OD \u003csub\u003ebioflocculant\u003c/sub\u003e represented the average optical density of the bioflocculant sugars\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003eThe bioflocculant produced from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain F29 was characterised using Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy coupled to energy dispersive X-ray analysis (EDX), high-performance liquid chromatography (HPLC) coupled to mass spectrometry, and the phenol‒sulfuric acid method. These investigations revealed the typical elemental compositions, the functional groups and the physical characteristics of the bioflocculant.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eFourier transform infrared (FTIR) spectroscopy\u003c/h2\u003e \u003cp\u003eThe FTIR result of the bioflocculant from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain F29, at 4000\u0026thinsp;\u0026minus;\u0026thinsp;400 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e frequency range, is shown on Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The functional groups of the bioflocculant from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain F29, were determined by interpreting the peaks in both the functional group and the fingerprint regions of the FTIR spectrum. The FTIR showed a strong absorption peak at 3241.10 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (broad), that correlated with O-H stretching of the hydroxyl group of a carboxylic acid/carboxylate (COOH) or a concentrated aromatic alcohol (-OH). This broad, strong, FTIR absorption peak at 3241.10 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e also fell within the 3200\u0026ndash;3300 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e range that correlated with N-H moieties that participated in hydrogen bonding. The absorption at 1635.05 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was medium and was possibly a result of vibrations from C\u0026thinsp;=\u0026thinsp;C alkene bond stretching in disubstituted (cis) compounds. This absorption at 1635.05 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e also indicated the presence of carbonyl (C\u0026thinsp;=\u0026thinsp;O) groups in sugars or/ and amide/peptide bonds. Furthermore, the absorption at 1635.05 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e fell within the 1500\u0026ndash;1660 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e range that is typical for vibrations from saturated nitro compounds. The strong absorption peak at 599.55 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was attributed to C-I, C-Br or C-Cl stretching (typically 600\u0026ndash;700 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) in a halo compound and was also due of the bending of carbonyl (C\u0026thinsp;=\u0026thinsp;O) groups in an amide/peptide bond. Furthermore, this strong FTIR absorption peak at 599.55 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, fell within the 390\u0026ndash;610 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e range for P\u0026mdash;Cl bonds; 550\u0026ndash;670 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e range typical for stretching vibrations from magnesium oxide bonds; and the 590\u0026ndash;600 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e range for sulfones. The absorption peak at 474.39 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was from the S‒S stretch correlated with polysulfides, while the FTIR of 418.53 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was from the vibrations of a metal-chloride (M-Cl) bond.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eScanning electron microscopy (SEM) and energy dispersive X-ray analysis/spectroscopy (EDS/EDX)\u003c/h3\u003e\n\u003cp\u003ePlate 1 shows an SEM image of the bioflocculant from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain F29, while Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the EDS/EDX result. The SEM image showed a high-resolution three-dimensional image of the scanned surface of a narrow stretch of the surface of the bioflocculant. This surface looked clumped and flaky in topography. The X-ray energy emitted by the SEM electron beam was captured and read by an in-built analyser (EDX.) The elemental composition of the bioflocculant was recorded as a percentage of its total weight, and the following results were obtained: chlorine (56.00%), carbon (20.50%), sodium (12.50%), oxygen (4.00%), phosphorus (3.00%), magnesium sulfur (2.43%), magnesium (1.06%), potassium (0.32%) and nitrogen (0.30%). The amount of chlorine was slightly greater than half the total weight of the bioflocculant. The presence of carbon and oxygen suggested the presence of carbonyl and carboxylic bonds, as found in sugars and carbonyl bonds present in proteins and peptides.\u003c/p\u003e\n\u003ch3\u003eHigh-performance liquid chromatography (HPLC) and mass spectrometry (MS)\u003c/h3\u003e\n\u003cp\u003eFigure 3 shows the total ion chromatograph of the HPLC and MS performed on the bioflocculant. The monosaccharide composition of the bioflocculant was revealed by HPLC, and the mass was determined by measuring the ionised atoms of the bioflocculant. The different masses that corresponded to different retention times were compared to a standard for identification of the monosaccharide. There were many peaks for a single simple sugar compound because of the different injection rates used in the HPLC and the existence of stereoisomers. Additionally, the total number of peaks for each type of simple sugar moiety were numbered. The total ion chromatogram showed that the bioflocculant from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain F29 consisted of monosaccharides; namely, glucose, galactose, inositol and mannose, D-ribose, arabinose, rhamnose and xylose. There was a total of sixteen recorded peaks, with glucose possessing four peaks (1, 2, 9 and 11); galactose with three peaks (5, 14 and 15); inositol with two peaks (3 and 16); rhamnose with two peaks (4 and 12); and mannose with two peaks (6 and 10). Arabinose, xylose and ribose had one peak each (7, 8 and 13). The presence of many peaks for a single simple sugar was due to the different rates of injection used in the HPLC process and the possible presence of stereoisomers. Glucose and galactose are stereoisomers.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eThe phenol‒sulfuric acid method\u003c/h2\u003e \u003cp\u003eThis analytic tool was used to determine the concentration of carbohydrate sugars in the bioflocculant from a standard glucose curve. The concentration of sugars in the bioflocculant was 0.0059 g/L.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePlate 1: Scanning electron microscopy (SEM) micrograph of the bioflocculant produced from\u003c/b\u003e \u003cb\u003ePseudomonas aeruginosa\u003c/b\u003e \u003cb\u003estrain F29\u003c/b\u003e\u003c/p\u003e \u003cp\u003eKey: F29 Bioflocculant from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain F29; WD: Working distance; Mag: Magnification; Hv: High voltage; HFW: Horizontal field switch; MM: millimetres KV: Kilovolts; \u0026micro;m: Micrometres; PA: Pascals\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eKey: y-axis- absorbance (2-log%T); x-axis-wavelength measured in cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eKey- C: carbon; O: oxygen; P: phosphorus; Cl: chlorine; S: sulfur; Na: sodium; Mg: magnesium; K: potassium; N: nitrogen; KeV (x-axis): energy of the X-rays; y-axis: peak intensity (counts); Wt (%): percentage weight; F29: \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain F29\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTime (s)\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 3: Total ion chromatogram of the bioflocculant from\u003c/b\u003e \u003cb\u003ePseudomonas aeruginosa\u003c/b\u003e \u003cb\u003eF29\u003c/b\u003e\u003c/p\u003e \u003cp\u003eKey: RT: retention time; s: seconds; glu: glucose; xyl: xylose; man: mannose; rha: rhamnose: rib: D-ribose; gal: galactose; ara; arabinose; ino; inositol\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe physical characteristics, the composition and relative distribution of the chemical elements, and the functional groups of the bioflocculant that was produced from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain F29, were determined, using Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM) coupled to energy dispersive X-ray spectroscopy (EDS or EDX), high-performance liquid chromatography (HPLC) coupled to mass spectrometry (MS), and the phenol-sulfuric acid method.\u003c/p\u003e \u003cp\u003eFourier transform infrared (FTIR) spectroscopy of the bioflocculant from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain F29, revealed the presence of certain functional groups corresponding to the given absorption peaks. The capacity of a bioflocculant to exert its coagulative-flocculative functions, the ambit of its resistance to denaturation, and its amenability to different spatial configurations, depend on the type of the functional group it possesses (Rao \u003cem\u003eet al\u003c/em\u003e., 2013). In fact, Okaiyeto \u003cem\u003eet al\u003c/em\u003e. (2015) reported that the functional groups in bioflocculants, provide dedicated areas for the chemical interactivity of positively charged ions and colloids suspended in a liquid medium.\u003c/p\u003e \u003cp\u003eThe presence of a carboxylic acid/carboxylate compound and/or a concentrated aromatic alcohol, in the bioflocculant from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain F29, was alluded to from the strong absorption peak at 3241.10 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; this peak was generated from the stretching of O-H (hydroxyl) functional groups. This finding was in partial agreement with the research by Okaiyeto \u003cem\u003eet al\u003c/em\u003e. (2013) on a purified bioflocculant, who reported that the peak at 3412 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, was attributed to a hydroxyl group. This strong absorption peak at 3241.10 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was also attributed to the presence of amino groups, and was within the protein amide A band of 3500\u0026ndash;3300cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Ji \u003cem\u003eet al\u003c/em\u003e., 2020).\u003c/p\u003e \u003cp\u003eThe medium absorption peak at 1635.05 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in the bioflocculant from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain F29, was attributed to C\u0026thinsp;=\u0026thinsp;C alkene bending in compounds, whose two alkyl groups are oriented on the same side of the alkene (cis isomer). In a study by Hurairah \u003cem\u003eet al\u003c/em\u003e. (2023) on coagulation-flocculation, the seed peel extract from \u003cem\u003eArchidendra jiringa\u003c/em\u003e, that was found to be effective as an aid in the elution of lead from man-made residual water, was noted to possibly contain alkene compounds (or aromatic compounds), as indicated by the weak FTIR bands at 3019 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 3001 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn the spectrum of the bioflocculant from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain F29, the absorption at 1635.05 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, also indicated the presence of carbonyl (C\u0026thinsp;=\u0026thinsp;O) groups, typically within the range of 1900\u0026ndash;1600 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Smith, 2017), that revealed the presence of carbohydrate sugars (Roberts and Caserio, 1977) in the bioflocculant. Together with the second functional group commonly found in carbohydrates, and detected at 3412 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, viz, the hydroxyl group, the presence of carbohydrates in the bioflocculant from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain F29, was confirmed.\u003c/p\u003e \u003cp\u003eIn another light, the carbonyl (C\u0026thinsp;=\u0026thinsp;O) groups indicated by the medium absorption at 1635.05 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, also fell within the amide I band, documented as typical for peptides or protein macromolecules, while the strong absorption peak, observed at 599.55 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, fell into another typical range for peptides or proteins, the amide VI band, that comprises \u0026ldquo;out-of-plane\u0026rdquo; bending of carbonyl (C\u0026thinsp;=\u0026thinsp;O) groups in amide/peptide bonds (Krimm and Bandekar, 1986; Bandekar, 1992; Li, 2016). Additionally, Li (2016), documented that the amide band I is the most readily detected, out of the nine amide bands typically characteristic for a protein macromolecule, and thus can be used to study its composition and surroundings. Yatsyna \u003cem\u003eet al\u003c/em\u003e. (2016) stated, in their research, that amide IV, V and VI bands, can be accurately used to identify the shape of the amide functional groups, and the framework of bound atoms present in a protein macromolecule. In addition, Yatsyna \u003cem\u003eet al\u003c/em\u003e. (2016) reported that, amide IV, V and VI bands, also reveal that, amide bonds, as may occur in peptides whose building blocks are arranged in rings, possess amino acid molecules oriented on the same side of the amide/peptide bond (\u0026ldquo;cis-conformation\u0026rdquo;). In addition, amide A, I, II and III bands, were documented, by Ji \u003cem\u003eet al\u003c/em\u003e. (2020), as the amide bands used to ascertain the construction of a protein macromolecule. The presence of a protein (or polypeptide) component in the bioflocculant from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain F29, was therefore, reasonably established, by the amide I, VI and A bands that were detected in it. Additionally, Wang \u003cem\u003eet al\u003c/em\u003e (2021) reported that amidation boosted the flocculation of superfine pyrite. Therefore, the inherent amide bonds present in the bioflocculant from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain F29, were more than likely, partly responsible for its flocculation bioactivity.\u003c/p\u003e \u003cp\u003eThe absorption at 1635.05 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e fell within the 1660\u0026ndash;1500 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e range that is typical for vibrations from saturated nitro compounds. From an extensive search of documented studies, it would appear that there is no information on the presence of nitro compounds in either conventional flocculants or bioflocculants. While research such as that by Smith (2020) may have suggested the inherent potential explosivity of the bioflocculant from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain F29, the study by Noriega \u003cem\u003eet al\u003c/em\u003e. (2022) on the vast array of the activities of nitro compounds, suggested that the bioflocculant from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain F29, may exhibit antimicrobial, antiparasitic and antitumour bioactivities in addition to certain other activities. Nonetheless, Noriega \u003cem\u003eet al\u003c/em\u003e. (2022) also stated that the sum total of the bioactivities of the nitro moiety, whether advantageous or harmful, is completely contingent on its chemical reduction (by receiving as many as 6 electrons) to the amide compound. As stated previously, the amide condensation reaction has been observed to enhance flocculation (Wang \u003cem\u003eet al\u003c/em\u003e., 2021). This amide bond, characterizes the amide/peptide moiety. Furthermore, Kurniawan \u003cem\u003eet al\u003c/em\u003e. (2022) reviewed research that called attention to certain mechanisms by which moieties such as the amide moiety, subserve the coagulation-flocculation brought about by the Moringa plant. Therefore, it is inferable that the nitro moiety may display flocculation via the amide compounds obtained from the nitro-reduction pathway it undergoes, and that the net flocculation exhibited by a nitro compound-containing polymeric flocculant, such as the bioflocculant from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain F29, would be partly due to this nitro moiety. Yet again, research such as by Kovacic and Somanathan (2014) and Gupta and Ronen (2024) have reported some nitro compounds as harmful contaminants in the environment and in wastewaters, in need of remediation. Ma \u003cem\u003eet al\u003c/em\u003e. (2020) observed that environmental contaminants such as chromium and nitrobenzene, were flocculated out of the experimental aqueous mixtures of these contaminants that they had created, via activated biomass. Consequently, it would be worthwhile to observe if the flocculation triggered by a nitro compound-containing flocculant such as the bioflocculant from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain F29, is effective in remediating toxic nitro compounds, from wastewater. If effective, it may be quite instructive to determine how this flocculation compares with effective, non-nitro compound-containing flocculants in common use.\u003c/p\u003e \u003cp\u003eThe strong FTIR absorption peak at 599.55 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was also within the 670\u0026ndash;550 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e range, typical for stretching vibrations from magnesium oxide bonds (Agyapong \u003cem\u003eet al\u003c/em\u003e., 2023). Although studies such as by Liu \u003cem\u003eet al\u003c/em\u003e. (2023) and by Das \u003cem\u003eet al\u003c/em\u003e., (2023), reported on the flocculation property of magnesium oxide, the study by Sofi \u003cem\u003eet al\u003c/em\u003e. (2021) that reported on some other properties of magnesium oxide, may give better insight into its presence in the bioflocculant produced from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain F29. It may be that, as reported by Sofi \u003cem\u003eet al\u003c/em\u003e. (2021), for pigments and zero-resistance materials, magnesium oxide enhanced and improved the properties and performance of the bioflocculant produced from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain F29. These enhancements and improvements would no doubt, have boosted the overall properties of the bioflocculant produced from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain F29, and not only bioflocculation.\u003c/p\u003e \u003cp\u003eThe strong FTIR absorption peak at 599.55 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, fell within the 600\u0026ndash;590 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e range for sulfones (Socrates, 2004). There seems to be an apparent lack of documentation on the existence of naturally-occurring sulfonated bioflocculants. However, lignosulfonates, that are bye products in the pulp and paper-making industry, have been observed to possess flocculation properties (Anukam \u003cem\u003eet al\u003c/em\u003e., 2021; Chan \u003cem\u003eet al\u003c/em\u003e., 2021). As such, lignosulfonates are strictly-speaking, man-made compounds. Tang \u003cem\u003eet al\u003c/em\u003e. (2020), functionalized a bioflocculant, chitosan, with a sulfone. This synthetic, chemically modified, sulfone-containing bioflocculant, CS-g-P(AM-AMPS), was observed to effectively remediate heavy metals from wastewater, by the binding of the sulfone moiety to the heavy metal ions, to form a stable ring structure, in addition to heavy metal-binding to other moieties, and simultaneous sedimentation (Tang \u003cem\u003eet al\u003c/em\u003e., 2020). The presence of sulfones in the bioflocculant produced from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain F29, was therefore, very likely, contributory to its overall flocculation capacity.\u003c/p\u003e \u003cp\u003eThe strong absorption peak at 599.55 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was additionally, attributed to the presence of halogen compounds (carbon-iodide, carbon-bromide or carbon-chloride bonds) (Aldrich, 2019), in the bioflocculant from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain F29. Maliehe \u003cem\u003eet al\u003c/em\u003e. (2016), also reported the presence of halo compounds with carbon-chloride bonds at the 599.14 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e absorption peak, and carbon-iodide bonds at the 509.5 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e absorption peak in their research on the bioflocculant TMT\u003csup\u003e1\u003c/sup\u003e. Bod\u0026icirc;rlău \u003cem\u003eet al\u003c/em\u003e. (2009) reported the stretching from a carbon-chloride (C-Cl) bond at 700\u0026ndash;600 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, in their research on amalgams made from two categories of wood, Tripathy and De (2006) described polyamines as polymeric compounds that possess flocculating activity and are formed by a condensation reaction between halogenated compounds and amines. Therefore, halo compounds can be said to possess inherent coagulating-bioflocculating properties and their presence in the bioflocculant produced from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain F29, may be partly responsible for its flocculation capacity.\u003c/p\u003e \u003cp\u003eThe strong FTIR absorption peak at 599.55 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was within the 610\u0026ndash;390 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e range for P-Cl bonds (Socrates, 2004). However, ascertaining the exact type of phosphorus-chlorine compounds in the bioflocculant from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain F29, fell outside the scope of this study. Roshchin and Molodkina (1977) documented that phosphorus chlorides particularly the oxychloride, trichloride and pentachloride, are noxious substances that are considered occupational dangers. However, Kudzin \u003cem\u003eet al\u003c/em\u003e. (2021) used gaseous forms of phosphorus trichloride to formulate a novel antimicrobial compound. Additionally, both phosphorus trichloride and phosphorus pentachloride, have been reported as chlorinating substances (Dillon \u003cem\u003eet al\u003c/em\u003e., 1976; Xiao and Han, 2019). Zhang \u003cem\u003eet al\u003c/em\u003e. (2018) reported that crystalline ferric chloride is a moderately strong electron pair-acceptor that is used as both catalytic and chlorinating substances, in the manufacture of carbon-based compounds. Nonetheless, research such as by Ettaloui \u003cem\u003eet al\u003c/em\u003e. (2021) has reported on the coagulation-flocculation property of ferric chloride. Therefore, it may be that the phosphorus chlorides present in the bioflocculant from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain F29, also possess coagulation-flocculation properties, that synergistically contribute to its total flocculation capacity. Although a phosphorus-chlorine (P-Cl) bond on FTIR, can also emanate from a phosphoryl chloride, the absence of the phosphoryl moiety in the bioflocculant from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain F29 bond, negated the possibility of the presence of a phosphoryl chloride.\u003c/p\u003e \u003cp\u003eThe FTIR of 418.53 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was from the vibrations of a metal-chloride (M-Cl) bond (Socrates, 2004), in the bioflocculant from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain F29. Three metals are naturally present in the bioflocculant, viz sodium potassium, and magnesium, and any, or all of them, could have formed the corresponding chloride salt. The study by Zhao and Zhang (2017) reported on the use of the chlorides of sodium, potassium and magnesium as aids to maximize the medium for the production of a bioflocculant from \u003cem\u003eBacillus subtilis\u003c/em\u003e. However, the use of sodium chloride, potassium chloride and magnesium chloride either as flocculants or flocculant aids have been well documented (P\u0026eacute;rez \u003cem\u003eet al\u003c/em\u003e., 2021; Zhou \u003cem\u003eet al\u003c/em\u003e., 2022; Ozkan and Yekeler, 2004). Therefore, any of these chlorides, if present in the bioflocculant from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain F29 would likely have contributed to its flocculation property.\u003c/p\u003e \u003cp\u003eThe bioflocculant from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain F29 also contained polysulfides. This was alluded to, by the peak at 474.39 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, that was attributed to S‒S disulfide bond stretching. Asmare \u003cem\u003eet al\u003c/em\u003e. (2021) used the coagulation-flocculation induced by a mixture of calcium polysulfide and ferrous sulphate, for remediating waste water from a leather-making industry. They discovered that although the use of a high concentration of calcium polysulfide (4% v/v), mixed with ferrous sulphate, did not eliminate the chemical oxygen demand entirely, it still resulted in a relatively high reduction (86.13%) in waste, and achieved all other indices of proper wastewater treatment. This finding highlights the relative effectiveness of polysulfide compounds in the coagulation-flocculation process.\u003c/p\u003e \u003cp\u003eHigh-performance liquid chromatography (HPLC) and mass spectrometry (MS) analyses, revealed different monosaccharides, indicating that the bioflocculant produced from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain F29 possessed a heteropolysaccharide component. Polysaccharides have been observed to possess bioflocculating properties (Salehizadeh \u003cem\u003eet al\u003c/em\u003e., 2018). In a research study conducted by Ma \u003cem\u003eet al\u003c/em\u003e. (2022), the heteropolysaccharide bioflocculant BP50-2, produced from discarded banana skin, demonstrated effective agglomerating activity. Therefore, the polysaccharide component of the bioflocculant produced from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain F29 would more likely have contributed to the net coagulation-flocculation capacity of the bioflocculant.\u003c/p\u003e \u003cp\u003eScanning electron microscopy of the bioflocculant produced from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain F29, revealed clumped and flaky surfaces. Xiong \u003cem\u003eet al\u003c/em\u003e. (2010) reported that the nature of the surface of a bioflocculant holds immense significance with regard to its flocculating capacity. The topographic layout of a bioflocculant, thus determines its potency or lack thereof (Tsilo \u003cem\u003eet al\u003c/em\u003e., 2022). EDX of the bioflocculant produced from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain F29, revealed a relative abundance of chlorine (56.00%) and carbon (20.50%). Sodium (12.50%) and oxygen (4.00%) were next in abundance. Phosphorus (3.00%) and sulfur (2.43%) were also present in the bioflocculant produced from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain F29, with the least abundant elements being magnesium (1.06%), potassium (0.32%) and nitrogen (0.30%). The carbon and oxygen compositions hinted at the presence of polysaccharides (Awuchi and Amagwula, 2021) and proteins (Biswas, 2020) in the bioflocculant produced from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain F29. The bioflocculant in the research conducted by Tsilo \u003cem\u003eet al\u003c/em\u003e. (2022), had a much higher oxygen (43.76%) and phosphorus (14.44%) content, and a much lower chlorine content (0.31%), when compared with the same elements in the bioflocculant produced from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain F29. The potassium content (0.34%) was almost the same as that present in the bioflocculant produced from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain F29 (0.32%). Calcium was absent, in the bioflocculant produced from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain F29, but was relatively abundant (20.35%) in the bioflocculant studied by Tsilo \u003cem\u003eet al\u003c/em\u003e. (2022). These differences in bioflocculant composition were probably due to species differences and the type and ratio of the constituents of the bacterial medium that was used in bioflocculant production.\u003c/p\u003e \u003cp\u003eSome metal cations have been observed to aid in floc-formation (Kuriyama \u003cem\u003eet al\u003c/em\u003e., 1991; Tawila \u003cem\u003eet al\u003c/em\u003e., 2019). Sodium alginate, a compound found in nature, was observed to possess good flocculating activity during sewage treatment, in research conducted by Tian \u003cem\u003eet al\u003c/em\u003e. (2020), while sodium polyacrylate was used to aggregate tiny hematite particles and thereby cause them to drift away from the intermixed quartz particles (Cheng \u003cem\u003eet al\u003c/em\u003e., 2022). Ozkan and Yekeler (2004), in their research to determine the floc-forming properties of strontium sulphate (\u0026ldquo;celestite\u0026rdquo;), by utilising two classes of flocculants, discovered that magnesium 2\u0026thinsp;+\u0026thinsp;ions from magnesium chloride, worked best on the strontium sulphate, than did calcium 2+ (from calcium chloride) and aluminium 3+ (from aluminium chloride). Also, in research conducted by Hejazi (2013), potassium compounds were combined with sodium dodecyl sulphate, to trigger floc formation in a mixture that contained megestrol acetate particles. The non-metal, chlorine, was utilised as an aid in flocculation, in addition to its use as a deodoriser and a disinfectant, in water purification experiments (Weston, 1924). Ou \u003cem\u003eet al\u003c/em\u003e. (2016), in an experiment conducted, observed that the chlorine-containing compound, sodium chloride (common salt) displayed good floc-forming activity against small-grained particles of industrial kaolinite, and this activity was directly proportional to the quantity per unit volume, of both flocculant and industrial kaolinite. Therefore, it may be deduced that the presence of the metals, sodium, potassium, magnesium, and the non-metal, chlorine, in the bioflocculant produced from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain F29, would have contributed, to some significant degree, to its bioflocculating activity. The non-metal elements, phosphorus, sulfur and nitrogen have been stated as constituents of certain carbohydrates (Gangasani \u003cem\u003eet al\u003c/em\u003e., 2022), and the bioflocculant produced from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain F29, possessed these elements in varying proportions. Nevertheless, these elements- phosphorus, nitrogen and sulfur - have been described by research such as by Kakade \u003cem\u003eet al\u003c/em\u003e. (2022), as lesser constituents of carbohydrates, and occur in those carbohydrates that have been modified. On the other hand, phosphorus, sulfur and nitrogen have also been documented as constituents of proteins (Biswas, 2020). Although, nitrogen was described by Biswas (2020) as a main constituent of a protein macromolecule, with phosphorus and sulfur present in relatively less proportions, the bioflocculant from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain F29 possessed relatively more abundance of phosphorus (3.00%) and sulfur (2.43%), than nitrogen (0.30%). In this manner, the protein component of the bioflocculant from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain F29 could more likely be described as atypical.\u003c/p\u003e \u003cp\u003eThe existence of a protein/proteins were alluded to, from the occurrence of phosphorus, nitrogen, sulfur, and amide/peptide bonds within amide I, VI and A bands (Li, 2016; Yatsyna \u003cem\u003eet al\u003c/em\u003e., 2016; Ji \u003cem\u003eet al\u003c/em\u003e., 2020; Kakade \u003cem\u003eet al\u003c/em\u003e., 2022), in the bioflocculant from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain F29. However, the more specific differentiation between a protein and that of a polypeptide, in the bioflocculant, could not be exactly ascertained. This was because the use of a more diagnostic method, such as x-ray crystallography, that can provide a stereographic view of a protein (Alberts \u003cem\u003eet al\u003c/em\u003e., 2002), fell outside the scope of this study. Additionally, the discovery that more than 75% of the bioflocculant from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain F29, consisted of carbon and chlorine (with chlorine accounting for more than half of the total amount of elements present), strongly indicated that the bioflocculant, was largely an organochlorine compound (organochloride). The sodium, potassium and magnesium ions, accounted for the total metal composition of the bioflocculant from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain F29. This metal composition was 13.32%, with sodium ion accounting for 90% of all metals present. Therefore, with the confirmed presence of metals, carbohydrate sugars, proteins and organochlorines, in the bioflocculant from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain F29, it was deduced that this bioflocculant was a metal-containing polymeric compound composed mainly of carbohydrates, proteins/polypeptides and organochlorines; possibly a \u003cb\u003e\u0026ldquo;\u003c/b\u003emetalloglyco-protein/polypeptide-organochlorine\u0026rdquo; bioflocculant.\u003c/p\u003e \u003cp\u003eLiterature is rife with documentation on polyfunctional compounds. However, it was particularly intriguing and puzzling to discover multiple moieties associated with a variety of well-documented bioactivities, and chemical compounds with diverse applications, packed into one macromolecule namely, the bioflocculant from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain F29. It would appear that this bioflocculant was produced by \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain F29, to execute a wide range of functions. The incorporation, into the bioflocculant, of multiple functional groups that provided dedicated reactive zones that determined its chemical reactiveness, characteristics and biological functions (Ertl \u003cem\u003eet al\u003c/em\u003e., 2020), was probably therefore, a response of \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain F29, to varied stimuli it had received in its environment. Further studies on this polyfunctional bioflocculant from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain F29, could provide valuable insight into its range of chemical properties, bioactivities and industrial applications.\u003c/p\u003e \u003cp\u003eAn extensive search of documented research, failed to reveal any report of either metal-containing \u0026ldquo;glycoprotein/polypeptide-organochlorines\u0026rdquo; (\u0026ldquo;metallo- glycoprotein/polypeptide-organochlorines\u0026rdquo;), or \u0026ldquo;glycoprotein/polypeptide-organochlorines\u0026rdquo;. There seemed to be an absence of documentation on the discovery of these categories of glycoconjugates. There was a report by Fujimori \u003cem\u003eet al\u003c/em\u003e. (2012) on \u003cem\u003ePseudomonas\u003c/em\u003e species producing the vaporescent organochloride compound, chloromethane. However, an extensive search of documented studies did not reveal any report of an organochloride bioflocculant, a \u0026ldquo;glyco-protein/polypeptide-organochlorine\u0026rdquo; bioflocculant, or a \u0026ldquo;metallo- glycoprotein/polypeptide-organochlorine\u0026rdquo; from \u003cem\u003ePseudomonas\u003c/em\u003e species or any other microorganism. Additionally, there was also an apparent lack of documentation on the natural occurrence of saturated nitro compounds, sulfones, polysulfides, phosphorus chlorine compounds, magnesium oxide and metal chlorides, in a bioflocculant. From available documentation, this is the first report of a polyfunctional \u0026ldquo;metalloglyco-protein/polypeptide organochlorine\u0026rdquo; bioflocculant that naturally contains saturated nitro compounds, sulfones, polysulfides. phosphorus chlorine compounds, magnesium oxide and metal chlorides; and of a bioflocculant produced from porcine faeces in Africa.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eA bioflocculant produced from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain F29 (accession number OQ734844) was characterised using investigative procedures. These procedures revealed that the bioflocculant was possibly a novel glycoconjugate, a \u0026ldquo;metallo-glycoprotein/polypeptide-organochloride\u0026rdquo;, that contained metals and naturally-occurring saturated nitro compounds, sulfones, polysulfides, phosphorus chlorine compounds, magnesium oxide and metal chlorides. From available documentation, this is the first report of a polyfunctional \u0026ldquo;metallo-glycoprotein/polypeptide-organochlorine\u0026rdquo; bioflocculant that naturally contains saturated nitro compounds, sulfones, polysulfides, phosphorus chlorine compounds, magnesium oxide, and metal chlorides; and of a bioflocculant produced from porcine faeces in Africa. Further research on this bioflocculant should prove helpful in establishing the ambit of its properties, activities and applications.\u003c/p\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAUTHORS CONTRIBUTION\u003c/h2\u003e \u003cp\u003eI, Ikechukwu Kenneth M. Okorie (corresponding author), carried out all the work in this study as my Masters (MSc.) research project. Professor Adeniyi A. Ogunjobi supervised my Master\u0026rsquo;s research study.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eSOURCE OF FUNDING\u003c/h2\u003e \u003cp\u003eThis study was self-funded by me, Ikechukwu. Kenneth M. Okorie. No external funding was received.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCOMPETING INTEREST\u003c/h2\u003e \u003cp\u003eBoth authors declare no financial or nonfinancial competing interest.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003ch2\u003eCONFLICTING INTEREST\u003c/h2\u003e \u003c/p\u003e\u003cp\u003eBoth authors declare no conflicting interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eACKNOWLEDGEMENTS\u003c/h2\u003e \u003cp\u003eThe corresponding author would like to thank Professor Adeniyi A. Ogunjobi for supervising this study. Many thanks also go to Professor Tonye G. Okorie for editing and proofreading the article and to Dr. Augustine Akpoka for his useful suggestions. Mr. Gabriel O. Bamidele helped greatly with the laboratory work.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbu Tawila, Z. M., Ismail, S., Dadrasnia, A. and Usman, M. M. 2018. Production and characterization of a bioflocculant produced by Bacillus salmalaya 139SI-7 and its applications in wastewater treatment. \u003cem\u003eMolecules\u003c/em\u003e 23. 10: 2689. http://dx.doi.org/10.3390/molecules23102689\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAgilent Technologies. 1999. \u003cem\u003eAgilent 1100 Series High Value System:User\u0026rsquo;s Guide\u003c/em\u003e. 11/99 ed. Waldbronn: Agilent Technologies.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAgyapong, P. O., Gikunoo, E., Arthur, E. K., Anang, D. A., Agyemang, F.O., Foli, G. and Baah, D. S. 2023. 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Bioflocculants in anaerobic membrane bioreactors: A review on membrane fouling mitigation strategies. \u003cem\u003eChemical Engineering Journal\u003c/em\u003e : 150260. https://doi.org/10.1016/j.cej.2024.150260\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYatsyna, V., Bakker, D. J., Feifel, R., Rijs, A. M. and Zhaunerchyk, V. 2016. Far-infrared amide IV-VI spectroscopy of isolated 2-and 4-Methylacetanilide. \u003cem\u003eThe Journal of chemical physics\u003c/em\u003e 145. 10. http://dx.doi.org/10.1063/1.4962360\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZoaka, M. H., Faruk, A. U., Ibn Abbas, M., Buba, F. M., Adamu, A. and Ismail, H. Y. 2025. 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Optimization of microbial flocculant-producing medium for Bacillus subtilis. \u003cem\u003eIndian journal of microbiology\u003c/em\u003e 57. 1: 83\u0026ndash;91. https://doi.org/10.1007/s12088-016-0631-3\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhong, C., Cao, G., Rong, K., Xia, Z., Peng, T., Chen, H. and Zhou, J. 2018. Characterization of a microbial polysaccharide-based bioflocculant and its anti-inflammatory and pro-coagulant activity. \u003cem\u003eColloids and Surfaces B: Biointerfaces\u003c/em\u003e 161: 636\u0026ndash;644. http://dx.doi.org/10.1016/j.colsurfb.2017.11.042\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou, X., Liu, Z., Wang, W., Miao, Y., Gu, L., Li, Y., Liu, X., Jiang, L., Hou, J. and Jiang, Z. 2022. NaCl induces flocculation and lipid oxidation of soybean oil body emulsions recovered by neutral aqueous extraction. \u003cem\u003eJournal of the Science of Food and Agriculture\u003c/em\u003e 102. 9: 3752\u0026ndash;3761. https://doi.org/10.1002/jsfa.11723\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"University of Ibadan","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"characterisation, novel “metalloglyco-protein/polypeptide organochlorine” bioflocculant, polyfunctional, Pseudomonas aeruginosa strain F29, antibiotic resistance","lastPublishedDoi":"10.21203/rs.3.rs-7453669/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7453669/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAntibiotic resistance has reached universal proportions, and the discovery of effective alternatives to the common antibiotics currently used, could aid in solving this problem. The aim of this study was to characterise a bioflocculant produced from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain F29, accession number OQ734844, that exhibited effective antibacterial activity against two antibiotic resistant bacteria, viz, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e SO183, and an identified strain of \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, in another study. FTIR detected saturated nitro compounds, sulfones, polysulfides, phosphorus-chlorine bonds, magnesium oxide bonds and metal-chloride bonds. FTIR also detected the following functional groups: carboxyl, amide/peptide, aromatic alcohol, alkene, and halo. SEM showed a clumped and flaky bioflocculant surface, while EDX detected chlorine (56.00%), carbon (20.50%), sodium (12.50%), oxygen (4.00%), phosphorus (3.00%), sulfur (2.43%) magnesium (1.06%), potassium (0.32%) and nitrogen (0.30%). HPLC and MS detected varied peaks of glucose, galactose, inositol and mannose, D-ribose, arabinose, rhamnose and xylose. The phenol sulfuric acid method calculated the concentration of these sugars as 0.0059 g/L. The bioflocculant is a polymeric compound composed mainly of carbohydrates, proteins/polypeptides and organochlorines, possibly a novel \u0026ldquo;metalloglyco-protein/polypeptide organochlorine\u0026rdquo; bioflocculant. The presence of the metals: sodium, potassium and magnesium; the non-metals: phosphorus, sulfur and nitrogen; and multiple moieties, likely contributed to the antibacterial activity of the bioflocculant produced from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain F29. From available documentation, this is the first report of a polyfunctional \u0026ldquo;metalloglyco-protein/polypeptide organochlorine\u0026rdquo; bioflocculant, that naturally contains saturated nitro compounds, sulfones, polysulfides, phosphorus chlorine compounds, magnesium oxide and metal chlorides; and of a bioflocculant produced from porcine faeces in Africa.\u003c/p\u003e","manuscriptTitle":"Characterization of a novel polyfunctional “metalloglyco-protein/polypeptide-organochlorine” bioflocculant containing saturated nitro compounds, sulfones, polysulfides, phosphorus chlorine compounds, magnesium oxide, and metal chlorides, produced from Pseudomonas aeruginosa strain F29, isolated from porcine faeces in Nigeria","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2026-01-05 01:31:02","doi":"10.21203/rs.3.rs-7453669/v2","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}},{"code":1,"date":"2025-08-26 10:31:59","doi":"10.21203/rs.3.rs-7453669/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ad010486-0919-4cbc-93a0-0d3950294f44","owner":[],"postedDate":"January 5th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":59490797,"name":"Biopolymers"},{"id":59490798,"name":"Chemical Biology"},{"id":59490799,"name":"Biological Chemistry"},{"id":59490800,"name":"Drug Discovery, Design, \u0026 Development"}],"tags":[],"updatedAt":"2025-08-26T10:31:59+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-05 01:31:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v2","identity":"rs-7453669","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7453669","identity":"rs-7453669","version":["v2"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}