Characterisation of a novel metal-containing “glyco-protein/polypeptide-organochlorine” bioflocculant produced from Pseudomonas aeruginosa strain F29 isolated from pig fecal matter collected from a mixed animal farm in Ibadan, Oyo State, Nigeria

doi:10.21203/rs.3.rs-5167346/v1

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Characterisation of a novel metal-containing “glyco-protein/polypeptide-organochlorine” bioflocculant produced from Pseudomonas aeruginosa strain F29 isolated from pig fecal matter collected from a mixed animal farm in Ibadan, Oyo State, Nigeria

https://doi.org/10.21203/rs.3.rs-5167346/v1

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Antibiotic resistance has reached global proportions, and the discovery of effective alternatives to the common antibiotics in current use, 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 possessed 69% flocculating activity, and that had been observed to demonstrate biocontrol activity against Staphylococcus aureus SO183 at concentrations of 0.090 g/L and 0.150 g/L, and against identified Pseudomonas aeruginosa, at a concentration of 0.150 g/L. Pseudomonas aeruginosa strain F29, was isolated from pig fecal matter collected from a mixed animal farm in Ibadan, Nigeria via the pour plate method, identified through biochemical tests and confirmed through molecular studies. Fourier transform infrared spectroscopy (FTIR) of the bioflocculant, revealed the presence of carboxyl, amide/peptide, aromatic alcohol, alkene, halo and polysulfide functional groups. Scanning electron microscopy (SEM) revealed a clumped and flaky bioflocculant surface, while energy dispersive X-ray spectroscopy (EDX) revealed the presence of chlorine (56.00%), carbon (20.50%), sodium (12.50%), oxygen (4.00%), phosphorus (3.00%), sulphur (2.43%) magnesium (1.06%), potassium (0.32%) and nitrogen (0.30%). High performance liquid chromatography (HPLC) and mass spectrometry (MS) revealed that the bioflocculant possessed varied peaks of glucose, galactose, inositol and mannose, D-ribose, arabinose, rhamnose and xylose. The phenol sulfuric acid method revealed that the concentration of these sugars was 0.0059 g/L. The bioflocculant was a metal-containing polymeric compound composed mainly of carbohydrates, proteins/polypeptides and organochlorines, possibly a metal-containing “glyco-protein/polypeptide organochlorine.” From available documentation, this is the first report of this type of bioflocculant.

Drug Discovery, Design, & Development

bioflocculant

characterisation

Pseudomonas aeruginosa strain F29

antibiotic resistance

metal-containing “glyco-protein/polypeptide organochlorine”

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). It 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 cause growth suppression in them (WHO, 2021). Alternative antibacterial agents are therefore being investigated. It has been reported that bioflocculants possess not only antibacterial activity but also other antimicrobial properties (Abu Tawila et al., 2018).

Bioflocculants are microbial extracellular polymeric substances produced as secondary metabolites, that have the capacity to cause flocculation in a given medium (Dih et al., 2019). Bioflocculants are produced as a result of cell breakdown and cell secretion by bacteria, fungi and algae, among other microbes (Alias et al., 2022). Bioflocculants, also called biocoagulants (Kurniawan et al., 2022), aggregate particles and remove these particles from the liquid medium in which they were formerly suspended. Broadly, flocculants have the capacity to clump small-sized substances suspended in a medium; these clumped substances are called flocs (Wang et al., 2022), and sediment over time. The specific mechanism of action by which bioflocculants operate is still unknown. A bioflocculant may exert its action by constructing a bridge between particles of the medium and its own molecules; they may also kick-start a neutralisation of charges present in the medium (Lai et al., 2018). Other hypotheses exist, nevertheless, the antibacterial activity of bioflocculants may be deduced from their inherent capacity to form flocs, that later sediment. The bacterial cell wall components may therefore be clumped together by the bioflocculant, and this action would more likely, produce tears in the bacterial cell membrane. The compromise in bacterial cell membrane integrity, if progressive enough, could inadvertently lead to an influx of noxious substances from the surroundings, a destabilisation of the internal milieu of the bacterial cell, and resultant bacterial cell lysis and death.

Bioflocculants are composed of cellulose, proteins, nucleic acids, lipids, glycoproteins and polysaccharides, which render them readily biodegradable and less injurious to the environment (Alias et al., 2022). Bioflocculants are polymeric substances produced during the bacterial cell growth phase (Tsilo et al., 2022), and each monomer contributes to the overall characteristics and mechanisms of action of the bioflocculant. Examples of naturally- occurring flocculants include chitosan and tannins (Othmani et al., 2020). Another example of a naturally-occurring flocculant is cellulose (Fauzani et al., 2021), and it is produced by plants. Nevertheless, there are different kinds of bacterial species that also produce cellulose (here known as bacterial or microbial cellulose), that provides mechanical support (Choi et al., 2022), in addition to possessing bioflocculant characteristics, such as that produced by plants. Bioflocculants made mainly of proteins, are easily affected by thermal fluctuations, because proteins naturally denature at high extremes of temperature (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 the discipline of molecular biomonitoring (Littlefair et al., 2022). An increase in lipid (glycerol and fatty acid) saturation was observed to start off the aggregation of cells—the coagulation-flocculation process—in cell membrane experiments on Saccharomyces cerevisiae (Degreif et al., 2017). This would imply that bioflocculants containing lipids with single bonds between the constituent fatty acid molecules, potentially possess greater bioflocculating activity than those containing lipids with double bonds. Glycoproteins are a complex of carbohydrates and proteins. 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 assemblage/coagulation-flocculation (Michaels, 1954; Gao et al. 2006). Bioflocculants can be used as antibiotics, anti-algal agents and antiviral drugs (Abu Tawila et al., 2018). In the discipline of immunology, bioflocculants can be employed for infectious disease and immune disorder diagnoses via flocculation tests (Sadeghalvad and Rezhaei, 2022). Bioflocculants can also be used to induce stabilisation, viscosification, and emulsification in the food industry and in drug manufacturing (Zhong et al., 2018).

Glycoconjugates are a diverse group of compounds possessing carbohydrate moieties that are joined to other types of chemicals through covalent bonds (Sharon, 1986). These other chemicals are usually relatively more abundant within the compound, than the carbohydrate moiety. The carbohydrate component of a glycoconjugate is termed a “glycan” or a “saccharide”, and is composed of many monosaccharides covalently joined via glycosidic bonds, to form oligosaccharides (where there are less than 12 monosaccharides) or polysaccharides (where there are 12 or more monosaccharides (Lebrilla et al. 2022). Indeed, carbohydrates almost never exist as simple sugars, in the natural world (Lebrilla et al. 2022). Polysaccharides have been observed to possess bioflocculating activity that is probably due to a loss or gain of electrons by the atoms and molecules present in the bioflocculating system (“ionisation”), which in turn, triggers a pull between the polysaccharide and the flocs, in order to cancel out the charges on the flocs (“charge neutralisation”) and aggregate them (Li et al. 2014; Vishali and Karthikeyan, 2018; Alazaiza et al. 2022). Another mechanism of polysaccharide bioflocculating activity, “bridging”, is the linking together of polysaccharide macromolecules with the solid flecks dispersed in the mixture being acted upon, resulting in the formation of clumps in the mixture (Hogg, 2013; Ma et al. 2022). In glycoconjugates, the macromolecules that are joined as conjugates of glycans are called “aglycones” and are frequently proteins and fatty compounds (Lebrilla et al. 2022). The chemical bonding of glycans and aglycones to form glycoconjugates occurs through the procedure of glycosylation that involves the bio-catalysed, non-polar linking of moieties present in both reactants (Crich, 2010; Reis et al., 2023). Glycoconjugates form a large part of both human and animal cells and are frequently located on their surfaces (Shivatare et al., 2022). They are also present in microorganisms such as bacteria (Hirai, 2022). Their functions include controlling the process of macroautophagy, managing the growth and development of blood vessels, overseeing the functioning of the immune system (Fahie and Zachara, 2016), and regulating stem cells in the nervous system (Yagi and Kato, 2017). Synthetic glycoconjugates have been employed in drug research and manufacturing (Kumbhar and Bhatia, 2023). They have also been used as vaccines to combat bacterial diseases (Mettu et al., 2020). While the carbohydrate component of glycoconjugates, in general have been utilised as markers for disease processes such as cancer (An et al., 2009), glycoconjugates, as a unit, have been used as biological indicators for the abuse of alcohol (Waszkiewicz et al., 2011). Glycoconjugates are commonly divided into so-called major groups, some of which are glycoproteins, lipopolysaccharides and glycolipids (Sharon, 1986). Nevertheless, there is still the possibility of discovering novel glycoconjugates. In 2021, the existence of glyco-ribonucleic acids” (“glycoRNAs”), was first reported (Flynn et al., 2021).

An organochlorine compound (also called a chlorinated hydrocarbon, an organic chlorine compound, or an organochloride) is a carbon-based compound in which a single chlorine atom or multiple chlorine atoms are covalently linked to one or more carbon atoms (Gupta, 2018; Euro Chlor, 2019), and influence the chemical properties of the compound (Gupta, 2018). Organic compounds are complexes that contain carbon particles molecularly bound to other types of chemical particles; however not all carbon-based complexes, such as cyanides, other chemicals and itself, has produced quite a large number of established complexes (Cleveland, 2015). Carbon is present in both living and non-living matter (Kempe, 1979; Prajapati et al. 2023). The versatility of, and the intrigue that is the element carbon, are demonstrated in its formation of two allotropes that are diametrically opposed in their degree of hardness; the exceptionally hard diamond and the reasonably soft graphite. Though the use of carbon cuts across many applications such as energy production (Cleveland, 2015), steelworks (Dutta and Choksi, 2021), and carbon-14 dating (Hajdas, 2009), certain gaseous carbon compounds have been implicated in the plight of global climate change (Shahzad, 2015). Chlorine is a non-metal element with nuclear charge number of 17 and a nucleon number of 35.45 (chlorine-35 isotope) (Kendrick, 2016), that exists as a chartreuse-coloured, acrid, caustic gas, at a temperature range of 20°C to 25°C, and as a golden-coloured liquid, when this gaseous form is subjected to high pressures or low temperatures (below 34°C) (ATSDR, 2014). The subcooled liquid chlorine is stored in durable, iron-carbon alloy barrels (ATSDR, 2014). In the natural world, chlorine does not exist as a free element (Peterson et al. 2007), but is found in both carbon-containing (“organic chlorine”) and non-carbon-containing (“inorganic chlorine”) complexes in the earth, the atmosphere, and the waters (Friend, 1990; Pszenny et al., 1993; Öberg, 1998; Khalil and Rasmussen, 1999; Keene et al. 1999; Winterton, 2000). The applications of chlorine and its compounds are found in both domestic and industrial spheres. These include their use as food additives and preservatives, as bleaching agents and decontaminants, for paper making, in drug manufacturing and synthetic polymer production and for water purification processes (Schmittinger, 2007; Tundo, 2012; Kendrick, 2018). With regards to water purification, the procedure of chlorine pre-oxidation (“pre-chlorination”), can be utilised to reduce both the quantity of impurities present (and thus any malodour) and the amount of chlorine required for the actual clumping of impurities (Weston, 1924). Therefore, chlorine can be used as an aid for coagulation-flocculation. Chlorine-containing compounds such as the chlorides of calcium, manganese and even sodium (common salt), have also been observed to possess good floc-forming activities when used as the sole coagulant-flocculant or in conjunction with other coagulant-flocculants (Kahn, 1958; Verma et al., 2012; Ou and Liang, 2016; Nakamura et al., 2020). The bonding of carbon and chlorine atoms produces an organic compound - an organochlorine - that has held immense importance to man, animals, plants and the world they inhabit. Organochlorines are a part of the much broader group of organohalogen compounds (Gribble, 1996), that are carbon-containing compounds, in which halogens (examples include iodine, chlorine, fluorine, bromine and astatine) are found (Euro Chlor, 2019). Organochlorines can be man-made or produced naturally. Examples of man-made organochloride compounds include aromatic organochlorines such as dichlorodiphenyltrichloroethane (DDT) and endosulfan; and aliphatic organochlorines such as vinyl chloride (Kaur et al., 2022). These compounds are not readily broken down in their surroundings and are therefore called persistent organic pollutants (Jayaraj et al., 2016); in this regard, they constitute an environmental hazard. Nevertheless, organochlorines have many applications that help humans: they are used in the manufacturing of pesticides such as DDT; plastics, such as vinyl chloride (Kaur et al., 2022); electronics, paper and drugs (Fauvarque, 1996). Although man-made organochlorines, particularly organochlorine pesticides, have been shown to be very injurious to the ecosystem (Jayaraj et al., 2016), natural organochlorines still exist and are produced from geological processes such as the eruption of volcanoes and the burning of forest trees (Gribble, 2010), and from life forms such as such as bacteria, white ants and vegetation (Moore, 2003).

A pure metal is a type of matter which, when in the compact form, is composed of an assemblage of atomic particles, each of which possesses centrally-placed, positively-, and neutrally-charged, subatomic particles, with surrounding, fixed and free negatively-charged subatomic particles (Delfino and Saccone, 2009). Metals also exist in the liquid form, at temperatures next to or within the range of 20°C and 25°C, an example of which is mercury (Guo et al., 2021). The inter-atomic linkages present in metals (“metallic bonds”) occur between the proton and the mobile electron subatomic particles in each atom, and determine the observable and quantifiable characteristics of any metal, such as its pliability, sheen, thermic energy conveyance, charged particles transmission, and durability (Saleh, 2021). Then again, metal atoms possess the capacity to combine with other types of metal atoms and certain non-metal atoms, to form complexes with applications in human endeavours (Minay, and Boccaccini. 2005). In this regard, some non-carbon-based, metal-containing compounds have been synthesised for use as flocculating agents and flocculating aids. These flocculants exert their action through some mechanisms. One such mechanism of floc-formation, for some synthetic, non-organic metal-containing compounds, involves the dissociation of their positively-charged metal ions, such as iron 3 + and aluminium 3 + ions, and their subsequent binding to hydroxyl groups in the aqueous medium; the resultant hydrated oxides of the metals, counterbalance the negative charges present on the dispersed particles, and induce the assemblage of suspended particles into flocs (Duan and Gregory, 2003; Hargreaves et al., 2016). Synthetic, non-carbon-based, metal-containing flocculants, possess several disadvantages such as causticity, expensiveness, non-corrodibility, the need for larger flocculant quantities per unit volume of flocculating system, contamination of the environs and threats to man and animal healthiness (Bojórquez-Quintal et al., 2017; Kurniawan et al., 2020; Sun et al., 2021). Magnesium chloride and calcium chloride have been documented as efficacious, non-carbon-containing flocculating agents (Ozkan and Yekeler, 2004; Chatsungnoen and Chisti, 2019). In addition, positively-charged ions such as calcium 2+, magnesium 2+, manganese 2+, aluminium 3 + and monovalent sodium ions, have been observed to enhance floc-formation in certain flocculation activity experiments (Kuriyama et al., 1991; Abu Tawila et al., 2019).

Fourier transform infrared (FTIR) spectroscopy is a form of spectroscopy that fundamentally utilises the infrared light absorption property of a majority of the molecules in a compound (Mishrah et al., 2018). In FTIR, the infrared radiation, which is not entrapped but leaves the compound being investigated, is registered and used for analysis, in a manner similar to that used in other forms of spectroscopy (Sigma‒Aldrich, 2023). The frequencies of exiting infrared radiation, are noted between 4000 and 400 cm^− 1 (Mishrah et al., 2018). The infrared wavelength of each molecule in the compound being analysed is unique to that molecule and thus sets it apart from others (Undavalli et al., 2021). In this manner, the functional groups present are characterised.

A scanning electron microscope (SEM) is a scientific device that uses a concentrated stream of dynamic electrons rather than a beam of radiant energy, to produce a highly magnified, detailed picture of a selected part of the surface of inorganic or organic matter. The contact of the electron stream with the surface of the compound produces secondary electrons without active energy loss, while back-scattered electrons are produced when this beam comes in contact with the atoms of the compound, with the attendant loss of energy (Nanakoudis, 2019). These electrons are what the SEM utilises to construct an image .(Nanakoudis, 2019). The chemical constituents are subsequently analysed using energy dispersive X-ray spectroscopy (EDX).

Energy dispersive X-ray spectroscopy (EDX) is a research procedure that analyses the elemental or chemical composition of a sample (Ismail et al., 2019). It identifies the type of chemicals in a compound and their percentage distribution in the compound; it therefore, serves both qualitative and quantitative functions (Nasrollahzadeh et al., 2019). EDX functions by utilising the X-rays produced by electron beam-sample interactions; these X-rays are captured by an EDX detector for chemical constituent analyses (Raval et al., 2019).

The combination of SEM and EDX has been used in diverse fields of human endeavour. Examples include forensic science for injury studies (Gentile et al., 2020), environmental science and monitoring for microplastic surveys (Wirnkor et al., 2019) and archaeology and anthropology for dating earthenware (Zuluaga et al., 2011).

High-performance liquid chromatography (HPLC) is an investigative technique that disassociates, identifies and assesses the amount of the constituents of a composite (Pitigoi 2022). It comprises a column that houses the mobile phase, a pump that drives this mobile phase through where it is located, and an in-built detector that displays the retention times of the purified disassociated constituents of the composite (Malviya et al., 2010). The disassociation of the constituents of the composite being investigated, occurs as a result of dissimilarities in their molecular masses as they meet with the solid adsorbent compound present in the column, as they move along (Pitigoi, 2022). The types of HPLC used include normal-phase HPLC, reverse-phase HPLC (Deshmukh et al., 2019), affinity HPLC, ion-exchange HPLC, size-exclusion HPLC, chiral-phase HPLC, adsorption HPLC (Dong, 2006), isocratic-separation HPLC and gradient-separation HPLC (Sadapha and Dhamak, 2022). The HPLC product can be quantified through mass spectrometry (MS).

Mass spectrometry (MS) is a chemical analysis tool that determines the nature and quantity of certain samples as a result of differences in the mass-to-charge ratio of their constituent ions (Rockwood et al., 2018). This technique utilises ionisation, ion disaggregation in an electromagnetic field and impingement of these disaggregated ions on device detectors (Reusch, 2013). It can be used in many disciplines, including environmental sciences, medicine, pharmacy and forensics (Lee et al., 2021).

The phenol‒sulfuric acid method measures the concentration of the total sugar content of a compound utilising the optical density readings from reactions of phenol and sulfuric acid mixed into the test compound and into glucose (Chaplin, 1986). It makes use of a standard glucose curve.

Antibiotic resistance has reached global proportions, and the discovery of effective alternatives to the common antibiotics currently in use, 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 (NCBI, 2023), that possessed 69% flocculating activity, and that had been observed to possess biocontrol activity against Staphylococcus aureus SO183 at concentrations of 0.090 g/L and 0.150 g/L and against identified Pseudomonas aeruginosa at a concentration of 0.150 g/L in a biocontrol activity study conducted as a master’s project (Okorie, 2023). This research characterised the bioflocculant produced from Pseudomonas aeruginosa strain F29 using FTIR, SEM coupled to EDX, HPLC coupled to MS and the phenol‒sulfuric acid method.

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 techniques 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. The bioflocculant sample was thinned between two potassium bromide (KBr) plates for FTIR analysis. Using an FTIR set of instructions from the NanoScience Technology Centre (NTSC) (2008) as a reference point, the computer on the FTIR device was switched on and the sample holder was wiped clean with acetone. The appropriate details for the FTIR scan were entered into the computer. The addition of computer commands enabled the recording of the generated infrared spectra, after which the data from these spectra was analysed using the spectroscopic software Win-IR Pro Version 3.0. The data was saved, the computer was logged off and the FTIR spectroscope was shut down. The sample holder was then completely wiped clean. The interpretation of the infrared (IR) spectra was performed using IR Spectra Table and Charts from Sigma Aldrich Company (Sigma‒Aldrich, 2023) and FTIR results analyses from Aksnes and Aksnes (1963) Bunaciu et al. (2014), Smith (2017), Nandiyanto et al. (2019) and Ji et al. (2020).

Scanning electron microscopy (SEM) and energy dispersive X-ray analysis/spectroscopy (EDS/EDX)

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 (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)

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 Sharma et al. (2020), Anumula (1994) and Guzzetta (2001) 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, 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 read at 490 nm. The test was performed in triplicate. 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 glucose standard 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 represents the optical density (absorbance) at a wavelength of 490 nm; m represents the mass; x represents the concentration of the bioflocculant sugars; and c represents the mathematical (velocity) constant (Jain et al., 2017).

Substituting values, the exact concentration of bioflocculant sugars was calculated from the following formula:

OD _{bioflocculant}= 0.3492x + 1.2234

where OD _{bioflocculant} represents the average optical density of the bioflocculant sugars

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), which correlated with O-H stretching of the hydroxyl group of a carboxylic acid/carboxylate (COOH) or a concentrated aromatic alcohol (-OH). 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. 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. This strong absorption peak at 599.55 cm^− 1 was also due of the bending of carbonyl (C = O) groups in an amide/peptide bond. The absorption peak at 474.39 cm^− 1 was from the S‒S stretch correlated with polysulfides, while that at 418.53 cm^− 1 was unrelated to any charted standard compound.

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: oxygen (4.00%), carbon (20.50%), chlorine (56.00%), phosphorus (3.00%), sodium (12.50%), magnesium (1.06%), nitrogen (0.30%), potassium (0.32%) and sulphur (2.43%). 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 were a total of sixteen recorded peaks, with glucose possessing four peaks (1, 2, 9 and 11); galactose having three peaks (5, 14 and 15); inositol having two peaks (3 and 16); rhamnose having two peaks (4 and 12); and mannose having 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 method 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.

The physical characteristics, the functional groups, the composition and the relative distribution of the elements 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 produced 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 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 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 3300 to 3500 cm^− 1 (Ji et al., 2020). The medium absorption peak at 1635.05 cm^− 1 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 to 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 – 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). 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 employed 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. 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 calcium polysulfide mixed with ferrous sulphate, as a coagulation-flocculation procedure for remediating waste water from an industry that produced hide. They discovered that though 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. The strong absorption peak at 599.55 cm^− 1 was also attributed to the presence of halogen compounds (carbon-iodide, carbon-bromide or carbon-chloride bonds) 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 TMT¹. Bodîrlău et al. (2009) reported the stretching from a carbon-chloride (C-Cl) bond at 600–700 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. The presence of halo compounds in the bioflocculant produced from Pseudomonas aeruginosa strain F29 could have been partly responsible for its effective flocculation capacity.

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. and Farnood, 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 coagulative -flocculative 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 carbon (20.50%) and chlorine (56.00%). Sodium (12.50%) and oxygen (4.00%) were next in abundance. Phosphorus (3.00%) and sulphur (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 commensurate 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 was probably due to species differences and the type and ratio of components in the bacterial medium used in bioflocculant production. Some metal cations have been observed to aid in floc-formation (Kuriyama et al., 1991; Abu 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, 1954). 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, sulphur 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 sulphur – have been described by Madson (2016) and Kakade et al. (2022), as lesser constituents of carbohydrates, and occur in those carbohydrates that have been modified. On the other hand, phosphorus, sulphur 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 sulphur present in relatively less proportions, the bioflocculant from Pseudomonas aeruginosa strain F29 possessed relatively more abundance of phosphorus (3.00%) and sulphur (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. Although the presence of phosphorus, nitrogen, sulphur, and amide/ peptide bonds (within amide I, VI and A bands), in the bioflocculant from Pseudomonas aeruginosa strain F29, generally alluded to the presence of a protein/ proteins, the more specific differentiation between the presence of a protein and that of a polypeptide, could not be exactly ascertained. This was because the use of a more diagnostic method, like x-ray crystallography that can provide a stereographic view of a protein (Alberts et al., 2002), fell outside the scope of this study. The discovery that more than 75% of the bioflocculant consisted of carbon and chlorine (with chlorine accounting for more than half of the total amount of elements present), strongly indicated that the bioflocculant from Pseudomonas aeruginosa strain F29, 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. In addition, with the confirmation of the presence of carbohydrate sugars and proteins 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 metal-containing “glycoprotein/polypeptide-organochlorine”.

An extensive search of documented research, failed to reveal any report of metal-containing “glycoprotein/polypeptide-organochlorines”. There seemed to be an absence of documentation on the discovery of this category of glycoconjugates. There was a report by Fujimori et al. (2012) of Pseudomonas species producing the vaporescent organochloride compound, chloromethane. However, an extensive search of documented studies did not reveal any report of either an organochloride bioflocculant or a “glycoprotein/polypeptide-organochlorine” bioflocculant, from Pseudomonas species or any other microorganism. From available documentation, this is the first report of a metal-containing “glycoprotein/polypeptide-organochlorine” bioflocculant.

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 metal - containing “glycoprotein/polypeptide-organochloride”. From available documentation, this is the first report of a metal-containing “glycoprotein/polypeptide-organochlorine” bioflocculant. Further research on this bioflocculant should prove helpful in establishing the full ambit of its activities and properties.

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.

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.

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 interests.

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Plate 1 is available in the Supplementary Files section.

The authors declare no competing interests.

Plate1.png
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

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Characterisation of a novel metal-containing “glyco-protein/polypeptide-organochlorine” bioflocculant produced from Pseudomonas aeruginosa strain F29 isolated from pig fecal matter collected from a mixed animal farm in Ibadan, Oyo State, Nigeria

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INTRODUCTION

MATERIALS AND METHODS

RESULTS

Fourier Transform Infrared (FTIR) Spectroscopy

Scanning electron microscopy (SEM) and energy dispersive X-ray analysis/spectroscopy (EDS/EDX)

High-Performance Liquid Chromatography (HPLC) and Mass Spectrometry (MS)

The Phenol‒Sulfuric Acid Method

DISCUSSION

CONCLUSION

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References

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