Kaolin Flocculation Research Articles

Synthesis of multi-stimuli-responsive flocculants using reversible addition-fragmentation chain transfer polymerization: Investigating the performance and mechanism of kaolin coagulation

Coumarin-containing amphiphilic block copolymers with different compositions, as a new class of pH- and photo-responsive flocculants, were synthesized through reversible addition-fragmentation chain transfer polymerization of dimethyl aminoethyl methacrylate (DMAEMA), methyl methacrylate, and 7-acryloyloxy 4-methylcoumarin. Proton nuclear magnetic resonance spectroscopy was used to study the structure and composition of the copolymers. Zeta potential results show that the copolymers are applicable as amphoteric flocculants. The optimal flocculant concentration was calculated using the Jar analysis for kaolin flocculation, and the results show that the flocculation dependence on the flocculent concentration decreases with increasing its molecular weight. The dominance of charge neutralization in the flocculants with lower molecular weights and the dominance of adsorption bridging and hydrophobic association in the flocculants with higher molecular weights were indicated. These results are in agreement with the rheological data. To investigate the governing mechanism more precisely, the flocculation rate was calculated using turbidity measurements at different pH values over time. The highest final flocculation rate (99.25 %) corresponds to pH 4.5, which caused by the protonation of the amine groups of the DMAEMA repeating units. The dependence of settling rate on pH indicates the pH-dependent performance of the flocculants. Finally, fluorescence microscopy image of the flocculants shows its proper functioning of in the vicinity of kaolin, which has a promising industrial application.

Depth dewatering of kaolinite cake by SDBS/PEI synergy and a mesoscopic mechanism study

The objective of this study was to elucidate the synergistic dewatering mechanism of mixed sodium dodecylbenzene sulfonate (SDBS) and polyethyleneimine (PEI) on kaolinite filter cakes and to test the dewatering effectiveness of a custom-built pressure dewatering device. The dewatering experiments showed that while the reagent dose was reduced by 50%, the dewatering rate decreased to 19.78%, surpassing the optimal dose of a single reagent. During the natural compression stage of the filter cake, the deformation reached its maximum under the same pressure difference. During the transitional period, the closure of the filter cake pores occurred at a slower rate, facilitating better moisture drainage. Under strong compression, the ultimate deformation reached its maximum. PEI acted as a bridging agent for kaolin flocculation and served as a medium for SDBS on the kaolin surface. A small amount of PEI reduced the electrostatic repulsion of SDBS, increasing its adsorption capacity. PEI adsorbed on the kaolin surface through electrostatic and hydrogen bonding, while SDBS could adsorb onto the PEI reagent layer through electrostatic and polar interactions, exposing its alkyl chains on the surface and thereby facilitating flocculation and generating a hydrophobic reagent layer. This study could provide a new method and insights for the efficient pressurized dewatering of kaolin.

Effect of bulk nanobubbles on flocculation of kaolin in the presence of cationic polyacrylamide

The study explores the impact of nanobubble flotation technology on fine mineral processes, focusing on its interaction with cationic polyacrylamide (CPAM) in kaolin flocculation. Nanobubbles influence particle size and promote aggregation. Experimental procedures involve bulk nanobubble preparation, kaolin suspension, and CPAM solutions, with analysis of sedimentation rates, turbidity, and zeta potential. Results show accelerated sedimentation and reduced turbidity with nanobubbles compared to traditional methods. Zeta potential measurements and DLVO theory support nanobubbles' role in reducing electrostatic interaction, facilitating flocculation. This research advances understanding in nanobubble-mediated mineral processing, highlighting eco-friendly flocculants and practical implications for optimization.

Analysing the role of Fe (II) on flocculation of sand-clay mixtures under estuarine mixing

Estuaries are fragile environment that are most affected by climate change. One of the major consequences of climate change on estuarine processes is the enhancement in salt intrusion leading to higher salinity values. This has several implications on the estuarine sediment dynamics. Of the various factors that affect the flocculation of cohesive sediments, salinity and turbulence have been recognized as to have great significance. Many of the estuaries are contaminated with heavy metals, of which, the concentration of Iron (Fe (II)) are generally on the higher range. However, the influence of Fe (II) on the flocculation of cohesive sediments at various estuarine mixing conditions is not well known. The present study investigated the influence of Fe (II) on the flocculation of kaolin at various concentration of Fe (II), salinity and turbulence shear. The results indicated that Fe (II) and salinity have a positive influence on kaolin flocculation. The increase in turbulence shear caused an initial increase and then a decrease in floc size. In case of sand-clay mixtures, that are observed in mixed sediment estuarine environments, a reduction in the floc size was observed, which is attributed to the breakage of flocs induced by the shear of sand. Breakage coefficient, which is a measure of break-up of flocs, is generally adopted as 0.5 assuming binary breakage. The present study revealed that the breakage coefficient can take values from 0 to 1 and is a direct function of Fe (II) and salinity and an inverse function of turbulence and sand concentration. Thus, a new model for breakage coefficient with the influencing parameters has been proposed, which is an improvement of existing model that is expressed in terms of turbulence alone. Sensitivity analysis showed that the proposed model can very well predict the breakage coefficient of Fe (II) – kaolin flocs. Thus, the model can quantify the breakage coefficient of flocs in estuaries contaminated with Fe (II) that is a vital parameter for population balance models.

Cytotoxicity and Antibacterial Efficacy of Betaine- and Choline-Substituted Polymers.

Cationic charge has been widely used to increase polymer adsorption and flocculation of dispersions or to provide antimicrobial activity. In this work, cationization of hydroxyethyl cellulose (HEC) and polyvinyl alcohol (PVA) was achieved by covalently coupling betaine hydrochloride and choline chloride to the polymer backbones through carbonyl diimidazole (CDI) activation. Two approaches for activation were investigated. CDI in excess was used to activate the polymers' hydroxyls followed by carbonate formation with choline chloride, or CDI was used to activate betaine hydrochloride, followed by ester formation with the polymers' hydroxyls. The first approach led to a more significant cross-linking of PVA, but not of HEC, and the second approach successfully formed ester bonds. Cationic, nitrogen-bearing materials with varying degrees of substitution were obtained in moderate to high yields. These materials were analyzed by Fourier transform infrared spectroscopy, nuclear magnetic resonance, polyelectrolyte titration, and kaolin flocculation. Their dose-dependent effect on the growth of Staphylococcus aureus and Pseudomonas aeruginosa, and L929 mouse fibroblasts, was investigated. Significant differences were found between the choline- and betaine-containing polymers, and especially, the choline carbonate esters of HEC strongly inhibited the growth of S. aureus in vitro but were also cytotoxic to fibroblasts. Fibroblast cytotoxicity was also observed for betaine esters of PVA but not for those of HEC. The materials could potentially be used as antimicrobial agents for instance by coating surfaces, but more investigations into the interaction between cells and polysaccharides are necessary to clarify why and how bacterial and human cells are inhibited or killed by these derivatives, especially those containing choline.

Compressive Yield Stress of Flocculated Kaolin Suspensions in Seawater.

The mining industry has resorted to using seawater while trying to find a solution to the water shortage, which is severe in some regions. Today, the industry looks to tailings dams to recover more water and, thus, increase recirculation. The migration of interstitial water due to the consolidation of particle networks can give rise to large water mirrors in different dam areas. These pools can contain enough water to be recovered and recirculated if the external stress caused by the weight of the pulp exceeds the compressive yield stress. The density and rheological properties of the discarded pulps determine the feasibility of water expulsion during tailings consolidation. As these conditions are largely established in the thickening stage, it is necessary to revisit operations, looking at the dam as a water source. Thus, a thorough understanding of the compressive properties that determine the level of consolidation of typical pulps and their relationships to aggregate properties, such as size and fractal dimension, is crucial. Here, the effect of two types of water, industrial water and synthetic seawater, on kaolin flocculation, sedimentation rate, yield stress, and compressive yield stress were studied. In addition, the relationship of these properties with the flocculant dose and the resulting aggregate size and fractal dimension was examined. One promising finding to practitioners was that salt and small doses of high molecular weight flocculant improved the consolidation of kaolin slurries under compression. These conditions generated low compressive yield stress compared to fresh water and water with low salt content, favoring the consolidation of the pulps and the release of water.

The last 25 years of research on bioflocculants for kaolin flocculation with recent trends and technical challenges for the future.

The generation of kaolin-containing wastewater is an inevitable consequence in a number of industries including mining, wastewater treatment, and bitumen processing. In some cases, the production of kaolin tailings waste during the production of bitumen or phosphate is as high as 3 times greater than the actual produced product. The existing inventory of nearly five billion barrels of oil sands tailings alone represents a massive storage and reclamation challenge, as well as a significant economic and environmental liability. Current reclamation options like inorganic coagulants and organic synthetic polymers may settle kaolin effectively, but may themselves pose an additional environmental hazard. Bioflocculants are an emerging alternative, given the inherent safety and biodegradability of their bio-based compositions. This review summarizes the different research attempts towards a better bioflocculant of kaolin, with a focus on the bioflocculant source, composition, and effective flocculating conditions. Bacillus bacteria were the most prevalent single species for bioflocculant production, with wastewater also hosting a large number of bioflocculant-producing microorganisms while serving as an inexpensive nutrient. Effective kaolin flocculation could be obtained over a broad range of pH values (1-12) and temperatures (5-95°C). Uronic acid and glutamic acid were predominant sugars and amino acids, respectively, in a number of effective bioflocculants, potentially due to their structural and charge similarities to effective synthetic polymers like polyacrylamide. Overall, these results demonstrate that bioflocculants can be produced from a wide range of microorganisms, can be composed of polysaccharides, protein or glycoproteins and can serve as effective treatment options for kaolin. In some cases, the next obstacle to their wide-spread application is scaling to industrially relevant volumes and their deployment strategies.

Application of Extracellular Polymeric Substances Extracted from Wastewater Sludge for Reactive Dye Removal

This study aimed to investigate the adsorption of three commercial reactive dyes using extracellular polymeric substances (EPS) extracted from the waste sludge of a beer wastewater treatment plant in Hanoi, Vietnam. EPS was extracted from sludge by the HCHO-NaOH method and was characterized by measuring kaolin flocculation activity, dry weight, chemical composition, and functional groups. Adsorption of dyes on EPS was conducted by Jartest at different pH values, contact times, and EPS dosages. The EPS was composed of 25% sludge by weight. The FTIR analysis showed the presence of amine and carboxyl groups in the EPS structure. The removal efficiencies of reactive dyes were high at pH values below 6, a contact time of 30 to 60 min, and EPS dosage of 200 – 250 mg/L. At optimum condition, removal efficiencies of 85%, 99%, and 99% were obtained for Reactive Yellow 176 (RY 176), Reactive Blue 21 (RB 21), and Reactive Red 241 (RR 241), respectively. The adsorption process could be described by both Langmuir and Freundlich models. The maximum dye adsorption capacities for RY 176, RB 21, and RR 241 were 0.50 g/g, 0.72 g/g, and 0.95 g/g, respectively. It is concluded that EPS in wastewater sludge could be utilized as an effective adsorbent for dye removal, thereby enhancing the value of sludge in wastewater treatment.

Floc structure and dewatering performance of kaolin treated with cationic polyacrylamide degraded by hydrodynamic cavitation

At present, efficient dewatering of mineral-processing–plant tailings still presents a serious challenge, especially for tailings with a high proportion of clay minerals. In this study, cationic polyacrylamide (CPAM) was degraded using hydrodynamic cavitation and was used for the flocculation and dewatering of kaolin. The properties of CPAM after degradation were characterized using a rheometer. At the same time, focused beam reflectance measurement, particle video microscopy, and low-field nuclear magnetic resonance were used to study the flocculation and filtration characteristics of kaolin treated by degraded CPAM. The results showed that with an increase in the cavitation time, the viscosity of CPAM decreases gradually and a flocculant mixture system with multimolecular weight is formed. In the process of kaolin flocculation, the pretreated CPAM makes the flocs smaller and denser, resulting in the acceleration of kaolin filtration.

Analysis of Kaolin Flocculation in Seawater by Optical Backscattering Measurements: Effect of Flocculant Management and Liquor Conditions

Optical backscattering (OBS) signal values were used to evaluate the flocculation of kaolin slurries and seek the implications of using seawater. Two anionic flocculants were applied to kaolin suspensions at several dosages and in water of varying pH and electrolyte concentration. An OBS height scan method was used to estimate the degree of aggregation, supernatant quality, and solids concentration of the sediments. The residual solids of the supernatant depended of the extent of particle coagulation before flocculant was added, where the pH and salinity displayed a significant impact on flocculation. The OBS results were highly sensitive to the presence of fine particles, which was estimated in parallel from the focused beam reflectance measurements (FBRM.) In seawater, without flocculant added, the samples had increased root-mean-square scattering (Frms) values and larger final sediment volume than samples prepared in water with lower electrolyte concentration. This indicates a higher initial state of aggregation of the particles in seawater. Then, the aggregation degree was best linked to the square-weighted chord length distribution of the FBRM data, which intensifies the sensitivity to coarse aggregates.

Synthesis of cationic polyacrylamide via inverse emulsion polymerization method for the application in water treatment

In order to improve the flocculent efficiency of wastewater treatment, a cationic flocculant poly (acrylamide-[2-(methacryloyloxy) ethyl] trimethyl ammonium chloride) (P (AM-DMC)) (CPAM) has been synthesized successfully via an inverse emulsion polymerization. Acrylamide (AM) and [2-(methacryloyloxy) ethyl] trimethyl ammonium chloride (DMC) were served as monomers. The molecular structure of CPAM was characterized by Fourier transform infrared spectra (FT-IR) and 1H nuclear magnetic resonance spectrum (1H-NMR). The morphology of CPAM particles has been investigated by transmission electron microscope (TEM). Results showed that CPAM was the copolymer of AM and DMC and the particles of CPAM were uniform spheres (the size was about 200 nm). The synthetic conditions of CPAM have been studied and optimized by single-factor experiments. An optimized product was obtained at an intrinsic viscosity of 560 mL/g with a total monomer concentration of 25% and initiator concentration V50 of 0.2% (based on the total monomer mass). The amount of emulsion was 6% and the HLB (Hydrophile-Lipophile-Balance) of emulsion was 7.3. In addition, the flocculation property of CPAM was evaluated with kaolin suspension using jar test, and the result demonstrated that the flocculation property of CPAM performed better than kaolin flocculation.

Flocculation of kaolin from aqueous suspension using low dosages of acrylamide‐based cationic flocculants

ABSTRACTFlocculation of kaolin from aqueous streams has become very important in view of its wide range of applications. In this work, cationic flocculants based on copolymers of acrylamide (AM) and 3‐acrylamidopropyltrimethylammonium chloride (APTMAC) were synthesized with two different mole ratios of monomers, 80 mol % of AM (CP‐8020) and 40 mol % of AM (CP‐4060). The chemical structures of copolymers were confirmed by 1H and carbon‐13 nuclear magnetic resonance (13C NMR) spectroscopy. The molecular weight (MW) and zeta potential of the copolymers were determined. High MW was obtained for copolymer with high content of AM (CP‐8020) and high zeta potential was observed for copolymer with high content of APTMAC (CP‐4060). Flocculation of kaolin suspension was performed using both CP‐8020 and CP‐4060 and the flocculation was correlated to the zeta potential and MW of the copolymers. The optimum dosages of flocculants were determined. The mechanism of flocculation was discussed in terms of charge neutralization and bridging. The flocs of kaolin were characterized in terms of moisture content and size. To the best of our knowledge, this copolymer system was used for the first time for kaolin flocculation and found out to be efficient. © 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2019, 136, 47286.

DMC-grafted cellulose as green-based flocculants for agglomerating fine kaolin particles

Novel cellulose based flocculants C-g-P (DMC) with various chain architectures are synthesized through a situ graft copolymerization. The cationic ammonium chloride group (DMC) is grafted onto cellulose by two separate inverse emulsion polymerization with γ-methacryloxypropyl trimethoxy silane (KH-570) and double bond addition reactions, which is a new and simple method to employ KH-570 as a bridge for the connection of cellulose matrix and DMC group. The effects of pH, flocculant dose, standing time on turbidity of kaolin suspensions and particle sizes have been studied systematically. In addition, the response surface methodology (RSM) study confirms that PAC and C-g-P (DMC) have synergy in turbidity removal with a higher removal efficiency of 98.32%. Moreover, C-g-P (DMC) 1 has higher removal efficiency with 96.5% at a low dosage of 0.6 mg L−1 and better floc properties than C-g-P (DMC) 2 and C-g-P (DMC) 3, suggesting that the length and quantity of cationic branch chains play a crucial role in Kaolin flocculation due to their dramatically enhanced bridging effects.

Effects of microbial cells and their associated extracellular polymeric substances on the bio-flocculation of kaolin and quartz

Microbes can function as bio-surfactants to selectively flocculate or disperse particles, and separate minerals. In the present study, Bacillus licheniformis cells and metabolite were used to change the settling behavior of kaolin and quartz. Moreover, to perform a more detailed study on the metabolite, settling effect of its protein and polysaccharide constituents were also investigated separately. In the best condition, about 40% improvement in kaolin settling was observed using bacterial cells and metabolite at pH=7 and 3, respectively. Also, enhancement in quartz sedimentation using the same bio-flocculants was >50% at pH=1–3. In addition, polysaccharide was more effective in kaolin flocculation, while protein was more influential in quartz agglomeration. Results of adsorption tests suggested that all of the bio-surfactants were more inclined to be adsorbed on quartz, and generally, increasing the pH value decreased the reagent adsorption and mineral flocculation. Moreover, although reducing the mineral concentration from 50 to 20g/l improved the settling of both kaolin and quartz, the highest difference (50%) in their sedimentation occurred at the higher mineral concentration. Finally, according to the statistics, clay concentration was the most influential parameter on kaolin, and the dispersion pH was the most effective variable in quartz bio-flocculation.

Fourier Transform Infrared Spectroscopy and Liquid Chromatography–Mass Spectrometry Study of Extracellular Polymer Substances Produced on Secondary Sludge Fortified with Crude Glycerol

This study was conducted to characterize the extracellular polymeric substances (EPS) produced by growing Cloacibacterium normanense in wastewater sludge alone and fortified crude glycerol. The EPS highest concentration of 17.5 g/L was produced using 25 g/L sludge suspended solids supplemented with 25 g/L of crude glycerol. Galactose and Glucose was the main compound of EPS produced with or without crude glycerol, respectively. EPS FTIR spectra revealed a variation in the different functional groups such as amines, carboxyl, and hydroxyl groups for EPS produced. Different functional groups were observed in the EPS produced with or without glycerol. The EPS exhibited kaolin flocculation activity up to 95% and was stable at high temperature. The high viscosity bioflocculant properties of EPS make it suitable for potential industrial applications

Revealing the characteristics of a novel bioflocculant and its flocculation performance in Microcystis aeruginosa removal.

In the present work, a novel bioflocculant, EPS-1, was prepared and used to flocculate the kaolin suspension and Microcystis aeruginosa. We focused on the characteristics and flocculation performance of EPS-1, especially with regard to its protein components. An important attribute of EPS-1 was its protein content, with 18 protein types identified that occupied a total content of 31.70% in the EPS-1. Moreover, the flocculating activity of these protein components was estimated to be no less than 33.93%. Additionally, polysaccharides that occupied 57.12% of the total EPS-1 content consisted of four monosaccharides: maltose, D-xylose, mannose, and D-fructose. In addition, carbonyl, amino, and hydroxyl groups were identified as the main functional groups. Three main elements, namely C1s, N1s, and O1s, were present in EPS-1 with relative atomic percentages of 62.63%, 24.91%, and 10.5%, respectively. Zeta potential analysis indicated that charge neutralization contributed to kaolin flocculation, but was not involved in M. aeruginosa flocculation. The flocculation conditions of EPS-1 were optimized, and the maximum flocculating efficiencies were 93.34% within 2 min for kaolin suspension and 87.98% within 10 min for M. aeruginosa. These results suggest that EPS-1 could be an alternative to chemical flocculants for treating wastewaters and cyanobacterium-polluted freshwater.

Preparation, characterization, and flocculation performance ofP(acrylamide‐co‐diallyldimethylammonium chloride) by UV‐initiated template polymerization

ABSTRACTThis study prepared TPDA, a high‐intrinsic‐viscosity cationic polyacrylamide, through ultraviolet (UV)‐initiated template polymerization. Acrylamide (AM) and diallyldimethylammonium chloride (DMD) served as monomers, and poly sodium polyacrylate (PAAS) served as the template. The structure of TPDA was characterized by Fourier‐transform infrared spectroscopy, proton nuclear magnetic resonance spectroscopy, and thermogravimetric analysis. The synthetic conditions of TPDA were studied and optimized by single‐factor experiments. An optimized product was obtained at an intrinsic viscosity of 11.3 dL g−1and a conversion rate of 97.2% with a total monomer concentration of 20%, DMD concentration of 30%, initiator concentration of 0.045%, pH of 8, EDTA concentration of 0.3%, and UV irradiation of 90 min. Results showed that TPDA was the copolymer of AM and DMD with a micro‐block structure at the molecular chain. Given its high intrinsic viscosity and cationic block structure, TPDA performed better in kaolin flocculation than that prepared without template addition. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci.2015,132, 41747.

Preparation of polyacrylamide grafted collagen extracted from leather wastes and their application in kaolin flocculation

ABSTRACTThis is probably the first report on the synthesis of a series of novel collagen‐based flocculants (CP11, CP12, and CP13) by grafting polyacrylamide (PAM) chains onto the collagen backbone, which was directly extracted from leather shavings via alkali hydrolysis. The results from FTIR, XRD, 1H NMR, and TGA well supported that PAM chains had been successfully grafted onto collagen backbone. In addition, the micrographs of SEM revealed that the PAM grafted collagen possessed much more porous and looser surface structures in comparison with virgin collagen. Zeta potential measurement showed that the introduction of branched PAM chains helped to improve the positively charge density. Furthermore, CP12 performed the best in the kaolin flocculation with the highest flocculation rate about 24%·min−1 and could induce the generation of much larger and denser floccs for the fast settling of kaolin particles. The corresponding flocculation mechanism was also presented by analyzing the collected flocs. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015, 132, 41556.

Extracellular polymeric substances production kinetics of 13 sludge isolates using wastewater sludge as raw material and its flocculation potential

The kinetics of batch fermentation of 13 extracellular polymeric substances (EPS) producing bacterial strains (9 Bacillus, 2 Serratia and 2 Yersinia) were carried out using sterilized sludge as a raw material. The most of Bacillus (µmax: 0.11–0.27 h−1), Serratia (µmax: 0.23–0.27 h−1) and Yersinia (µmax: 0.18–0.19 h−1) strains had capability to grow and produce EPS (1.36–2.12 g/L) in the sterilized sludge. In general, EPS production was mixed growth associated for all the bacterial strains cultivated independently. Bacillus sp. 7, Serratia sp. 2 and Yersinia sp. 2 produced higher concentration (1.95–2.12 g/L) of EPS than the other remaining bacterial strains. Protein and carbohydrate contents of EPS remained constant during fermentation. Broth EPS (B-EPS) exhibited high kaolin flocculation activity (≥75%) in most of the cases except Bacillus sp. 1, Bacillus sp. 5 and Bacillus sp. 9, respectively. In general, high flocculation activities (FAs) (≥75%), were attained using 1.31–1.70 mg B-EPS/g kaolin, 0.45–0.97 mg protein/g kaolin and 0.11–0.21 mg carbohydrates/g kaolin. The study suggests that further systematic exploration is required for optimizing the process of EPS production. EPS produced in the sludge can potentially be used for different water and wastewater treatments.

Guar gum and guar gum-oligomeric poly(vinyl alcohol) blends as novel flocculants for kaolinated waste water

This is probably the first report on kaolin flocculation done with aqueous guar gum (GG) at various pH. Dynamic light scattering and reduced viscosity proved the polyelectrolytic feature (zeta potential) of aqueous GG which changed on changing pH. Interestingly, the molecular size of GG did not always increased with rising zeta potential due to strong intermolecular repulsion leading to macromolecular recoiling. Best pH range for settling was 4.0–5.0 which included isoelectric point (IEP) of kaolin. Post-settling turbidity was also acceptable at that range. Optimized GG was further blended with oligomeric poly (vinyl alcohol) (PVA) (Mn 14,000) to prepare a new set of flocculant. Oligomeric PVA was previously optimized at similar pH as excellent kaolin flocculant in our earlier investigation. All GG–PVA blends including neat GG exhibited faster settling than neat PVA. However, settling times of all blend compositions were slightly greater than that of neat GG except for few cases where the post settling turbidity was found significantly lower than all our previous investigations.