- Email: [email protected]
Increasing and eliminating the (Fecal coliforms, thermotolerant coliform and fecal streptococcus) bacteria by resin of the poly (para carboxy acid phenol-d -Glucose) to clean up waste water
Human Microbiome Journal 11 (2019) 100053
Contents lists available at ScienceDirect
Human Microbiome Journal journal homepage: www.elsevier.com/locate/humic
Original Article
Increasing and eliminating the (Fecal coliforms, thermotolerant coliform and fecal streptococcus) bacteria by resin of the poly (para carboxy acid phenol-D-Glucose) to clean up waste water Safia Miloudia,b, Messaoud Chaibc, Moulkheir Ayata,d, Abdelkader Rahmounid,
T
⁎
a
Department of Chemistry, Faculty of Sciences, Dr. Moulay Taher University, BP138 City, Nasr Saida 20000, Algeria Department of Chemistry, Faculty of Sciences, University Djillali Liabes, Sidi Bel Abbes 22000, Algeria c Department of Chemistry, Faculty of Science Exact, University Ibn-Khaldoun, Bp 78, Tiaret 14000, Algeria d Department of Chemistry, Faculty of Sciences, Oran1 University Ahmed Benbella, Bp 1524, El’Menouer, 31000 Oran, Algeria b
A R T I C LE I N FO
A B S T R A C T
Keywords: Poly (para-carboxy acid phenol-D-Glucose) Fecal coliforms Thermotolerant coliform Fecal streptococcus Antibacterial resin Polycondensation reaction Homogeneous catalyst
In this research paper we represent a novel synthetic antibacterial phenolic polymer (pAP-DG) containing carboxylic acid group in para position. The study of poly (p-carboxylic acid phenol D-Glucose) as an antibacterial resin was focused on the development of antimicrobial polymers to clean up the water of pathogenic bacteria. The polycondensation reaction was realized between p-carboxy acid phenol (pAP) and D-Glucose (DG) as monomers; using sulfuric acid as homogeneous catalyst. The obtained polymer was used via fecal coliform (TC), thermotolerant coliforms (CTT) and fecal streptococci (ST) bacteria to evaluate the water quality. The antibacterial activity of the synthesized resin was carried out by the contact method so the capacity of this resin was confirmed. In general, the polymer has a good antimicrobial activity against the microorganisms tested; the resulting polymer was characterized by various spectroscopy methods such as: 1H NMR, 13C NMR, DSC and FTIR and a mechanism has been proposed for this polycondensation reaction.
1. Introduction Phenolic resins generate a wide variety of polymeric materials [1,2]; each polymer is composed of a multitude of structures and a variety of raw materials with the use of catalysts. Polymer demand has grown steadily for many years then it has every day enriched by new applications of specific macromolecular materials. Phenolic resins [3,4] are among the oldest industrial polymer that are still used due to some of their properties such as dimensional stability and chemical resistance as well as their low cost [5,6]. The use of polymers and resins of the treatment of industrial effluent in general and which have antibacterial activity in particular is the interest of so many researches [7,8]. Polyphenols are commercial products synthesized by condensing phenol with formaldehyde at different molar ratios depending on the type of the desired polymer by reacting phenol on the ortho and para positions in the boxing ring with bifunctional formaldehyde where the reactions produce a mixture of methylol phenols [9,10]. The composition of the mixture can be varied by altering the phenol to formaldehyde ratio. Some phenolic compounds (resins with phenol derivatives containing
⁎
one, two or three hydroxyls groups) are bactericides are also brought back to the antibacterial activities due to the groups of hydroxyl [11,12]. The introduction of sugar into the structure brings new properties to polymers which makes them important for specific applications of higher added value and could accommodate higher production costs [13]. Glucose was used to replace the toxic formaldehyde to synthesize a novel Novolacs type phenolic resins catalyzed by strong acid (H2SO4) in temperature between (120–150 °C) and the condensation polymerization could succeed with solutions of phenol and glucose at varied molar ratios (1:0.4 to 1:0.9) through the Friedel-Crafts reaction [14,15]. But in this study our resin was prepared by polycondensation of para carboxy acid-phenol (pAP) with D-Glucose (DG) in an acid medium (H2SO4) as homogeneous catalyst (see Fig. 1). The bactericidal activity of the poly (p-carboxy acid phenol-D-Glucose) increased with increasing phenolic hydroxyl groups in the resin.
Corresponding author. E-mail address: [email protected] (A. Rahmouni).
https://doi.org/10.1016/j.humic.2018.11.003
Available online 24 January 2019 2452-2317/ © 2019 Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
Human Microbiome Journal 11 (2019) 100053
S. Miloudi, et al.
OH H2C
O
C
OH
+
H
H
C
OH
HO
C
H
H
C
OH
H
C
OH
COOH
+ H2SO4
HO
H
HO
H
H
- H 2O
OH
HO
H
OH CH
CH2OH
para-carboxy acid phenol
D- Glucose
COOH
n Fig. 1. Schematic representation of the synthesis of polymer [(pAP-DG)].
2. Experimental part
3. Results and discussion
2.1. Materials
The polycondensation reaction of pAP with DG was examined in the presence of H2SO4 as a catalyst at 130 °C (see Fig. 1).
Para-carboxy acid phenol and D-Glucose (99%) were purchased from Aldrich (Paris, France) purified by fractional distillation under reduced pressure. Tetrahydrofuran (THF) Sigma-aldrich, Dimethyl sulfoxide (DMSO) Panreac, acetone, methanol, solution of sodium hydroxide, sulfuric acid (98%) and distilled water were used for solutions’ preparation
3.1. Determination of the pKa of the polymer The aqueous solution was prepared by mixing 25 mg of resin in aqueous solution NaNO3 (0.01 M) with magnetic stirring for 24 h for deprotonated the carboxylic groups of resin. Finally, the excess of the free protons in the solution were titrated by a standard solution of NaOH (0.01 M) at controlled temperature (25 °C). The variation of pH with VNaOH added and its derivative curve were shown in Fig. 2(a) and (b). The pKa corresponds to the volume of half-equivalence (V = 62 ml) was 4.28 which allow to determinate the total concentration CA of reactive carboxylic sites of polymer [16]. All solutions were maintain at 25 °C and contain high purity NaNO3 (0.01 M) as supporting electrolyte.
2.2. Methods The polymers were characterized by FT-IR spectroscopy scanned on SHIMADZU spectrometer using KBr pellets at waves ranging from 400 to 4000 cm−1. The spectra of nuclear magnetic resonance of proton (1H NMR) and carbon (13C NMR) were recorded in a 300 MHz Bruker spectrometer, in deuterium acetone solvent using tetra methyl silane (TMS) as internal standard Thermal analysis DSC is a Differential scanning calorimetry apparatus (200PC NETZCH, 204F1PHONIX240-12–0110-L DSC) the measurements were carried out with a heating rate of 10 °C /min under 50 cm3/min nitrogen flow.
3.2. Spectroscopy characterization of polymer The structure of the resulting polymer was confirmed by 1H NMR, C NMR and Infrared spectroscopy (IR) measurements. The assignments of peaks of polymer were given in Tables 1 and 2. we can see from Figs. 3 and 4, the appears of a new peak at 1.42 ppm and 25.25 ppm corresponds to the methylene bridge. the same results obtained by Nonaka et al [17]. The polymers were characterized by IR and the spectra were scanned on SHIMADZU spectrometer using KBr pellets at wave numbers from 400 to 4000 cm−1 (Fig. 5). The infrared spectra of the synthesized resins in the present investigation were dominated by the large OH stretch which encompasses signals related to the hydroxyl [18]. Absorption bands characteristic of the resin (pAP-DG), and their functions were shown in the Table 3. The position of the absorption band around 1660 cm−1 corresponding to the stretching vibration of C]O indicates that the group C]O and conjugated double bond (aromatic ring). Broadband between 3065 and 3400 cm−1 corresponding to the OH function is attributable to the presence of carboxylic acid and alcohol. The ]CHR] bond of the bending vibration indicates the presence of ]CHR] (methylene bridg) [19]. 13
2.3. Polymerization procedure In a necked flask equipped with a reflux condenser and a thermometer introduced (0.1 mol) p-carboxy acid phenol and (0.05 mol) of DGlucose with 0.01 g initiator and the mixture was stirred for 1 h at 130 °C .The polycondensation reaction of para carboxy acid phenol and D-Glucose was carried out in a homogeneous system using sulfuric acid (H2SO4) Riedel- de Haein as a catalyst. After 2 h recuperating the contents of the flask and added 1.7 ml of sodium hydroxide solution (1 M) for neutralization and then heated to remove water. The reaction scheme was shown in Fig. 1. The resulting polymer was dissolved in acetone and precipitated in Tetrahydrofuran (THF) Sigma-aldrich then dried at room temperature. The purification procedure was repeated several times in order to obtain a highly purified polymer to reach a higher yield of 80%. The product was characterized by FT-IR, 1H NMR, 13 C NMR spectroscopy to confirm the structure. 2
Human Microbiome Journal 11 (2019) 100053
S. Miloudi, et al. 2,0
12
1,8 1,6
10 1,4
8
1,0
pH
d(pH/dV)
1,2
0,8
6
0,6 0,4
4
0,2 0,0
2
-0,2 -20
0
20
40
60
80
100
120
140
160
0
180
100
200
300
400
500
volume (ml)
volume (ml)
(a)
(b)
Fig. 2. (a) Plots of the experimental results for the pH titration for the polymer (pAP-DG), (b) the pH variation vs. the volume of NaOH (0.1 M) for the compound (pAP-DG).
4. Thermal analysis
low turbidity waters (< 1 NTU). Without chemical pretreatment, the removal by filtration averaged 69% (0.51 logs) for Giardia and 28% (0.14 logs) for turbidity. Adding alum and polymer filter aids increased the removal efficiency to more than 95% (1.30 logs) for Giardia, 99% (2 logs) for total coliforms bacteria and 70% (0.52 logs) for turbidity. Other process variables such as filtration mode (direct and in-line filtrations), filter media (monomedium with sand, and dual-media with sand and anthracite) and temperature (5 °C and 18 °C) did not significantly affect the filtered water quality [22]. Varying the filtration rate from 5 to about 20 m/h had little effect on removal of Giardia, total coliforms bacteria and heterotrophic bacteria, but increased turbidity in the filtered water [23].
Thermal properties of the copolymers were investigated by DSC. The DSC analysis of (pAP-DG) copolymers indicates two closely neighboring melting endotherms at temperatures between 160 °C and 210 °C and the glass transition temperature Tg of the resulting copolymers was observed in the temperature ranges of 50–70 °C as mentioned in Fig. 6 which means that the polymer is semi-crystalline [20,21]. 5. Study of pretreatment for removal of microbes The importance of chemical coagulation pretreatment for removal of microbes by granular filtration has been emphasized by numerous studies. Al-Ani et al. (1986) conducted a pilot-scale filtration study for Table 1 1 H NMR Chemical shifts and signal assignation of poly (pAP-DG). Structure
3
δ (ppm), intensity
Attribution
(2.05–2.23,d) (1.42,d) (4.25,s) (6.7–6.93,s), (11.15, s) (3.61–3.66, m) (1.78–1.82, m)
Hm Hd Ha Hc aromatic Hb H n, l, j, g, f H e ,h, i, k
Human Microbiome Journal 11 (2019) 100053
S. Miloudi, et al.
Table 2 13 C NMR Chemical shifts and signal assignation of (PAP-DG) polymer. Structure
HO
i CH2
HO
Attribution
δ (ppm)
Ca Cb Cc Cd COOH Ce,f,g,h,i
205.32 135.86 119.06 25.25 130.35 28.16–29.81
C h HO C
g
OH
Cf HO OH
a
C e CH
d
c b COOH
n
Fig. 3. 1H RMN spectrum of the (pAP-DG) polymer. 4
Human Microbiome Journal 11 (2019) 100053
S. Miloudi, et al.
Fig. 4.
13
C RMN spectrum of the (pAP-DG) polymer.
5.1. Action of resin to removal of microbial pathogens by granular filtration
Table 3 Different IR absorption bands of the synthesized poly(pAP-DG).
Removal of microbial pathogens by granular filtration does not rely on physical processes alone. Comparing the pore size of granular filters with the size of most types of microbe (as in Fig. 7), it is evident that effective removal of microbes by granular filtration cannot rely on physical straining alone, at least at the initial stage of a filter run [24]. The removal of particles by granular filtration is considered to involve two steps: transport of particles from suspension to filter medium, followed by attachment of particles to the medium. The transport step depends on the physical and hydrodynamic properties of the system.
Chemical shift
Absorption Band
Nature
vOH
3200–3500 3241 3065 2859 1660 1611–1578 1363 1232 1103–1014 890–952 758
OH bond OH acid Stretching vibration Aromatic Aliphatic Carboxylic acid benzene ring Phenolic Phenolic Aromatic Para substituted Ortho substituted
vCH vCH v(C]O) v(C]C) dip(OH) dip(C]O) dip(CH) dop(CH) dop(CH)
5.2. Biological activity In general to make a well-defined study of the biological activity of
Fig. 5. Infrared spectrum of poly(pAP-DG) in the region 400–4000 cm−1. 5
Human Microbiome Journal 11 (2019) 100053
S. Miloudi, et al.
Fig. 6. Thermogramm of the (pAP-DG) polymer.
5.3. Antimicrobial activity
CT SF CTT
90
The antibacterial activity was tested by the contact method [25,26] with the use of polymeric material (pAP-DG) which is insoluble in water. The number of the bacterial colonies was counted after the test tubes incubation at 37 °C for 24 h under (200 rpm). The percent reduction of the bacterial: CTT, CT and SF cells were calculated by the following formula:
80
% reduction
70
% reduction = [(NRb − NRt)/NRb]·100.
60
where NRt signify the number of the bacterial cells counted after adding the biocide resin and NRb is the number of the bacterial cells before adding biocide resin [27].The number of bacteria cell per (ml) was calculated by multiplying the number of colonies by the dilution factor and the result is summarized in Table 4.The CTT cells disappeared completely (reduction 100%) but the number of fecal coliforms and thermotolerant coliforms cells were reduced only to 98.5% and 95.11% respectively after contact with (pAP-DG) for 24 h. This study shows clearly that the reduction of pathogenic bacteria is better with the resin which contains an aromatic atoms, groups acid (eCOOH) and hydroxyl groups (eOH) [28,29].
50 40 30 20 0,20
0,25
0,30
0,35
0,40
0,45
pore size of granular filters (μm) Fig. 7. Comparing the pore size of granular filters with the % reduction of CT, SF and CTT.
Table 4 Antibacterial activities of pAP-DG resin (MIC 0.01 g/ml). (MIC minimum inhibitory concentration was determinate by agar dilution method).
the water and to rinse all the microbe it is necessary to go through several stages such as: coagulation, flocculation and sedimentation. Our study uses only an effective and selective step that is to introduce a polymer which has several hydroxyl groups such as poly (para-carboxy acid phenol-D-Glucose).
6
24 h
48 h
72 h
CT Blank SF CTT
404 1536 3392
1248 5285 >
1287 > >
(% reduction CT poly(pAP-DG) SF CTT
46 (88.6%) 616 (59.9%) 1810 (46.6%)
61 (95.11%) 79 (98.5%) 00 (100%)
202 (84.3%) 00 (100%) 00 (100%)
Human Microbiome Journal 11 (2019) 100053
S. Miloudi, et al.
Fig. 8. Proposed mechanism for obtained (pAP-DG) copolymer catalyzed by H2SO4.
6. Mechanism of polymerization
ion pairs with functional groups of the monomer or the polymer chain can affect the propagation rate and in some cases induces side reactions that can cease the polymerization [32]. In a side reaction-free cationic polymerization; the termination is a simple rapid reaction wherein cations are quenched through acidic hydrogen or another suitable electrophile [33]. Hence, it is important to match the reactivity of the initiator with the propagating species in order to have fast and homogeneous initiation [34]. The proposed cationic polymerization mechanism is as shown in Fig. 8. In order to obtain polymers with viable molecular weights of a few thousand, the polymerization temperature
In general, the cationic polymerization like in other polymerization methods consists of three main reactions: (a) initiation, (b) propagation, and (c) termination, as described in Fig. 5. However, termination is brought about intentionally using a suitable electrophile, which can be useful for end group modification [22]. The initiation reaction is generally fast and is not reflected in the overall rate of the polymerization [30]. The kinetics of the polymerization is predominantly controlled by the propagation step [31]. The interaction of propagating 7
Human Microbiome Journal 11 (2019) 100053
S. Miloudi, et al.
must be particularly low; to reduce the relative kinetic contribution of the transfer reaction with respect to chain propagation. The driving forces for the reaction are the high reactivity of the double bond of monomer and the high reactivity of catalyst [35].
Springer-Verlag Phenolic Resins: Chemistry, Applications and Performance (1985). [7] Edberg SC, Allen MJ, Smith DB. National field evaluation of a defined substrate method for the simultaneous evaluation of total coliforms and Escherichia coli from drinking water: comparison with the standard multiple tube technique. Appl Environ Microbiol 1988;54:1595–601. [8] Fricker CR, DeSarno M, Warden PS, Eldred BJ. False negative β-D-glucuronidase reactions in membrane lactose glucuronide agar medium used for the simultaneous detection of coliforms and Escherichia colifrom water. Lett Appl Microbiol 2008;47:539–42. [9] Hshieh FY, Beeson HD. Flammability testing of flame-retarded epoxy composites and phenolic composites. Fire Mater 1997;21:4129. [10] Seo K, Kim J, Bae JY. Towards the development of thermally latent novolac based char formers for ABS resins. Polym Deg Stab 2006;91:1513–21. [11] Young-Sik Young-. Leeand young seop Byoun. J Environ Chem Eng Howan Univ Gunson Korea 2002;19:573–718. [12] Nayaka PL, Lanka S. Laboratory of polymer and fiber department of chemistry Ravenshaw College. [13] Ley AN, Bowers RJ, Wolfe S. Indoxyl-β-glucuronide, a novel chromogenic reagent for the specific detection and enumeration of Escherichia coli in environmental samples. Can J Microbiol 1988;34:690–3. [14] Rao AM, John VT, Gonzalez RD, Akkara JA, Kaplan DL. Catalytic and interfacial aspects of enzymatic polymer synthesis inreversed micellar systems. Biotechnol Bioeng 1993;41:531–40. [15] Williams MM, Braun-Howland EB. Growth of Escherichia coli in model distribution system biofilms exposed to hypochlorous acid or monochloramine. Appl Environ Microbiol 2003;69:5463–71. [16] Rahmouni A, Harrane A, Belbachir M. Thermally stable forms of pure polyaniline catalyzed by an acid-exchanged montmorillonite clay called maghnite-H+ as an effective catalyst. Int J Polym Sci 2012;7:846710. [17] Nonaka T, Uemura Y, Ohse K, Jyono K, Kurihara S. J Appl Polym Sci 1997;66:1621. [18] Ayat M, Rahmouni A, Belbachir M. Bull Chem React Eng Cat 2016;11:376–88. [19] Tejado A, Kortaberria G, Pena C, Labidi J, Echeverria JM, Mondragon J. Appl Polym Sci 2007;106:2313. [20] Ayat M, Belbachir M, Rahmouni A. J Mol Struct 2017;1139:381–9. [21] Lee S, Teramato Y, Shiraishi N. J Appl Polym Sci 2002;83:1473. [22] Speight VL, Kalsbeek WD, DiGiano FA. Randomized stratified sampling methodology for water quality in distribution systems. J Water Resour Plann Manag 2004;130:330–8. [23] U.S. EPA National primary drinking water regulations: analytical techniques; coliform bacteria - final rule. U. S. Environmental Protection Agency. Fed Regist, 1992;57(112):24744. [24] Snead MC, Olivieri VP, Kawata K, Krusé CW. The effectiveness of chlorine residuals in inactivation of bacteria and viruses introduced by post treatment contamination. Water Res 1980;14:403. [25] Schoenen D, Kolch A. Photoreactivation of E. coli depending on light intensity after UV irradiation. Zentralbl. Hyg., 192, 565–570 (1992). [26] Evans TM, Waarvick CE, Le Seidler RJ, Chevallier MW. Failure of the most probable number technique to detect coliforms in drinking water and raw water supplies. Appl Environ Microbiol 1981;41:130–8. [27] U. S EPA National primary drinking water regulations: revisions to the total coliform rule; proposed rule. U. S. Environmental Protection Agency, Washington, DC (EPA-HQ-OW-2008-0878) (2010). [28] Stevens MN, Ashbolt NJ, Cunliffe D. Microbial indicators of drinking water quality – a NHMRC discussion paper. Canberra, Australia: National Health and Medical Research Council; 2001. [29] Dufour A, Strickland E, Cabelli V. Membrane filter method for enumerating Escherichia coli. Appl Environ Microbiol 1981;41:1152–8. [30] Xu CC, Wang M, Yung Z, Leith M. Synthesis of novolac-type phenolic resins using glucose as the substitute for formaldehyde. J Appl Polym Sci 2010;118:1191–7. [31] Gran G. Determination of the equivalence point in potentiometric titrations: part 2. Analyst 1952;77:661–71. [32] Dorner SM, Anderson WB, Gaulin T, Candon HL, Slawson RM, Payment P, et al. Pathogen and indicator variability in a heavily impacted watershed. J Water Health 2007;5:241–57. [33] Dutka BJ, El-Shaarawi A. Microbiological water and effluent sample preservation. Can J Microbiol 1980;26:921–9. [34] Fricker CR, DeSarno M, Warden PS, Eldred BJ. False negative β-D-glucuronidase reactions in membrane lactose glucuronide agar medium used for the simultaneous detection of coliforms and Escherichia coli from water. Lett Appl Microbiol 2008;47:539–42. [35] Foppen JWA, Schijven JF. Evaluation of data from the literature on the transport and survival of Escherichia coli and thermotolerant coliforms in aquifers under saturated conditions. Water Res 2006;40:401–26.
7. Conclusions New synthetic methods must include health and environmental point of views so, it is possible to synthesis macromolecules with predominant structures, discrete molecular weights and with specific functional groups, while our study is based on the use of monomer intoxic as D-Glucose instead of monomer toxic as formaldehyde. The poly (para carboxyl acid phenol-D-Glucose) polymer was successfully synthesized from para carboxyl acid phenol and D-Glucose by condensation reaction. This polymer with hydroxyls and acids handed groups are used widely as anti-bacterial. The structure of the polymer was confirmed by FT-IR and NMR spectroscopy. Synthesized polymer has good antibacterial properties and was found to have a novel and remarkable ability to remove bacteria. The Thermotolerant coliforms were completely removed (100% reduction) but fecal coliforms; fecal streptococcus coliforms were reduced only to (84.3%). The bactericidal activity of the poly (para carboxy acid phenol-D-Glucose) increased with increasing phenolic hydroxyl groups in the resin, In particular Poly (para carboxy acid-D-Glucose) has the highest antibacterial activity toward CTT. In conclusion and based on our results this polymer can be used as a material for waste water purification infected by pathogenic bacteria. Therefore, it has become essential to decrease the toxicity of catalyst changing it with heterogeneous catalyst. Conflict of interest All authors declare that there are no conflict of interest. Acknowledgment All our gratitude to the anonymous referees for their careful reading of the manuscript and valuable comments which helped in shaping this paper to the present form. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.humic.2018.11.003. References [1] Steven MP. Polymer chemistry: an introduction. 2e éd. New York: Oxford University Press; 1990. [2] Odian“la G. Polymérisation principes et applications. 3e éd. New York: Wiley et Sons; 1991. [3] Gibson SL, Riffle JS. Chemistry and properties of phenolic resins and networks. In: Rogers ME, Long TE, editors. Synthetic methods in step-growth polymers. John Wiley & Soms, Inc.; 2003. [4] Hirano K, Asami M. Phenolic resins-100 years of progress and their future. React Funct Polym 2013;73(2):256–69. [5] Gibson SL, Riffle JS, Rogers ME, Long TE. Chemistry and properties of phenolic resins and networks. Synthetic Methods in Step-growth Polymers. John Wiley & Soms Inc.; 2003. [6] Knop A, Pilato LA. ISBN 3-540-15039-0. Berlin/Heidelberg/lew York/Tokyo:
8
Contents lists available at ScienceDirect
Human Microbiome Journal journal homepage: www.elsevier.com/locate/humic
Original Article
Increasing and eliminating the (Fecal coliforms, thermotolerant coliform and fecal streptococcus) bacteria by resin of the poly (para carboxy acid phenol-D-Glucose) to clean up waste water Safia Miloudia,b, Messaoud Chaibc, Moulkheir Ayata,d, Abdelkader Rahmounid,
T
⁎
a
Department of Chemistry, Faculty of Sciences, Dr. Moulay Taher University, BP138 City, Nasr Saida 20000, Algeria Department of Chemistry, Faculty of Sciences, University Djillali Liabes, Sidi Bel Abbes 22000, Algeria c Department of Chemistry, Faculty of Science Exact, University Ibn-Khaldoun, Bp 78, Tiaret 14000, Algeria d Department of Chemistry, Faculty of Sciences, Oran1 University Ahmed Benbella, Bp 1524, El’Menouer, 31000 Oran, Algeria b
A R T I C LE I N FO
A B S T R A C T
Keywords: Poly (para-carboxy acid phenol-D-Glucose) Fecal coliforms Thermotolerant coliform Fecal streptococcus Antibacterial resin Polycondensation reaction Homogeneous catalyst
In this research paper we represent a novel synthetic antibacterial phenolic polymer (pAP-DG) containing carboxylic acid group in para position. The study of poly (p-carboxylic acid phenol D-Glucose) as an antibacterial resin was focused on the development of antimicrobial polymers to clean up the water of pathogenic bacteria. The polycondensation reaction was realized between p-carboxy acid phenol (pAP) and D-Glucose (DG) as monomers; using sulfuric acid as homogeneous catalyst. The obtained polymer was used via fecal coliform (TC), thermotolerant coliforms (CTT) and fecal streptococci (ST) bacteria to evaluate the water quality. The antibacterial activity of the synthesized resin was carried out by the contact method so the capacity of this resin was confirmed. In general, the polymer has a good antimicrobial activity against the microorganisms tested; the resulting polymer was characterized by various spectroscopy methods such as: 1H NMR, 13C NMR, DSC and FTIR and a mechanism has been proposed for this polycondensation reaction.
1. Introduction Phenolic resins generate a wide variety of polymeric materials [1,2]; each polymer is composed of a multitude of structures and a variety of raw materials with the use of catalysts. Polymer demand has grown steadily for many years then it has every day enriched by new applications of specific macromolecular materials. Phenolic resins [3,4] are among the oldest industrial polymer that are still used due to some of their properties such as dimensional stability and chemical resistance as well as their low cost [5,6]. The use of polymers and resins of the treatment of industrial effluent in general and which have antibacterial activity in particular is the interest of so many researches [7,8]. Polyphenols are commercial products synthesized by condensing phenol with formaldehyde at different molar ratios depending on the type of the desired polymer by reacting phenol on the ortho and para positions in the boxing ring with bifunctional formaldehyde where the reactions produce a mixture of methylol phenols [9,10]. The composition of the mixture can be varied by altering the phenol to formaldehyde ratio. Some phenolic compounds (resins with phenol derivatives containing
⁎
one, two or three hydroxyls groups) are bactericides are also brought back to the antibacterial activities due to the groups of hydroxyl [11,12]. The introduction of sugar into the structure brings new properties to polymers which makes them important for specific applications of higher added value and could accommodate higher production costs [13]. Glucose was used to replace the toxic formaldehyde to synthesize a novel Novolacs type phenolic resins catalyzed by strong acid (H2SO4) in temperature between (120–150 °C) and the condensation polymerization could succeed with solutions of phenol and glucose at varied molar ratios (1:0.4 to 1:0.9) through the Friedel-Crafts reaction [14,15]. But in this study our resin was prepared by polycondensation of para carboxy acid-phenol (pAP) with D-Glucose (DG) in an acid medium (H2SO4) as homogeneous catalyst (see Fig. 1). The bactericidal activity of the poly (p-carboxy acid phenol-D-Glucose) increased with increasing phenolic hydroxyl groups in the resin.
Corresponding author. E-mail address: [email protected] (A. Rahmouni).
https://doi.org/10.1016/j.humic.2018.11.003
Available online 24 January 2019 2452-2317/ © 2019 Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
Human Microbiome Journal 11 (2019) 100053
S. Miloudi, et al.
OH H2C
O
C
OH
+
H
H
C
OH
HO
C
H
H
C
OH
H
C
OH
COOH
+ H2SO4
HO
H
HO
H
H
- H 2O
OH
HO
H
OH CH
CH2OH
para-carboxy acid phenol
D- Glucose
COOH
n Fig. 1. Schematic representation of the synthesis of polymer [(pAP-DG)].
2. Experimental part
3. Results and discussion
2.1. Materials
The polycondensation reaction of pAP with DG was examined in the presence of H2SO4 as a catalyst at 130 °C (see Fig. 1).
Para-carboxy acid phenol and D-Glucose (99%) were purchased from Aldrich (Paris, France) purified by fractional distillation under reduced pressure. Tetrahydrofuran (THF) Sigma-aldrich, Dimethyl sulfoxide (DMSO) Panreac, acetone, methanol, solution of sodium hydroxide, sulfuric acid (98%) and distilled water were used for solutions’ preparation
3.1. Determination of the pKa of the polymer The aqueous solution was prepared by mixing 25 mg of resin in aqueous solution NaNO3 (0.01 M) with magnetic stirring for 24 h for deprotonated the carboxylic groups of resin. Finally, the excess of the free protons in the solution were titrated by a standard solution of NaOH (0.01 M) at controlled temperature (25 °C). The variation of pH with VNaOH added and its derivative curve were shown in Fig. 2(a) and (b). The pKa corresponds to the volume of half-equivalence (V = 62 ml) was 4.28 which allow to determinate the total concentration CA of reactive carboxylic sites of polymer [16]. All solutions were maintain at 25 °C and contain high purity NaNO3 (0.01 M) as supporting electrolyte.
2.2. Methods The polymers were characterized by FT-IR spectroscopy scanned on SHIMADZU spectrometer using KBr pellets at waves ranging from 400 to 4000 cm−1. The spectra of nuclear magnetic resonance of proton (1H NMR) and carbon (13C NMR) were recorded in a 300 MHz Bruker spectrometer, in deuterium acetone solvent using tetra methyl silane (TMS) as internal standard Thermal analysis DSC is a Differential scanning calorimetry apparatus (200PC NETZCH, 204F1PHONIX240-12–0110-L DSC) the measurements were carried out with a heating rate of 10 °C /min under 50 cm3/min nitrogen flow.
3.2. Spectroscopy characterization of polymer The structure of the resulting polymer was confirmed by 1H NMR, C NMR and Infrared spectroscopy (IR) measurements. The assignments of peaks of polymer were given in Tables 1 and 2. we can see from Figs. 3 and 4, the appears of a new peak at 1.42 ppm and 25.25 ppm corresponds to the methylene bridge. the same results obtained by Nonaka et al [17]. The polymers were characterized by IR and the spectra were scanned on SHIMADZU spectrometer using KBr pellets at wave numbers from 400 to 4000 cm−1 (Fig. 5). The infrared spectra of the synthesized resins in the present investigation were dominated by the large OH stretch which encompasses signals related to the hydroxyl [18]. Absorption bands characteristic of the resin (pAP-DG), and their functions were shown in the Table 3. The position of the absorption band around 1660 cm−1 corresponding to the stretching vibration of C]O indicates that the group C]O and conjugated double bond (aromatic ring). Broadband between 3065 and 3400 cm−1 corresponding to the OH function is attributable to the presence of carboxylic acid and alcohol. The ]CHR] bond of the bending vibration indicates the presence of ]CHR] (methylene bridg) [19]. 13
2.3. Polymerization procedure In a necked flask equipped with a reflux condenser and a thermometer introduced (0.1 mol) p-carboxy acid phenol and (0.05 mol) of DGlucose with 0.01 g initiator and the mixture was stirred for 1 h at 130 °C .The polycondensation reaction of para carboxy acid phenol and D-Glucose was carried out in a homogeneous system using sulfuric acid (H2SO4) Riedel- de Haein as a catalyst. After 2 h recuperating the contents of the flask and added 1.7 ml of sodium hydroxide solution (1 M) for neutralization and then heated to remove water. The reaction scheme was shown in Fig. 1. The resulting polymer was dissolved in acetone and precipitated in Tetrahydrofuran (THF) Sigma-aldrich then dried at room temperature. The purification procedure was repeated several times in order to obtain a highly purified polymer to reach a higher yield of 80%. The product was characterized by FT-IR, 1H NMR, 13 C NMR spectroscopy to confirm the structure. 2
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12
1,8 1,6
10 1,4
8
1,0
pH
d(pH/dV)
1,2
0,8
6
0,6 0,4
4
0,2 0,0
2
-0,2 -20
0
20
40
60
80
100
120
140
160
0
180
100
200
300
400
500
volume (ml)
volume (ml)
(a)
(b)
Fig. 2. (a) Plots of the experimental results for the pH titration for the polymer (pAP-DG), (b) the pH variation vs. the volume of NaOH (0.1 M) for the compound (pAP-DG).
4. Thermal analysis
low turbidity waters (< 1 NTU). Without chemical pretreatment, the removal by filtration averaged 69% (0.51 logs) for Giardia and 28% (0.14 logs) for turbidity. Adding alum and polymer filter aids increased the removal efficiency to more than 95% (1.30 logs) for Giardia, 99% (2 logs) for total coliforms bacteria and 70% (0.52 logs) for turbidity. Other process variables such as filtration mode (direct and in-line filtrations), filter media (monomedium with sand, and dual-media with sand and anthracite) and temperature (5 °C and 18 °C) did not significantly affect the filtered water quality [22]. Varying the filtration rate from 5 to about 20 m/h had little effect on removal of Giardia, total coliforms bacteria and heterotrophic bacteria, but increased turbidity in the filtered water [23].
Thermal properties of the copolymers were investigated by DSC. The DSC analysis of (pAP-DG) copolymers indicates two closely neighboring melting endotherms at temperatures between 160 °C and 210 °C and the glass transition temperature Tg of the resulting copolymers was observed in the temperature ranges of 50–70 °C as mentioned in Fig. 6 which means that the polymer is semi-crystalline [20,21]. 5. Study of pretreatment for removal of microbes The importance of chemical coagulation pretreatment for removal of microbes by granular filtration has been emphasized by numerous studies. Al-Ani et al. (1986) conducted a pilot-scale filtration study for Table 1 1 H NMR Chemical shifts and signal assignation of poly (pAP-DG). Structure
3
δ (ppm), intensity
Attribution
(2.05–2.23,d) (1.42,d) (4.25,s) (6.7–6.93,s), (11.15, s) (3.61–3.66, m) (1.78–1.82, m)
Hm Hd Ha Hc aromatic Hb H n, l, j, g, f H e ,h, i, k
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Table 2 13 C NMR Chemical shifts and signal assignation of (PAP-DG) polymer. Structure
HO
i CH2
HO
Attribution
δ (ppm)
Ca Cb Cc Cd COOH Ce,f,g,h,i
205.32 135.86 119.06 25.25 130.35 28.16–29.81
C h HO C
g
OH
Cf HO OH
a
C e CH
d
c b COOH
n
Fig. 3. 1H RMN spectrum of the (pAP-DG) polymer. 4
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Fig. 4.
13
C RMN spectrum of the (pAP-DG) polymer.
5.1. Action of resin to removal of microbial pathogens by granular filtration
Table 3 Different IR absorption bands of the synthesized poly(pAP-DG).
Removal of microbial pathogens by granular filtration does not rely on physical processes alone. Comparing the pore size of granular filters with the size of most types of microbe (as in Fig. 7), it is evident that effective removal of microbes by granular filtration cannot rely on physical straining alone, at least at the initial stage of a filter run [24]. The removal of particles by granular filtration is considered to involve two steps: transport of particles from suspension to filter medium, followed by attachment of particles to the medium. The transport step depends on the physical and hydrodynamic properties of the system.
Chemical shift
Absorption Band
Nature
vOH
3200–3500 3241 3065 2859 1660 1611–1578 1363 1232 1103–1014 890–952 758
OH bond OH acid Stretching vibration Aromatic Aliphatic Carboxylic acid benzene ring Phenolic Phenolic Aromatic Para substituted Ortho substituted
vCH vCH v(C]O) v(C]C) dip(OH) dip(C]O) dip(CH) dop(CH) dop(CH)
5.2. Biological activity In general to make a well-defined study of the biological activity of
Fig. 5. Infrared spectrum of poly(pAP-DG) in the region 400–4000 cm−1. 5
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Fig. 6. Thermogramm of the (pAP-DG) polymer.
5.3. Antimicrobial activity
CT SF CTT
90
The antibacterial activity was tested by the contact method [25,26] with the use of polymeric material (pAP-DG) which is insoluble in water. The number of the bacterial colonies was counted after the test tubes incubation at 37 °C for 24 h under (200 rpm). The percent reduction of the bacterial: CTT, CT and SF cells were calculated by the following formula:
80
% reduction
70
% reduction = [(NRb − NRt)/NRb]·100.
60
where NRt signify the number of the bacterial cells counted after adding the biocide resin and NRb is the number of the bacterial cells before adding biocide resin [27].The number of bacteria cell per (ml) was calculated by multiplying the number of colonies by the dilution factor and the result is summarized in Table 4.The CTT cells disappeared completely (reduction 100%) but the number of fecal coliforms and thermotolerant coliforms cells were reduced only to 98.5% and 95.11% respectively after contact with (pAP-DG) for 24 h. This study shows clearly that the reduction of pathogenic bacteria is better with the resin which contains an aromatic atoms, groups acid (eCOOH) and hydroxyl groups (eOH) [28,29].
50 40 30 20 0,20
0,25
0,30
0,35
0,40
0,45
pore size of granular filters (μm) Fig. 7. Comparing the pore size of granular filters with the % reduction of CT, SF and CTT.
Table 4 Antibacterial activities of pAP-DG resin (MIC 0.01 g/ml). (MIC minimum inhibitory concentration was determinate by agar dilution method).
the water and to rinse all the microbe it is necessary to go through several stages such as: coagulation, flocculation and sedimentation. Our study uses only an effective and selective step that is to introduce a polymer which has several hydroxyl groups such as poly (para-carboxy acid phenol-D-Glucose).
6
24 h
48 h
72 h
CT Blank SF CTT
404 1536 3392
1248 5285 >
1287 > >
(% reduction CT poly(pAP-DG) SF CTT
46 (88.6%) 616 (59.9%) 1810 (46.6%)
61 (95.11%) 79 (98.5%) 00 (100%)
202 (84.3%) 00 (100%) 00 (100%)
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Fig. 8. Proposed mechanism for obtained (pAP-DG) copolymer catalyzed by H2SO4.
6. Mechanism of polymerization
ion pairs with functional groups of the monomer or the polymer chain can affect the propagation rate and in some cases induces side reactions that can cease the polymerization [32]. In a side reaction-free cationic polymerization; the termination is a simple rapid reaction wherein cations are quenched through acidic hydrogen or another suitable electrophile [33]. Hence, it is important to match the reactivity of the initiator with the propagating species in order to have fast and homogeneous initiation [34]. The proposed cationic polymerization mechanism is as shown in Fig. 8. In order to obtain polymers with viable molecular weights of a few thousand, the polymerization temperature
In general, the cationic polymerization like in other polymerization methods consists of three main reactions: (a) initiation, (b) propagation, and (c) termination, as described in Fig. 5. However, termination is brought about intentionally using a suitable electrophile, which can be useful for end group modification [22]. The initiation reaction is generally fast and is not reflected in the overall rate of the polymerization [30]. The kinetics of the polymerization is predominantly controlled by the propagation step [31]. The interaction of propagating 7
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must be particularly low; to reduce the relative kinetic contribution of the transfer reaction with respect to chain propagation. The driving forces for the reaction are the high reactivity of the double bond of monomer and the high reactivity of catalyst [35].
Springer-Verlag Phenolic Resins: Chemistry, Applications and Performance (1985). [7] Edberg SC, Allen MJ, Smith DB. National field evaluation of a defined substrate method for the simultaneous evaluation of total coliforms and Escherichia coli from drinking water: comparison with the standard multiple tube technique. Appl Environ Microbiol 1988;54:1595–601. [8] Fricker CR, DeSarno M, Warden PS, Eldred BJ. False negative β-D-glucuronidase reactions in membrane lactose glucuronide agar medium used for the simultaneous detection of coliforms and Escherichia colifrom water. Lett Appl Microbiol 2008;47:539–42. [9] Hshieh FY, Beeson HD. Flammability testing of flame-retarded epoxy composites and phenolic composites. Fire Mater 1997;21:4129. [10] Seo K, Kim J, Bae JY. Towards the development of thermally latent novolac based char formers for ABS resins. Polym Deg Stab 2006;91:1513–21. [11] Young-Sik Young-. Leeand young seop Byoun. J Environ Chem Eng Howan Univ Gunson Korea 2002;19:573–718. [12] Nayaka PL, Lanka S. Laboratory of polymer and fiber department of chemistry Ravenshaw College. [13] Ley AN, Bowers RJ, Wolfe S. Indoxyl-β-glucuronide, a novel chromogenic reagent for the specific detection and enumeration of Escherichia coli in environmental samples. Can J Microbiol 1988;34:690–3. [14] Rao AM, John VT, Gonzalez RD, Akkara JA, Kaplan DL. Catalytic and interfacial aspects of enzymatic polymer synthesis inreversed micellar systems. Biotechnol Bioeng 1993;41:531–40. [15] Williams MM, Braun-Howland EB. Growth of Escherichia coli in model distribution system biofilms exposed to hypochlorous acid or monochloramine. Appl Environ Microbiol 2003;69:5463–71. [16] Rahmouni A, Harrane A, Belbachir M. Thermally stable forms of pure polyaniline catalyzed by an acid-exchanged montmorillonite clay called maghnite-H+ as an effective catalyst. Int J Polym Sci 2012;7:846710. [17] Nonaka T, Uemura Y, Ohse K, Jyono K, Kurihara S. J Appl Polym Sci 1997;66:1621. [18] Ayat M, Rahmouni A, Belbachir M. Bull Chem React Eng Cat 2016;11:376–88. [19] Tejado A, Kortaberria G, Pena C, Labidi J, Echeverria JM, Mondragon J. Appl Polym Sci 2007;106:2313. [20] Ayat M, Belbachir M, Rahmouni A. J Mol Struct 2017;1139:381–9. [21] Lee S, Teramato Y, Shiraishi N. J Appl Polym Sci 2002;83:1473. [22] Speight VL, Kalsbeek WD, DiGiano FA. Randomized stratified sampling methodology for water quality in distribution systems. J Water Resour Plann Manag 2004;130:330–8. [23] U.S. EPA National primary drinking water regulations: analytical techniques; coliform bacteria - final rule. U. S. Environmental Protection Agency. Fed Regist, 1992;57(112):24744. [24] Snead MC, Olivieri VP, Kawata K, Krusé CW. The effectiveness of chlorine residuals in inactivation of bacteria and viruses introduced by post treatment contamination. Water Res 1980;14:403. [25] Schoenen D, Kolch A. Photoreactivation of E. coli depending on light intensity after UV irradiation. Zentralbl. Hyg., 192, 565–570 (1992). [26] Evans TM, Waarvick CE, Le Seidler RJ, Chevallier MW. Failure of the most probable number technique to detect coliforms in drinking water and raw water supplies. Appl Environ Microbiol 1981;41:130–8. [27] U. S EPA National primary drinking water regulations: revisions to the total coliform rule; proposed rule. U. S. Environmental Protection Agency, Washington, DC (EPA-HQ-OW-2008-0878) (2010). [28] Stevens MN, Ashbolt NJ, Cunliffe D. Microbial indicators of drinking water quality – a NHMRC discussion paper. Canberra, Australia: National Health and Medical Research Council; 2001. [29] Dufour A, Strickland E, Cabelli V. Membrane filter method for enumerating Escherichia coli. Appl Environ Microbiol 1981;41:1152–8. [30] Xu CC, Wang M, Yung Z, Leith M. Synthesis of novolac-type phenolic resins using glucose as the substitute for formaldehyde. J Appl Polym Sci 2010;118:1191–7. [31] Gran G. Determination of the equivalence point in potentiometric titrations: part 2. Analyst 1952;77:661–71. [32] Dorner SM, Anderson WB, Gaulin T, Candon HL, Slawson RM, Payment P, et al. Pathogen and indicator variability in a heavily impacted watershed. J Water Health 2007;5:241–57. [33] Dutka BJ, El-Shaarawi A. Microbiological water and effluent sample preservation. Can J Microbiol 1980;26:921–9. [34] Fricker CR, DeSarno M, Warden PS, Eldred BJ. False negative β-D-glucuronidase reactions in membrane lactose glucuronide agar medium used for the simultaneous detection of coliforms and Escherichia coli from water. Lett Appl Microbiol 2008;47:539–42. [35] Foppen JWA, Schijven JF. Evaluation of data from the literature on the transport and survival of Escherichia coli and thermotolerant coliforms in aquifers under saturated conditions. Water Res 2006;40:401–26.
7. Conclusions New synthetic methods must include health and environmental point of views so, it is possible to synthesis macromolecules with predominant structures, discrete molecular weights and with specific functional groups, while our study is based on the use of monomer intoxic as D-Glucose instead of monomer toxic as formaldehyde. The poly (para carboxyl acid phenol-D-Glucose) polymer was successfully synthesized from para carboxyl acid phenol and D-Glucose by condensation reaction. This polymer with hydroxyls and acids handed groups are used widely as anti-bacterial. The structure of the polymer was confirmed by FT-IR and NMR spectroscopy. Synthesized polymer has good antibacterial properties and was found to have a novel and remarkable ability to remove bacteria. The Thermotolerant coliforms were completely removed (100% reduction) but fecal coliforms; fecal streptococcus coliforms were reduced only to (84.3%). The bactericidal activity of the poly (para carboxy acid phenol-D-Glucose) increased with increasing phenolic hydroxyl groups in the resin, In particular Poly (para carboxy acid-D-Glucose) has the highest antibacterial activity toward CTT. In conclusion and based on our results this polymer can be used as a material for waste water purification infected by pathogenic bacteria. Therefore, it has become essential to decrease the toxicity of catalyst changing it with heterogeneous catalyst. Conflict of interest All authors declare that there are no conflict of interest. Acknowledgment All our gratitude to the anonymous referees for their careful reading of the manuscript and valuable comments which helped in shaping this paper to the present form. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.humic.2018.11.003. References [1] Steven MP. Polymer chemistry: an introduction. 2e éd. New York: Oxford University Press; 1990. [2] Odian“la G. Polymérisation principes et applications. 3e éd. New York: Wiley et Sons; 1991. [3] Gibson SL, Riffle JS. Chemistry and properties of phenolic resins and networks. In: Rogers ME, Long TE, editors. Synthetic methods in step-growth polymers. John Wiley & Soms, Inc.; 2003. [4] Hirano K, Asami M. Phenolic resins-100 years of progress and their future. React Funct Polym 2013;73(2):256–69. [5] Gibson SL, Riffle JS, Rogers ME, Long TE. Chemistry and properties of phenolic resins and networks. Synthetic Methods in Step-growth Polymers. John Wiley & Soms Inc.; 2003. [6] Knop A, Pilato LA. ISBN 3-540-15039-0. Berlin/Heidelberg/lew York/Tokyo:
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