Nghiên cứu tổng hợp và khảo sát khả năng diệt khuẩn của oligome trên cơ sở guanidin trong xử lý nước nhiễm khuẩn tt tiếng anh

+ Researching clarifying issues related to mechanisms, methods and synthesis conditions of oligomer based on guanidine, in order to obtain high efficiency OHMG.HCl with appropriate molec

NGUYEN VIET HUNG

SYNTHESIS AND INVESTIGATE THE ANTISEPTIC ACTIVITY

OF OLIGOMER BASED ON THE GUANIDINE FOR TREATING

THE INFECTED WATER SUPPLY

Major: Organic chemistry Code: 944 01 14

SUMMARY OF DOCTORAL THESIS IN CHEMISTRY

HA NOI - 2019

ACADEMY OF MILITARY SCIENCE AND TECHNOLOGY

Scientific Supervisors:

1 Prof Dr Nguyen Viet Bac

2 Associate Prof Dr Tran Van Chung

Reviewer 1: Prof Dr Vu Thi Thu Ha

Vietnam Institute of Industrial chemistry

Reviewer 2: Associate Prof Dr Chu Chien Huu

Academy of Military Science and Technology

Reviewer 3: Associate Prof Dr Dong Kim Loan

Vietnam National University, Ha Noi

The thesis was defended at the Doctoral Evaluating Council at Academy level held at Academy of Military Science and Technology at… date ……… , 2019

The thesis can be found at:

- Vietnam National Library

- Library of Academy of Military Science and Technology

INTRODUCTION

1 The necessaries

The issue of environmental sanitation has been of particular concern to the public as it directly relates to human health and environmental ecology Pollution of water resources due to the wastewater from factories and domestic effluents containing pathogenic microorganisms is the main cause of disease outbreaks affecting human health Currently, people mainly use traditional disinfectants such as hypochlorite, chlorine, chloramine B However, these disinfectants must be often used in large quantities, when disinfecting the water producing chlorine compounds so that are directly harmful to the health of humans and organisms [2], [4]

Therefore, new biocidal products, with the high disinfection effect, low toxicity, that do not create dangerous by-products in the process of sterilization, easy to use and friendly with the environment have been used by many domestic and foreign organizations There are three types of antimicrobial antigens that are being extensively studied, including antibacterial peptidomimetic polymers, amphiphilic polymers and oligomers, and nonionic polymers [51] Oligome hexamethylene guanidine hydrochloride (OHMG.HCl) is a cationic polymer being studied as a biocide in medical and industrial applications, due to the wide range of bactericidal, low-usage and highly active disinfectant The research on the synthesis of guanidine-based bactericidal oligomers has been started However, there have been no published guidelines on the method and conditions for the synthesis of bactericidal oligomers as well as the application of compounds based on guanidine These are the bases for PhD students to

select and propose thesis topic “ Synthesis and investigate the antiseptic activity of oligomer based on guanidine for treating the infected water supply”.

2 The goal of thesis

+ The synthesized and classification of bactericidal oligomer based on guanidine

to be applied in pratice

+ Evaluation of the efficiency, bactericidal activity of guanidine oligomer synthesized in treatment of infected water

3 The main research

methods and chemicals used in disinfection; Situation of research, application and methods of synthesis of guanidine based biocide

+ Researching clarifying issues related to mechanisms, methods and synthesis conditions of oligomer based on guanidine, in order to obtain high efficiency OHMG.HCl with appropriate molecular weights for the purpose of treating infected water

+ The study evaluated the disinfection ability of synthetic oligomers in infected water treatment

+ Study on the compare bactericidal effect and environmental friendliness of synthetic oligome with biocide based on chlorine compounds

4 Scientific and practical value of the thesis

+ Successfully synthesized and clarified the scientific basis of the reaction conditions of guanidine-based oligomer synthesis

+ Results of the study evaluating the disinfection ability of synthetic oligomers

in the treatment of infected water sources, showing efficacy and environmental friendliness compared with chlorine-based microbicides The results of the thesis contribute to the diversification of bioproducts used in the treatment of bacterial wastewater in Vietnam

5 Structure of the thesis

The thesis has 124 pages including:

Introduction: 3 pages; Chapter 1 Overview: 33 pages; Chapter 2 Subjects and methods of research: 13 pages; Chapter 3 Results and discussion: 45 pages; Conclusion:

2 pages; List of scientific works related to thesis: 1 page; References: 13 pages (105 references)

CHAPTER 1: OVERVIEW 1.1 Contaminated wastewater

The sources of biological pollution and bacterial contamination in the water environment are mainly cause by domestic wastewater, hospital wastewater, biological wastes and waste…

Current technologies for treating bacterial waste water include: Sequencing batch reactor (SBR); Membrane Bio Reactor (MBR); Moving Bed Biofilm Reactor (MBBR); Continuous biological treatment technology uses a variety of microorganisms (AAO: Anaerobic - Anoxic - Oxic) The main principle of method are biological wastewater treatment combined with the physical method [11] [14]

1.2 Disinfection methods for contaminated wastewater

Various methods of disinfection for wastewater include: chemical methods (using some strong oxidants such as chlorine and its compounds, ozone .); Physical methods (using UV rays) and some other methods (thermal sterilization, ultrasound, silver ion reduction )

1.3 Polymers and bactericidal oligomers based on guanidine compounds

Oligomer bactericides based on guanidine namely oligo (hexamethylene) guanidine hydrochloride - OHMG.HCl, IUPAC nomenclature are guanidine hydrochloride polyhexamethylene (PHMG) Oligo (hexamethylene) guanidine hydrochloride usually exists in the form of white or yellowish powder; good solubile in

water, alcohol, glycol, ether With the ability to kill bacteria at low levels, not toxic, odorless; OHMG.HCl has been widely used as a biocide in homes, hospitals, water purifiers in many countries around the world [41- 42], [54], [104]

- The bactericidal effect of the OHMG.HCl molecule is based on the positive charge of the guanidine moiety within each monomer The mechanism of action is: the adsorption of the positively charged N+ ions of the OHMG.HCl molecule on negative charge sits on the bacterial cell wall attracts or altering the osmotic pressure that disrup the cell membrane

hexamethylene diamine (HMDA) in the melting state This is a condensed reaction, occurring in the nucleophilic (AN) addition mechanism as shown in Figure 1.1

Figure 1.1: Straight chain OHMG.HCl synthesis reaction using HMDA

Conclusion of the chapter 1:

Through the overview of technology, methods and materials for synthesis of disinfectant OHMG.HCl, if performed under conditions of our country, it will be highly feasible Therefore, the research and production of OHMG.HCl and its application in treating waste water are the research objective of the thesis

CHAPTER 2: SUBJECTS AND METHODS OF RESEARCH

of microorganisms: Bacillus subtilis, E.coli, Coliform, mold; Environment of microorganisms growing: normal, LB broth and Hansen broth; Standard tube NaOH 1N: plastic tube, specific gravity 1,03 g/cm 3 , pH=14 (H 2 O, 20 o C), (Merck, Germany);

Standard tube HCl 1N : plastic tube, specific gravity 1,03 g/cm 3 , pH <1 (H 2 O, 20 o C), (Merck, Germany); The water of To Lich river, Duc Giang General Hospital wastewater,

Phung Khoang slaughterhouse wastewater; NaCl solution: phase at 0.2 N; 0.3 N; 0.4 N;

0.5 N; Solvents: Ethanol, Acetone, DMSO, ether

Organic synthesis tools include three-necked flask, reed condenser, gas absorber, stirrer, heating system, cooling system .; MSH 20 D/MP4 magnetic stirrer; Laboratory instruments: measuring cup 500ml; 250m, bottle contains sample

Electronic balance AAA250 - ADAM accuracy 0.0001g; PH Meter HI2215 pH / ORP Meter - HANNA; Ubbelohde capillary glaucoma

2.2.2 OHMG.HCl synthesis process

In a 500 mL three-neck flask, there is a

stirrer, thermometer and vacuum system,

NH3 absorption system Take 20 g (0.172

mol) hexamethylene diamine and 16.44 g

(HMDA:GHC = 1:1 ratio) Heat and

maintain the reaction at 100 °C for 1 h, then

raise the reaction temperature to range of

temperature for 3 to 8 h The resulte

OHMG.HCl product is a viscous liquid,

bright yellow and solidified after cooling Figure 2.1 Reaction equipment system

OHMG.HCl synthesis

The factors that affect the reaction process such as the proportion of reactants, time and reaction temperature were studied Efficiency, metabolism of reaction are determined by NH3 removal from the reaction system The synthesized oligomers are dissolved in distilled water and filtered through a Schott vacuum filter at 60 °C for 3 h; The formed gel is dried to constant weight [19-20], [45]

2.2.3 Methods of analysis, determination of physical and chemical properties

Analytical methods such as infrared spectroscopy (IR); nuclear magnetic resonance (1H NMR,13C NMR,COSY,HSQC); gel chromatography (GPC); Mass spectrometry (MS); Thermal analysis method (TGA); Liquid chromatography - mass spectrometry (LC- MS); Scan electron microscopy (SEM) and Transmission electron microscopy (TEM) were used

2.2.4 Methods of evaluation, testing of bactericidal activity

2.2.4.1 The solubility of the product

2.2.4.2 Methods of assessment of disinfection ability

+ Disk diffusion

+ The time for killing bacteria by minimum bacteria concentration (MBC)

2.2.5 Investigation of factors affecting the disinfection ability of OHMG.HCl for contaminated wastewater

The infected water samples are taken according to TCVN 6663-1: 2011, preserved and stored according to TCVN 6663-3: 2008 and analyzed and determined before and after treatment according to current Vietnamese standards, applying for wastewater treatment

CHAPTER 3: RESULTS AND DISCUSSION 3.1 Research results of syntheszed OHMG.HCl oligomer

3.1.1 Study on the ability to synthesize OHMG.HCl by molten condensation method

3.1.1.1 Investigation of OHMG.HCl synthesis

Table 3.1: Infrared spectral analysis of OHMG.HCl synthesis

Figure 3.1: Infrared spectrum of GHC Figure 3.2: Infrared spectrum of HMDA

Figure 3.3: Infrared spectrum

group - NH-C (NH) -NH-; peak 3.39 ppm (1H, J = 3,371 Hz)- corresponds to the group

– CH2-NH-; peak 1.81 ppm (2H, J = 1,996 Hz) - corresponds to the group (- CH 2-)n,

indicating the formation of OHMG.HCl in the synthesized product, similar to the results

of Martin Albert et al [70]

The results of this study have shown that OHMG.HCl can be synthesized from GHC and HMDA by the melting condensation reaction

3.1.1.2 The dependence of molecular weights on the basic properties of OHMG.HCl

Table 3.2: OHMG.HCl molecular mass was obtained in the study

* Effect of molecular weight to internal viscosity of OHMG.HCl solution

Table 3.3: Relationship of molecular weight to internal viscosity

of OHMG.HCl solution obtained in the study

2 4 6 8 10 12 200

Figure 3.5: Relational chart Mw to

internal viscosity of OHMG.HCl solution

The results showed that the internal viscosity of OHMG.HCl solution increased with the increase

of molecular weight Viscosity change occurs due to the in OHMG.HCl molecular size This leads to OHMG.HCl having a too large molecular weight, which in turn affects the molecular dynamics and solubility of the oligomer

* Effect of molecular weight to solubility of OHMG.HCl in various solvents

Table 3.4: Effect of molecular weight to solubility of OHMG.HCl

Sample average Molecular

weight M W (g/mol)

Solubility (g/100 g solution) Distilled water Ethanol Acetone

to select the optimal conditions to synthesize products with high water solubility, when they want to be used as biocides in wastewater disinfection technology

Table 3.5: Effect of molecular weight on E coli's ability to kill OHMG.HCl

(Antibacterial ring diameter - mm)

Table 3.5 shows that the high efficiency of E coli corresponds to the molecular

weight of OHMG.HCl reaches from 500 g/mol or more

3.1.2 Research and survey the factors affecting the synthesis of OHMG.HCl at scale

of 150 g /batch

3.1.2.1 Effect of reaction temperature

Carry out the reaction according to the conditions: molar ratio [HMDA]: [GHC]

= 1: 1 (Specifically, weighing 100 g HMDA and 82.2 g GHC respectively in the reaction system); Reaction time = 5 hours Surveying the effect of reaction temperature on the average molecular weight of OHMG.HCl in the range 120 °C - 180 °C The results are shown in Table 3.6 and Figure 3.6 graph

Table 3.6: Effect of reaction temperature to molecular weight

The results showed that the

reaction temperature Previous studies have shown [46] that the maximum bactericidal effect corresponds to the

OHMG.HCl from 500 to 650 g/mol

OHMG.HCl produceds gives Mw =

526 g/mol

3.1.2.2 Effect of reaction time

Table 3.7: Effect of reaction time to averagre molecular weight of OHMG.HCl

averagre Molecular weight

Reaction time, hours

Figure 3.7: Effect chart of reaction time

to Mw of OHMG.HCl

The results show that the

OHMG.HCl is dependent on the reaction time and increasing with time of reaction Previous research results [46], [70], [101] show that the maximum water-bactericidal efficiency of oligomers is the highest value with an average molecular weight of 500 - 650 g/mol From the results of the survey, the optimum time for OHMG.HCl synthesis is 5 hours

3.1.2.3 Effect of content of reactants

Bảng 3.8: Effect of content of reactants to averagre molecular weight of OHMG.HCl

OHMG.HCl from 500 to 650 g/mol (due to the solubility of the product in water) So that the optimum ratio of reactants is selected as [HMDA]: [GHC] = 1: 1.1

3.2 Results of analysis of the structure and properties of OHMG.HCl

3.2.1 Physical and chemical analysis of OHMG.HCl

3.2.1.1 Results of infrared spectrum

Table 3.9 shows the correlation between infrared absorption peaks and

functional groups in OHMG.HCl synthesized corresponding to the infrared spectrum of products imported from the Russian Federation

Table 3.9: Peak comparison results of OHMG.HCl infrared spectrum

Functional

group

Peak (cm -1 )

from the Russian Federation

Figure 3.9: Infrared spectrum of

OHMG.HCl from the Russian Federation

Figure 3.10: Infrared spectrum of OHMG.HCl synthesized

3.2.1.2 Results of 1 H-NMR spectrum and 13 C-NMR spectrum

Figure 3.13: Structural formula (expected) of OHMG.HCl synthesized

Based on the above formula, the result of complete reconciliation of the resonant signals in the 13C-NMR spectrum of OHMG.HCl from Figure 3.14 is shown in Table 3.10 as follows

Table 3.10: The results of the resonance coordinate signal in the 13 C-NMR spectrum

to the C IV 'atoms of guanidine atoms once that is also close to 156-159 ppm along with the main resonance of C IV

Through the 1HNMR spectrum and 13CNMR spectrum spectra, the presence of

the major derivative in the product was the linear oligo hexamethylene guanidine hydrochloride

3.2.1.3 Results of mass spectrum

Figure 3.14: Mass spectrum