Reasearch on the ability of decolorization of reactive dyes and degradation of herbicides / dioxin by microbes producing laccase

MINISTRY OF EDUCATION AND TRAINING VIET NAM ACADAMY OF SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY ----------------------------- PHUNG KHAC HUY CHU REASEARCH ON THE ABILITY OF DECOLORIZATION OF REACTIVE DYES AND DEGRADATION OF HERBICIDES/DIOXIN BY MICROBES PRODUCING LACCASE Major: Environmental Technique Code: 9.52.03.20 SUMMARRY OF ENVIRONMENTAL TECHNIQUE DOCTORAL THESIS Hanoi, 2018 The work was realized in Graduate University of Sicence and Technology – Vietnam Academy of Science and Technology Advisor 1: Assoc. Professor, Dr Dang Thi Cam Ha Advisor 2: Examiner 1:.. Examiner 2:.. Examiner 3: The thesis will be evaluated by doctoral committee at Graduate University of Science and Technology, Vietnam Academy of Science and Technology on date..month2018. The thesis is available at: - Library of Graduate University of Science and Technology - National Library of Vietnam 1 INTRODUCTION 1. The necessary of the thesis In recent years, pollution by chemicals including difficultly- decomposed organic substances (POPs) increasingly created by human beings, causing a lot of implications for human health and the environment. In such developing countries as Vietnam, the use and discharge of chemical waste in agriculture, medicine, industry and other manufacturing sectors without ability to be controlled has caused serious consequences to the environment and humans. In addition, herbicides containing dioxin in existence by the wars after more than 40 years and the nature of dioxins formed during the process of industrial development still continues to cause more serious consequences for the ecological environment and people in Vietnam. Vietnam is a tropical country of biodiversity in the top 10 of the world, especially microbial diversity. There are many species of microorganisms with differently-synthesized enzymes; two of which are oxidoreductive and peroxidase enzyme having included many high performance applications in the fields of economy and protecting human health. Being not only high-activity laccase but also capable of strong activity, just together with liberal oxygen as well as the help of fastening substance or fastening systems (mediator). Then this laccase has been in the greater consideration than any other. Therefore, the subject of study and research with the name "Research on the ability of decolorization of reactive dyes and degradation of herbicides/dioxin by microbes producing laccase" has been conducted. 2 2. Research objectives A selection of microbes capable of laccase, laccase-like strains borning from Ba Vi National forests, in the soil of contaminated herbicides/dioxin at the Bien Hoa formed airbase; Ability to degrade the herbicides containing dioxin and decolor type reactive dyes by laccase, laccase-like strains, and microbe strains were chosen to apply in the orientation in defense activities. 3. The implementation content Isolation, classification of fungi, ascomycetes that capable of producing high potential laccase-like and laccase-like from the Ba Vi National Forest and dioxin contaminated soil at Bien Hoa Airbase, Dong Nai Province; selection cultured medium highly capable of laccase, laccase-like biosynthesis, representatives from Basicomycetes and Streptomycetes are highly isolated; Research the chemical-physical characteristics, chemical- biological basis of laccase, laccase-like are purified; Performance evaluation of decolorization types of synthetic dyes, reactive dyes by laccase, laccase-like strain of microbes and laccase biosynthesis, laccase-like; Evaluate the biodegradable herbicides 2.4-D, 2, 4, 5-T pure and have in land pollution in Bien Hoa formed airbase by laccase, single race and mixed fungal strains laccase biosynthesis; Evaluate the performance of biodegradable type 2, 3, 7, 8-TCDD with single fungal strains and strain mixture laccase biosynthesis. 3 CHAPTER 1 LITERATURE REVIEW 1.1. Laccase, laccase-like microbial biosynthesis and laccase, laccase-like 1.1.1. General introduction of laccase Laccase was interested in research from many years with the content: Group microorganisms laccase synthesis, molecular structure and mechanism of catalytic activity, the study of gene, protein and chemical-physical characteristics of laccase. Laccase is, through the previous ones, shown to be the subject of quite detailed research. There has, so far, been little use of many microbial strains of laccase synthesis in dealing with environmental pollution in general and dyes, difficultly- decomposed organic pollutants in particular, because selecting the combination of many micro- organisms animals of likely higher laccase synthesis is not simple and requires much research effort. This is the opportunity for research on purpose of finding a way to make up the processing technology to environmental pollution in general and water treatment technology of textile waste and environmental pollution of dioxin-containing herbicides in particular. The results of the research on the ability of laccase need to continually complement the scientific base, a database of diverse levels of microbial strains laccase biosynthesis in general and on the geographical aspects different ecosystems in particular. 1.1.2. Introduction of laccase-like In the course of its growth, the fungal strains beyond biosynthesis enzymes are protein in nature, they also have 4 another ability of the biosynthesis of some biological agents with low molecular weights, the name of these substances depends on the source it is generated. The agents are likely the same as laccase oxidation but chemical-physical characteristics are very different. These compounds do not have a protein structure but just are paragraphs peptides being highly oxidized. A number of studies in Vietnam have discovered active substances generated from the strains of streptomycetes which was isolated from soil contaminated herbicides/dioxin at the Bien Hoa airbase, but detailed research to learn the characteristic -as well as the possibility of their application in this kind of decolorization dyes, decomposing the organic compound biodegradation is virtually published. Thus, to distinguish those names and the term of the previous research and conveniently in the name of research of the thesis, the active ingredient will be called laccase-like. 1.2. The waste water pollution characteristics and textile treatment technology This section outlines characteristics of textile waste water, the technology currently used for textile dyeing wastewater treatment. The overview of the research on the application of laccase, laccase biosynthesis microbial treatment of decolored dyes. 1.3. Pollution herbicides/dioxin in Vietnam and the treatment technology This section outlines the current state of pollution by dioxin-containing herbicides in the "hot spots" in Vietnam, stressing the current state of contamination in Bien Hoa airbase. The technology has been on world statistics and Vietnam in 5 dioxin polluted environment. The synthesis research focus on using extracellular enzymes in general and laccase in particular in the degradation of organic compounds and difficultly- decomposed organic compounds. CHAPTER 2 SUBJECTS AND RESEARCH METHODS 2.1. Research subjects 2.1.1. Microbial strains Make sure the fungal strains collected and isolated at an altitude above 600 m from Ba Vi National forests in three regions and in Ba Trai around the foot area of Ba Vi Hanoi; The strains of streptomycetes are collected from the batch of biological treatment of herbicides/dioxin-contaminated soil in the area XĐ-1 Dong Nai, Bien Hoa airbase; The strains of streptomycetes are collected from the polluted soil in Southwest new pollution area (Pacer Ivy) Dong Nai, Bien Hoa airbase; Basidiomycetes strain FBD154 was isolated from Bidoup Nui Ba, Lam Dong and FNBLa1 filamentous fungus was isolated from rice straw in Ninh Binh in the same collection of research groups in the Biotechnology Institute. 2.1.2. Examination object The herbicides 2.4-D, 2, 4, 5-T standard purity analysis (PA) of the Sigma and the toxic congener 2, 3, 7.8-TCDD; Polluted soil herbicides/dioxins were collected from two areas of Bien Hoa airbase, on toxic average of about 20,000 ng TEQ/kg; Some synthetic dyes in 2 groups of azo and anthraquinone; The commercial active dyes are collected from the plant X20/General Department of Logistics. 6 2.1.3. Cultivated mediums Gause M; potato extract (DCKT); PDA/B; Czapeck,; PDB- DT; MEG; Vis; TSH1 medium. 2.2. Research Methods Due to performance of many experiments to determine the characteristics, capabilities of laccase, laccae-like and synthetic microorganisms strains laccase, laccae-like in this kind of decolorization dyes and biodegradable herbicides/dioxin should experimental scheme described by the diagram in Figure 2.1. Figure 2.1. Experimental scheme diagram 2.2.1. Isolated and cultured of microorganisms 2.2.2. Classification of microorganisms 2.2.3. Biochemistry methode 2.2.4. Determination of decolorization ability 2.2.5. Determine the ability of decomposing herbicides/dioxin 2.3. Data processing methods Analysis data is processed by Microsolf excel/Microsoft office 10 software. 7 CHAPTER 3 RESEARCH RESULTS AND DISCUSSION 3.1. Isolation, selection, and identification of fungi and ascomycete strains biosynthesis capable of laccase, laccase-like 3.1.1. Isolation, selection and identification of fungi strains at high laccase Have collected 45 fungal basidiomycetes in nature and in the ground of decayed wood at the items sample areas. After four days, 22 strains of fungal has well-developed fibers, spread on the surface environment, fungi foam cotton fiber system is white, smooth and round red-brown on the creation of the environment containing the substances directive guaiacol. Laccase activity in situ was identified shortly after the fungal samples were returned to the laboratory to be conducted. The results obtained demonstrate the strains capable of biosynthesis enzymes in extracellular peroxidase (MnP, LiP) or called oxidoreductases (laccase). The results showed that there are about 16 samples of fungi collected in Ba Vi national forests for laccase biosynthesis. Based on results of activity measurements of in situ laccase and growth capabilities. Based on results of activity measurements of in situ laccase and the ability to grow fast, easy, FBV40 strains were selected to study the classification, enzyme properties, capable of decolorization dyes and biodegradable herbicides/dioxins. Such a review on the characteristics of samples, the morphology of bacteria, spores and the sequence of the ITS1-5, 8S-ITS2 FBV40 fungus strains (already registered on the GenBank sequence with code 8 MG243365) are gaining on the genus Rigidoporus and named Rigidoporus sp. FBV40. Figure 3.2. Phylogenetic tree of FBV40 fungal strain 3.1.2. Isolation and classification of ascomycetes capable of growing on herbicides/dioxin medium and laccase-like biosynthesis From the polluted soil source, 8 strains of actinomycetes were isolated, of which 2 strains are (XKBHN1, XKBHN2) from Western land- and the other 6 strains are XKBiR1 XKBiR2, XKBiR3, XKBiR4, XKBiR929, and XKBiR930 from the original soil (before processing the area Z1) contaminated herbicides containing dioxin in Bien Hoa airbase. Based on the morphological characteristics of bacteria, spores and comparing the sequences of 16S rRNA encoding gene segments, XKBHN1, strain XKBiR929 was placed in the genus Streptomyces and named Streptomyces sp. XKBHN1 and Streptomyces sp. XKBiR929. 9 Figure 3.5. Phylogenetic tree of XKBHNN1 and XKBiR929 3.1.3.2. The growth of XKBHN1 and XKBiR929 in chlorinated organic and aromatic hydrocarbons (PAHs) medium After 7 days of being cultured in conditions of 30ºC, being shaken in 120 rpm, two strains XKBHN1 and XKBiR929 were capable of growth on the medium containing soil extract contaminants 2,4,5-T; 2,4-D; DBF and PAHs. Strains of XKBiR929 biosynthesis laccase-like with highest activity is 867 U/l in the medium containing 200 ppm PAHs, Gauss M after 15 days. There has not, so far, been much research in reference to the ability of laccase biosynthesis-like, as well as the ability to metabolites to break down from the representatives of the genus Streptomyces on the medium containing contaminants as used in this study. 3.2. Purification and physic-chemical characteristics of laccase, laccase-like 3.2.1. Purification laccase of Rigidoporus sp. FBV40 Electrophoresis map shows purified enzymes have good quality, forming a single homogeneous band, the results 10 indicate that there are two protein bands obtained in 55 kDa in size and 60 kDa and called Lac1 and Lac2. The weight of two isozyme of fungal strains laccase obtained is consistent with the weight published on laccase and the research results of the thesis has also added information on the collection of fungi belonging to the genus in laccase isozyme of Rigidoporus. 3.2.2. Purification laccase-like of Streptomycese sp. XKBiR929 Molecular weight was determined by gel electrophoresis on 15-20% polyacrylamide (SDS-PAGE) to check the ability of the purification enzyme. Electrophoresis map doesn't show "protein" to be purified. Molecular weight of laccase-like can be determined smaller than 10kDa. Also as mentioned above, laccase-like is a product created by the strains of actinomycetes which are not proteins. Because, after boiling cultured for few hours, oxidation ability to blue ABTS is still observed. It's likely to be metabolites capable of joining the oxidation reactions of the aromatic ring organic compounds. To assert this observation, a lot of detailed study next to search would need to be carried out to better understand the chemical nature and the catalytic activity of the substance synthesized by strains of Streptomycete sp.XKBiR929. 3.2.3. The chemical-physical properties of purified laccase and laccase-like 3.2.3.1. Physical and chemical properties of purified laccase a) Influence of pH on laccase activity and stability The results showed that at pH 3, both Lac1 and Lac2 reached the most highest activity. Lac1 loses its activity at pH values of 1, 7, 8, and 9, while for Lac2 it is at pH of 1 and 8. 11 About the durability of laccase purification, at pH 5 Lac1 for active rest on 50% and in the pH value 3, 4 active rest respectively in turn is 21 and 29% after 5 h. Meanwhile at pH 6 Lac2, for active rest on 92% and at pH 3, 4, 9 active rest respectively in turn is 54.55 and 78% after 3 h. As such, it can be seen that the pH conditions of laccase activity up 2 isozyme from FBV40, especially vaccinations affect durability. b) Influence of temperature on the activity and thermal stability Influence of temperature on the activity and the stability of the Lac 1, Lac 2 from strain FBV40 has been implemented in about from 35 to 70oC, examination time to 140 minutes for Lac 1. The temperature from 30 to 90oC and thermal stability has examinated from 35 to 70oC with time stretching to 180 minutes for Lac 2. The results obtained both Lac1, Lac2, and reached highest activity at 60°C. In the range of temperatures from 65 to 70°C reduced Lac1 active respectively in turn from 15 to 23%. Lac2 reduce active from 20, 32 and 75% when the corresponding temperature is 70, 80 and 90°C. The most stable Lac1 activity at 35oC when still 18% compared with the original activity after 140 minutes, while it reaches 92% Lac2 active and 90% at 30oC and 40oC after 180 minutes. c) Specific substrates Lac1 and Lac2 can oxidize the organic compounds specificity of laccase such as ABTS, syringaldazine (Syrin), 2- 6, DMP and guaiacol (Gua). The activity Lac 1 with the organic chemicals 2.6-DMP, guaiancol and syringaldazine 12 versus ABTS is 76.5%, 0.54% and 0.13%, corresponding to Lac is 74% 0.3%, 2 and 0.2%. d) Influence of inhibitors and metal ions The effect of some particular inhibitors protein on the activity of Lac1, Lac 2 has been studied. The results are combined as follows: Inhibitors Comment on the level of being inhibition Lac 1 Lac 2 SDS Was completely inhibited at any concentration Was completely inhibited at any concentration EDTA 10 mM activity was completely inhibited; 5 mM activity was inhibited 65.5% and 2 mM activity was inhibited 28.5% 10 mM activity was inhibited by 92%, 5 mM was inhibited by 58.4% and 2 mM was inhibited by 31.6% Cl- 10 mM activity was completely inhibited; 5 mM activity was inhibited 72%; 2 mM inhibitory activity was 9.4% - L-cystein 5 mM and 10 mM activity were completely inhibited; 2 mM inhibitory activity 54.5% - Arginin - 10 mM activity was inhibited 48%; 5 mM activity was inhibited 36.5; 2 mM activity was inhibited 28.5%. Note: "-" not tested The influence of metal ions such as follows: ion Comment on the degree of being influence Lac 1 Lac 2 13 ion Comment on the degree of being influence Lac 1 Lac 2 Cu2+ Increased 109% activity at the concentration 2 mM Increased 112% to 184.6% activity at the concentrations from 1.0 to 5 mM, respectively Mg2+ Inhibitory activity at all the concentrations and ranged from 21.5 to 78.95%, respectively, from 0.5 mM to 5.0 mM Inhibitory activity at all the concentrations and ranged from 11.6% to 27%, corresponding to 0.5 mM to 5.0 mM Ni2+ In 1.0 mM 100.6%, increase in the concentration of the inhibitor and the rest are ranged from 5.3% to 44.4% respectively 0.5 mM to 5 mM Inhibitory activity in all the concentration and ranges from 12.8% to 30% with the concentration from 0.5 mM to 5 mM Mn2+ Inhibitory activity in all the concentration and range from 21.1% to 35% with the concentration from 0.5 to 5.0 mM Inhibitory activity in all the concentration and range from 11.6% to 18.9% concentrations from 0.5 to 5.0 mM Co2+ Inhibitory activity in all the concentration and range from 15.5% to 35.2% concentrations from 0.5 to 5.0 mM Cause inhibition of activity in all the concentration and range from 14.5% to 32.3% from 0.5 mM concentrations up to 5 mM Fe2+ Inhibitory activity in all the concentration and range from 71.6% to 93.3% concentrations from 0.5 mM Inhibitory activity in all the concentration and range from 95.9% to 100% with the corresponding 14 ion Comment on the degree of being influence Lac 1 Lac 2 to 5 mM concentrations from 0.5 mM to 5 mM Ca2+ Increase 107% and 128% of activity at the concentration of 0.5 and 2 mM Inhibitory activity at all the concentration and range from 16.2% to 23.3% from 0.5 mM concentrations up to 5 mM The results obtained from this study showed that the influence of metal ions onto activity of laccase biosynthesis by FBV40 strain are similar to the research internationally published beforehand, however there exist differences in the extent of inhibition between Lac1 and Lac2. 3.2.3.2. Kinetics of purified laccase Km and Vmax of Lac1 is respectively 0.3 µ M and 200.000 µM/min. Meanwhile with Lac2 is 0.4 µM and Vmax is 10,000 µM/min. 3.2.3.3. The Physical-chemical characterization of the crude laccase Research on the influence of env