Summarry of Environmental Technique Doctoral Thesis: Reasearch on the ability of decolorization of reactive dyes and degradation of herbicides/Dioxin by microbes producing Laccase

The research objective of the thesis is to select microorganisms capable of producing laccase, laccaselike from Ba Vi National Forest area, in herbicide / dioxin contaminated soil at Bien Hoa airport. Assessing the ability of decomposing herbicides containing dioxin and reactive dyes by laccase, laccase-like and VSV strains were selected to guide the application in defense activities.

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

The thesis is available at:

- Library of Graduate University of Science and Technology

- National Library of Vietnam

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 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

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

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

dioxin polluted environment The synthesis research focus on using extracellular enzymes in general and laccase in particular

in the degradation of organic compounds and decomposed organic compounds

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

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

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

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

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

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

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