MINISTRY OF EDUCATION VIET NAM ACADAMY OF AND …gust.edu.vn/media/26/uftai-ve-tai-day26171.pdf · LITERATURE REVIEW 1.1. Laccase, laccase-like microbial biosynthesis and 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…..month……2018.

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


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



and range from 11.6%

to 18.9%

concentrations from

0.5 to 5.0 mM

Co2+


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+


concentration and range from

71.6% to 93.3%

concentrations from 0.5 mM




to 100% with the

corresponding

14


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



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 environmental factors onto the

raw laccase activity has been carried out.

Table 3.10. Factors affecting on the crude laccase of FBV40

Factors affecting Change laccase activity

The influence of pH

pH 3 The highest activity

pH 2 The activity lost

pH 4 Active rest on 65% after 120 minutes

The influence of temperature

30 to 40oC The highest activity

50oC, 60 to 70oC

Be inhibited in turn is 15% after 50

minutes at 160oC and 40% after 79

minutes at 60oC and up to 98% after 20

15 Factors affecting Change laccase activity

minutes at 70oC

30oC and 40oC After 160 minutes of activity is almost

not be inhibited

Catalytic kinetics

Km 0.5 µM

Vmax 25,000 µM/phút

The influence of inhibitors

SDS With 5 mM severely inhibited completely

and 2 mM were 79% inhibition.

L-lysis With 5 mM severely inhibited completely

and 2 mM inhibits 86%.

EDTA With 5 mM severely inhibited is 92% and

2 mM were inhibited 10%

Effects of mental ions

Co2+

Inhibition of activity in all concentrations

and ranged from 44.7% to 79.5% with a

concentration of 2 to 10 mM

Mn2+

Inhibition of activity at all concentrations

and ranged from 47.3% to 80.2% at

concentrations of 2 to 10 mM

K+



concentration of 2 to 10 mM.

Na+


and ranged from 75.5% to 94% at a


Mg2+




Ca2+


and ranges from 41.5% to 68.5% with a

concentration of 2 to 10 mM

Fe2+


and ranged from 97% to 97.9% with a


16 Factors affecting Change laccase activity

Ni2+




Cu2+

Inhibition activity at all concentrations

ranged from 48.1% to 76.2% with a


The results obtained from the research shows that there

exists similar ability but also big differences in the level of

influence of the same element on crude laccase activity and

purified laccase strains of FBV40. The research results showed

that to apply purified laccase or crude one into environmental

pollution disposal, overall and detailed studies should be needed

to determine the factors and their influence levels; thereby to

improve the efficiency of their application.

3.2.3.4. Special chemical-physical properties of purified laccase-like

Purified laccase-like has the highest activity at pH 1, rising

fast and almost not being identified at pH 3. The purified

laccase-like activity of vaccine XKBiR929 hardly decreases

when being boiled in the time up to 3 hours. Even, when

fractional distillation takes off on the device filmed at a

temperature of 800C and a pressure of 0.8 atm, active laccase-

like of this strain still does not change. As such, laccase-like

created from XKBiR929 strain is very heat- durable. Based on

the first study, the influence of organic loading ability of

oxidation of laccase-like, calculated the value of it with value

Vmax is 142,857 µ M/min and Km is 0.43 µ M. Compared with

the value of laccase strains of fungi FBV40, the speed of

response of the laccase-like is just lower than Lac1(200,000 µ

17

M/min) and about 14 times higher than Lac2 (10,000 µ M/min)

and more than 5 times higher than the crude laccase (25,000 µ

M/min) . With such a value of Vmax and Km obtained shows

the very high oxygen capacity of laccase-like, especially in the

very low value of pH. It is a very notable feature of this thesis

that the active substances are found in Actinobacteria. The most

powerful inhibitor aganst laccase-like activity is L-lys. In

concentrations 10 mM, active laccase-like rest 14%, followed

by EDTA Active rest 66.7% and SDS having affected at least

with laccase activity-like rest 83.6%. Among the metal cation

activity test with laccase-like, cation Fe2 + -induced inhibition

of strong activity in all the concentration of a survey. Compared

with the effects of metal ions for laccase strains of FBV40, it

shows that this is a very special properties of laccase-like race

from XKBiR929. The results obtained by the thesis concerning

the characteristics of the laccasee-like are actinomycetales

created, which are also the first results obtained when surveying

the basic characteristics of the protein and showed the

outstanding characteristics of laccase-like.

3.3. Decolorization dyes and digradation herbicides/dioxin

3.3.1. Decolorization dyes by laccase, laccase-like

3.3.1.1. Decolorization synthetic dye color by crude laccase

Laccase of FBV40 strains has highly capability in the

category of basic colour azo like NY1 and the basic color as

NY7 anthraquinon as NY5 even if present and not present

mediator. This result is the first one of the ability to color sorts

NY1, NY5 and fungi in the genus NY7 Rigidoporus was

isolated from natural forests in Vietnam.

18 3.3.1.2. Decolorization reactive dyes used in the military by

crude laccase

a) Capable of decolorization a number of reactive dyes

Laccase of FBV40 strains shows decolorization ability

some commercial reactive dyes used to dye a few army military

sewing. The ability to decolorate types best for dyes MN.FBN

was 92.17% with dye, NN.SG was 91.84% after 3.5 h.

b) The selection of reactive dyes eliminated the highest

decolorization

Compare the capability of decolorization of crude laccase

vaccine FBV40 with MN.FBN dye 92.67% achieved the highest,

with the dye of NN.SG was 91.84%. The MN.FBN

decolorization fastness rate reached over 85% while with the dye

of NN.SG just get over 17% during 0.25 h. Especially without

mediator, laccase activity reduced at 62.9% when decolorization

MN.FBN dyes and 91.4% for NN.SG after 3.3 and 4 hours. The

dye MN.FBN was, thus, selected to further study.

c) Capable of decolorization dye MN.FBN with crude

laccase in the presence of D-glucose

After 138 hours, with a concentration of 50 mg/L of the

dye MN.FBN, the crude laccase FBV40 decolorated 99% and

97.6% in the presence of 0.1 g and 0.05 g D-glucose.

3.3.1.3. Decolorization the active dye MN.FBN by Lac1

After 24 h, in the presence of 500 ViO µ M, Lac 1 of

FBV40 strains decolorated 91% color of MN.FBN with initial

concentration of 50 mg/L. The results clearly indicate the role

and the possibility of very specific laccase strains FBV40 in loại

màu dyes in general, and the commercial activity of the plant

X20/General Logistics Bureau in particular.

19 3.3.1.4. Decolorization the active dye MN.FBN by purified

laccase-like

At pH 1, purified laccase-like of vaccine XKBiR929 with

the presence of fastening substance ViO decolorated 76.3 %

MN. FBN after 23 h. But only after 5h, laccase-like decolorated

75.8% with the original dye concentration of 100 mg/L. With

the usage of the other CGK: HBT, its capability of

decolorization reached 54.7% after 23 h, and hit 54.0% after

5h . The role of the CGK ViO for laccase-like strains

XKBiR929 in decolorization is not great (just add performance

increase was 22%), with CGK HBT performance only increased

the color type. The results of the research have opened a new

research direction to apply laccase-like in eliminating of organic

compounds in low pH conditions.

3.3.2. Decolorization reactive dyes by fungi FBV40 strain

3.3.2.1. The ability to decolorate reactive dyes used to dye

fabric for the military

FBV40 Strains are capable of decolorization MN. FBN at

the highest of 99% after 7 days being cultured. For MY dye

color. EG and MY. BES efficiency decolorization reached a

low level, just determined to be from 6 to 8%. Observe of

fruiting bodies growing in MY.EG and MY.BES dye solutions

showed color absorption on fruiting bodies, whereas MN.FBN

dye did not see the adsorption, in contrast with the color dyes

for MN. FBN did not see the adsorption on. From the above

phenomenon can identify strains FBV40 type coloring dye,

MN. FBN by the mechanism of decomposition, metabolism.

3.3.2.2. Decolorization dye MN. FBN in different concentrations

20

After 1 to cultured 3 days FBV40 strain type color dyes for

MN. FBN has a concentration of 100 mg/L achieved from

52.7% to 97.3 %, while in the concentration of the dye 50 and

200 mg/L , the type of color performance reach respectively

from 49.8 % to 94.3% and 35.2 % to 97.1 %. As such, FBV40

fungus strains have the ability to sort the highest color in the

dye concentration 100 mg/l.

3.3.2.3. Decolorization dyes MN.FBN with the presence of D-

glucose

After 1 day performance, efficency decolourization dyes

for MN. FBN by FBV40 have highly reached from 31 to 97%,

while the environment has no D-glucose, then the performance

is just 8%. The obtained results of the thesis, together with the

research of the author worldwide, D-glucose can be carbon

source or is the CGK appropriate to the process of coloring dye,

MN. FBN by FBV40 fungus strains of highly effective.

3.3.2.4. Decolorization dyes MN.FBN in the presence of other

sugars

With the use of the sugars as a source of carbon, the D-

glucose reaches the highest effiency decolorization of 96%,

reaching 90% lactose, mannose, gained 87% sacharose is 74%,

while the template does not add the sugars, only reached 28%

after 9 days. The research results were presented on again

affirmed and clearly demonstrated the role of carbon sources

suits in general as well as demonstrate the role of D-glucose in

decolorization reactive dyes MN. FBV40 strains of FBN.

3.3.2.5. Decolorization dyes MN.FBN in the presence of the

different nitrogen sources

21

(NH4)2SO4 in the FBV40 strains for cultivation medium, its

performance of decolorization reaches 67%,3 is 57% KNO and

NH4NO3 is 54% after 9 days compared with cultured form for

the certificate is 29%. The results obtained by the dissertation

shows, nitrogen source inorganic influence grew to the ability

and they play a very important role in the process of laccase

biosynthesis of fungal strains.

3.3.3.Degradation herbicides/dioxin by laccase and

biosynthesis of fungal laccase 3.3.3.1. Degradation herbicides/dioxin by crude laccase

a) Degradation of soil extracts

After 13 days of experiments capable of degrading 2.4-D

and the 2,4,5-T respectively reached 65.46% and 85.71% in

experimental formula in the presence of acetate buffer and

mediator ViO.

b) Degradation of 2,4,5-T pure

After 20 days, crude laccase strains of FBV40 are given the

ability to degrade for 2, 4, 5-T at different rates in the range

from 40.55 to 98.45% respectively with 2, 4, 5-T is in the form

of experiment is 1.401 mg to 0.037 mg.

c) Degradation of 2,4-D and 2,4,5-T in contaminated soils

After 27 days, 2.4-D in contaminated soils degraded 49.5%

and 2,4,5-T degraded 37.71% of initial concentrations in the very

highly contaminated soil 556.92 respectively and 981.88 ppm.

3.3.3.2. Degradation herbicides/dioxins by fungi biosynthesis laccase

a) Degradation 2,4,5-T in contaminated soil at Bien Hoa

formed airbase

2,4,5-T in soil have very high rate in reaching 473.5 mg/kg,

after 8 days FBV40 strains cultured biodegrade by more than

7% corresponds to 36 mg/kg, if measurement processing

22

performance/day for mass experiments, the FBV40 were

processed are 0, 045mg 2,4,5-T/day.

b) Degradation 2.4-D and the 2, 4, 5-T in polluted soil in

Bien Hoa formed airbase in single and mixed fungal strains

The performance of single FBV40 strains for 2.4-D, 2, 4, 5-

T in polluted soil in bien Hoa formed airbase in turn is 11.44

and 12.13% after 10 days processing with initial content in turn

is 62.95 mg/kg and 63.5 mg/kg. Also when using mixed strains

of FBV40, FBD154 and FNBLa1 with 1:1:1 rate by volume

then the degradation performance 2.4-D, 2,4,5-T increased

significantly and in turn reached 36.06% and 56.08%.

c) Degradation congener 2,3,7,8-TCDD in single and mixed

fungal strains

After 10 days, single race and racial mixtures have the

potential to biodegrade congener 2,3,7,8-TCDD with 44.68%

performance when the original toxic was 2,000 ng-TEQ/L and

mixed strains have a higher potential to 92.89% performance

after 10 days of cultivation.

THE CONCLUSION

1. Having selected strains of FBV40 from 22 strains

isolated from Ba Vi national forests, Hanoi with the highest

crude laccase activity being 107.708 U/l on the medium

following TSH1 8 on cultured. FBV40 strains are classified in

the genus Rigidoporus and named Rigidoporus sp.FBV40.

Isolation and classify two strains of Actinobacteria

Streptomyces, named Streptomyces sp.XKBHN1 and

Streptomyces sp.XKBiR929 laccase biosynthesis-like in the

medium containing PAHs, 2.4-D, 2, 4, 5-T and soil extract.

23

2. Having purified and identified characteristics of laccase

of FBV40 strains including 2 Lac1 and isozyme Lac2 that have

the molecular mass of 55 kDa and 60 kDa, both of whose has

different private properties. After 24 h of interaction, Lac1

eliminated 91% decolorization commercial dye MN.FBN at

concentrations of 50 mg/L. Laccase-like purified from

Streptomyces sp. XKBiR929 was very thermal stable, even at

1000C. Within 3 h the activity does not change; affected by

protein inhibitors such as L-lys, EDTA, SDS that is very

different from real laccase; decolorization 76% MN.FBN dye in

the initial concentration of 50 mg/L in 1 pH condition in the

presence of mediator ViO after 5 h.

3. Efficiency decolorization synthesis dye by crude laccase of

FBV40 in the concentration of 100 mg/L is 82% after 2,5 h, 85%

after 4 h for synthetic dye azo NY1, NY7 and 70 % after 48 hours

for anthraquinone dye NY5. There is an inverse ratio between the

rate relationship decrease laccase activity with the concentration of

fastening in the process of decolorization synthetic dyes.

4. After 138 h in the presence of 0.1 g of D-glucose, crude

laccase from Rigidoporus sp. FBV40 has decolorated 99%

MN.FBN dye and with the absence of D-glucose, just 44%. After a

day of implanting Rigidoporus sp. FBV40 in medium containing

100 mg/L MN.FBN dye and 0.5 g D-glucose 97.2% of the color

was removed.

5. The crude laccase of FBV40 degradated 40.55 to 98.45%

of 2, 4, 5-T purity in the laboratory with the weight of 2.5 mg.

When concentrations of 2,4-D, 2,4,5-T up to 556.92 mg/kg and

24

981.89 mg/kg, after 27 days, crude laccase degraded 49.5 and

37.7% . After 7 to 10 days of cultured strains Rigidoporus

sp.FBV40 biodegradable 11.44% (7.2 mg/kg) of 2.4-D

herbicides with initial concentrations of 62.95 mg/kg and

12.13% (7.7 mg/kg) 2, 4, 5-T with concentration is 63.5 mg/kg.

Make sure the mixture of FBV40, FBD154 and FNBLa1

biodegradable 56.07% (35.29 mg/kg) of 2.4-D and 36.06%

(22.89 mg/kg) 2, 4.5-T.

6. After 8 days of cultivation, strains FBV40 is degradable

44.7 percent congener 2,3,7,8-TCDD with toxicity 2,000 initial

ng-TEQ/L, while the mixture of races of the fungus is FBV40,

FBD154 and FNBLa1 degraded 92.9%.

Recommendation

Continue to do more research to develop the capabilities of

laccase, laccase-like and synthetic microorganisms strains laccase,

laccase-like so as to gradually perfect the innovative technology in

processing of organic pollutants hard degradation in general and

herbicides containing dioxin in particular, contributing to

completing the remedial program of chemical poisons remain

saved after the war in Vietnam and the treatment of pollution to

protect the environment.

NEWS CONTRIBUTIONS OF THE THESIS

1.The first study on laccase-like characteristics biosynthesized

by Streptomyces sp. XKBiR929 and its ability in decolorization of

the commercial active dye MN.FBN;

2. Demonstrate fungi strain Rigidoporus sp. FBV40 is

biosynthesized with crude laccase activity up to 107,708 U/L

25

and has high potential in decolorization of azo and

anthraquinone dyes used in the military.

3. State that Rigidoporus sp. FBV40 was also capable of

degradation 2,4-D, 2,4,5-T herbicides and the most toxic congener

of dioxins 2,3,7,8-TCDD. Additionally, clarify and confirm that

the mixture of different fungi strains (i.e. Basidiomycetes and

Ascomycetes) isolated from nature in Vietnam degradate not only

2.4-D, 2,4,5-T but also degradating 2,3,7,8-TCDD with higher

efficiency than the use of the single strain.

LIST OF PUBLISHED WORKS

1. Phung Khac Huy Chu, Đao Thi Ngoc Anh, Đinh Thi

Thu Hang, Đang Thi Cam Ha, Isolation, classification and study

of laccase-like biosynthesis possibility of some actinomyces in

herbicides/dioxin contaminated soil of Bien Hoa airbase, Dong

Nai, Province, Hue University Journal of Science, (2015), 110

(11): 29-41.

2. Phung Khac Huy Chu, Nguyen Hai Van, Dang Thi Cam

Ha, Purification and characterization of laccase involved in

the decolourization of synthetic dyes and 2,3,7,8-TCDD

congener degradation by the white rot fungus isolated from

bavi forest of Vietnam, Vietnam Journal of Science and

Technology, (2017), 55 (4C): 180-185.

3. Phung Khac Huy Chu, Đao Thi Ngoc Anh, Dang Thi

Cam Ha, Basidiomycetes fungus synthesis laccase that has

decolouration abitily of active dyes used to dye fabric sewing

ammunition uniform, Journal of Military Science and

Technology, (12-2017), 52: 169-179.

http://joshueuni.edu.vn/

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