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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
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 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+
Inhibition of activity at all concentrations
and ranged from 41.7% to 80.7% with a
concentration of 2 to 10 mM.
Na+
Inhibition of activity at all concentrations
and ranged from 75.5% to 94% at a
concentration of 2 to 10 mM.
Mg2+
Inhibition of activity at all concentrations
and ranged from 13.4% to 79.1% with a
concentration of 2 to 10 mM.
Ca2+
Inhibition of activity at all concentrations
and ranges from 41.5% to 68.5% with a
concentration of 2 to 10 mM
Fe2+
Inhibition of activity at all concentrations
and ranged from 97% to 97.9% with a
concentration of 2 to 10 mM.
16 Factors affecting Change laccase activity
Ni2+
Inhibition of activity at all concentrations
and ranged from 2.7% to 88.3% with a
concentration of 2 to 10 mM.
Cu2+
Inhibition activity at all concentrations
ranged from 48.1% to 76.2% with a
concentration of 2 to 10 mM.
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
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(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
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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.
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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.