Transcript Detoxification of herbicide/dioxin contaminated soil based

2ND Meeting of
The Agent Orange Working Group
Detoxification of herbicide/dioxin
contaminated soil based on microbial
diversity and their gene expression
Assc. Prof. Dr. Dang Thi Cam Ha
Institute of Biotechnology (IBT)
Vietnam Academy of Science and Technology
(VAST)
Hanoi July 2, 2010
DANANG “HOT SPOT’
Composition of contaminants in
former military soil
• Herbicide: 2,4,5-T: 2,4-D/ 50 : 50 (20
100 – 5 000 000 g/kg);
• 2,3,7,8-TCDD: >99%, several thousands
to hundred thousands pgTEQ/g ;
• Other contaminants: TCP, DCP, PAHs
etc.;
• Heavy compounds of diesel oil.
Các nguồn vào/ra
Đất như một “hộp đen”
các chất diệt cỏ
khác v.v.
Sinh khốibiomass
-Đa dạng VSV đất ở mức độ nào?
- Microbial diversity?
- Bằng cách nào đánh giá được đa dạng
VSV đất? Dection of this diversity by
what kind of methods?
Các chỉ thị sinh
học bioindicators
Vi sinh vật
không nuôi cấy
được-uncultural
microbes
Các vi sinh vật nuôi
cấy được –cultural
microbes
DNA/RNA
Đa dạng VSV: đa dạng về loài
VSV và khả năng trao đổi chất
của VSV
Các nguồn
ra
0,01-10% tổng số VSV
-Total microbes
Microbial diversity: species
and their metobolisms .
Biodegradation pathways
for detoxification of herbicide/dioxin
contaminated sites
• Oxidation
oxy hóa cắt vòng thơm
• Declorination
Loại clo
• Catalyzation (extracellular enzymes: Laccase + O2,
MnP and LiP + H2O2)
Xúc tác (enzyme ngoại bào: Laccase+O2, MnP và LiP + H2O2)
• Reductive declorination
Loại khử clo không cần O2
Bioremediation
Stimulation
Augmentation
Phytoremediation
Kích thích sinh học
Tăng cường sinh học
Xử lý bằng thực vật
All resolutions based on microbial activities:
oxydation, dehalogenation and catalysis
reaction.
Tất cả các giải pháp đều dựa trên hoạt động của
VSV tham gia và oxy hóa cắt vòng, loại khử clo
và phản ứng xúc tác bởi enzyme
There are six biotechnologies which may be engineered
for detoxification of soil contaminated with herbicides
and dioxin and mixtures of other toxicants
• Aerobic bioreactor followed by secure landfill for
sensitive land uses requiring very low cleanup criteria;
• Anaerobic bioreactor for chlorinated pesticides;
• Anaerobic bioreactor followed by aerobic bioreactor for
fast mineralization of certain contaminant mixtures
(anaerobic/aerobic cycling);
• Aerobic bioreactor, either biostimulated or bioaugmented
to achieve cleanup levels rapidly;
• Phytoremediation, either by passive land treatment or
monitored natural attenuation;
• Combination of all technologies, depending on site
characterization and the contaminated properties.
In Vietnam: Active landfill for huge complex
contaminated site was developed
Microbial diversity of contaminated
site and biotreatments
(Culturable and unculturable)
Herbicide/dioxin, DDT,HCH,TNT
degrading microbes
45 bacterial strains
• 12 Actinomycete strains
• 20 Filamentous fungal strains
• 20 Anaerobic communities
•
Herbicide/dioxin anaerobic
degrading community (SEM)
After 120 day inoculation, 17% toxicity was
reduced by strain Pseumonas
sp.SETDN1
Functional gene present and expression
In soil and in purified cultures
Dioxygenase gene in dioxin and dibenzofuran degrading
bacteria isolated from herbicide/dioxin contaminated site
carAa-Ps. CA10
car Aa-Ps. CA 10
dbfA1-Te. DMA
dbfA1-Rh. Y K2
dbfA1-Rh. DFA3
dbfA1-Rh. DFA 3
HDN3
HDN3
dbfA1-Ter. DBF63
dbfA1-Rh. YK2
DMA
dbfA-Te. DBF63
DM A
phdA-No. KP7
phdA-No. K P7
dfdA1-Te.YK3
dxnA1-Sp. RW1
psbAb-Rd. No.7
0.05
bphA1- Rh. TA 421
bphA1-Co. TK102
ditA1 Ps-BKME-9 (AF119621)
dxnA12-Sp. RW1
dodA Rh-SA0101(AB110633)
bphA1-Rh. TA412
narA Rh-1BN(AJ40162)
nidA My-MHP-1(AB179737)
phdA No-KP7(AB031319)
pdoA2 My-6PY1(AJ494743)
dbfA1 Rh-DFA3(AB181127)
dbfA1 Te-DBF63(AB095015)
ahDO Pa-YK5//clone C89 (AB201839)
0.05
Ao3
Ao3
ahDO Pa-YK5/clone C89(AB201841)
0.05
phnAc-Bu.RP007
bphA-Ag. IAM12620
Cây phát sinh chủng loại giữa một số
trình tự đại diện mã hóa enzym dioxygenase và trình tự nhân lên từ
chủng Ao3, DMA và HDN3 sử dụng cặp
mồi DIOXY-F và DIOXY-R
Numbers gene tfdA of copy
in
herbicide/dioxin
contaminated (Method:
MPN-PCR)
HDN1
HDN2
HDN3
HDN4
HDN5
HDN6
3,67 x 1,31 x 1,11 x 1,06 x 5,78 x 5,99
105
105
105
105
105
106
HDN7
HDN8
HDN9
x 2,85 x 5,27 x 5,99 x
105
105
106
Copy number catechol dioxygenase in
aerobic biotreating cells
Cell
C23O/g
M7.0
2,5 x106
M7.3
2,7 x 106
M8.0
3,7 x 105
M8.3
6,7 x 106
M9.0
2,1 x 105
M9.3
2,2 x 106
M10.0
4,6 x 105
M10.3
2,4 x 106
M11.0
2,6 x 106
M11.3
1,3 x 106
Herbicide/dioxin, DDT,HCH,TNT
degrading microbes
45 bacterial strains
• 12 Actinomycete strains
• 20 Filamentous fungal strains
• 20 Anaerobic communities
•
Detoxification of herbicide/dioxin at
different scale
1.
2.
3.
4.
5.
At the lab. Condition
At field trail (0.5- 100 m3), Danang hot spot
Bioreactors at lab. condition (50kg soil)
Active landfill at Bien Hoa, 3384 m3
VAST and EPA pilot scale (11 cells with 2
m3 soil)
Biotreatment at pilot scale in
lab. condition
Treatments
2,3,7,8 TCDD
concentration
(ppb)
0,1DN1
71.68
degradation
percentage in
comparison to
0,1DN1
0
0,1DN2
20.76
71.04
0,1DN3
43,32
39,36
0,1DN4
31.69
55.79
0,1DN5
48.14
32.84
1A
1B
Untreated soil sample (1A) and treatted
sample (1B)
•
•
These results indicate that different groups of microbes play different roles in the
detoxification of contaminated soil in an examined former military base.
After 8, 18 and 24 months in all treatments 50-70 % of toxicity were reduced.
Soil composition change before and after treatment
1.5DN5C (Before treatment)
1.5DN5T (After two year treatment)
Fold
I
2,4,5-T Methyl ester; 2,4-D methyl ester; Acetic
acid, (2,4-dichlorophenoxy)
2,4,5-T Methyl ester
51.0
IIA
Phenol, 2,4-dichloro-; Phenol, 2,4,5-trichloro-
Phenol, 2,4-dichloro-; Phenol, 2,4,5-trichloro-
1.6
IIB
Phenol, 4,5-dichloro-2-methoxy-; Phenol, 2,4,6trichloro-; Phenol, trichloro-; Phenol, 2,6bis(1-methylpropyl); Phenol, 2,3,4,6tetrachloro-; Phenol, 2,3,5,6-tetrachloro-;
Phenol, 2,3,5-trichloro-; Phenol, 2,3,6trichloro-
Phenol, 2,4,6-trichloro-; Phenol, trichloro-;
Phenol, 2,6-bis (1,1-dimethylethyl); Phenol,
dichloro-; Phenol, 2,6-dichloro-
5.4
III
Benzene, 1,2,4-trichloro-3-methoxy; 1,2Dimethoxy-4,5-dichloro-benzene; Benzene,
dichlorodimethoxy- ; Naphthalene, 1,3,7trichloro-; Benzene, 1,2,4-trichloro-5ethoxy-
Benzene, dichlorodimethoxy-;
Benzene, 1,2,3-trichloro-4-methoxy
20.3
IVA
1-Nonadecene; 9-Tricosene, (Z)-; Nonadecane,
2-methyl-; Heptadecane; Tetradecane;
Nonadecane; Eicosane; Pentadecane;
Octadecane; Hexadecane
1-Octadecene; 1-Nonadecene;
Heptadecane;Tetradecane;
Nonadecane; Eicosane; Octadecane; Docosane
1.1
IVB
9-Octadecenamide, (Z)-; 9-Octadecenoic acid,
(E)-; Dodecanoic acid; Dodecanoic acid, 1methylethyl ester;
Octadecanoic acid; n-Hexadecanoic acid;
Tetradecanoic acid
9-Octadecenoic acid, (E)-; Dodecanoic acid;
Dodecanoic acid, 1-methylethyl ester;
Octadecanoic acid; n-Hexadecanoic acid;
Tetradecanoic acid; Nonanoic acid;
Hexadecanoic acid, methyl ester; Z-7Hexadecenoic acid
2.9
•
•
Result of GC/MS analysis indicates that 51,7% of toxicity of several hundreds g
TEQ/g was removed after two year.
Composition change in soil sample before treatment in comparison to sample after
two year treatment. Not only 2,3,7,8 -TCDD congener was reduced, but also other
herbicide contents were decreased too. Comparing chemicals that analyzed in
1.5DN5 sample before treatment and two year treated sample, the change of
biodegrading
products was detected in treated sample. Some diesel oil
compositions were also degraded. This finding shows that bioremediation treatment
can be applied for soil with high concentration of 2,3,7,8 -TCDD, 2,4,5-T and 2,4-D.
Obtained data from GC/MS scanning analysis of the main existing chemicals in soil
before and after two year treatment, also shows that bioremediation treatment by
‘Active landfill” technology providing promising tool for detoxification of heavily
contaminated soils by dioxin and other toxic compounds.
35000000
30000000
Initial toxicity: 268.000
pgTEQ/g
25000000
•
20000000
15000000
10000000
5000000
0
I
IIA
IIB
1,5DN5T
III
1,5DN5C
1,5DN5T
IVA
1,5DN5C
IVB
• After two year treatment:
129.200 pgTEQ/g (51,7 %
removed)
Change in composition of 10DNT and 100DNT
treatments after 24 and 29 months
Group
Compouds
Samples of two year treatment from 100DNT
biotreatment
Detected compounds
I
Agent orange
Undetected
IIA
2,4-D-PCP vµ
2,4,5-PCP
IIB
Other PCP
III
Derivatives
Benzen
of
Pick
0
4000000
Phenol,
2,4-dichloro-;
Phenol, 2,4,5-trichloro-
573382
3500000
Phenol, trichloro-
172806
2500000
1,2-Benzenedicarboxylic
acid, diis
873193
2000000
3000000
1500000
1000000
IVA
Hydrocacbon
oxacycloheptandecan-2one; 7-Hexadecene, (Z)-
262889
IVB
Cacboxylic
acid
Chlorobutanol;
9Octadecenamide, (Z)-; nHexadecanoic acid; 1Octadecene;
Tetradecanoic
acid;
Erucylamide
3526843
500000
0
I
IIA
IIB
III
100DNT
IVA
IVB
• 10DNT , 29 residual: 3249 pgTEQ/g, 50-70% total toxicity removed (limit:1000
ppt)
• 100 DNT: toxicity 4499 pgTEQ/g, 50-70% total toxicity removed
Conclusion (Danang trals)
• Application of in situ bioremediation treatment not only dioxin
was reduced but other polluted components in soils, 2,4-D; 2,4,5T, TCP,DCP, PAHs also transformed or degraded. This
biotechnology can be applied for detoxification of heavy
herbicide/dioxin contamination in “Hot spots”.
• Results of biodiversity indicates that different microbial groups
and species play certain role and it changes during
bioremediation treatments;
• Indigenous microorganisms of long time exposure by dioxins
and other contaminants play leading role in detoxification of
2,3,7,8-TCDD, 2,4,5-T; 2,4-D and other chemicals in contaminated
soils;
• Obtained results driving us to develop further research for
enhancing bioremediation in detoxification process of dioxins
and other pollutants in former military bases in Vietnam.
• This year bioremediation in “active landfill” technology which
created by us is applying for in situ detoxification of heavy
herbicide/dioxin contaminated site at former military base of
Bien Hoa, Dong Nai province (firstly for one big cell 3384 m3).
DEVELOPMENT OF BIOTECHNOLOGY
Used aerobic and falcultative bioreactors
Reduction of toxicants
44,1% total toxicity was removed after 16
weeks
Total toxicity (pg
Removal
TEQ/g)
efficiency (%)
Initial
After 8 weeks treated in
aerobic bioreactor
After 16 weeks treated in
both aerobic and
facultative bioreactors
21.687,4
15.518,5
28,4
12.129
44,1
Average 85,3 pg TEQ/g/day
detoxified
PROJECT (Bien Hoa)
“ Application of active landfill for detoxification of
dioxin contaminated soil at Bien Hoa air base “
- Scale: 3,384 m3
- Time: March to April 2009
- Two institutions were involved this project: Chemical
command (MOD) and Institute of Biotechnology (IBT,
VAST)
Products: DHS1, DHS2 AND
SLOW-D
What have been researching and developing in IBTVAST concerning to the use of bioremediation for
detoxification of herbicide/dioxin and other POP
contaminated soil and sediment?
1.
2.
3.
4.
POP (2,3,7,8-TCDD is included) degrading microbes and
their degradation capability analyzed by HPLC, GC/MS, DRCalux and other methods;
Microbial community structure of contaminated soil and
sediment (Lakes) and their change during bioremediation
treatments of Danang and Bien Hoa former military bases;
Characterization of bacteria, Actinomyces, filamentous fungi
and some their functional genes (in a international GenBank
database with author names: Identification of ,
oxidoreductase, peroxidase enzymes from different POP
degrading microbes, consortium and whole community.
Detection of genus Dehalococcoides in different original
HDIX sediments and almost biotreatments at the field trail at
Danang and Bien Hoa sites. These anaerobic microbes have
been known with the name “dechlorinating workers”
Summary IBT-VAST's activity
• At laboratory scale: 32- 71% removal after a month
• Field trial without supplied oxygen (0.5 - 1.5 m3): 51, 7%
of initial concentration 268 ppb and 90 % (with lower
TEQ) after two year treatment.
• Active landfill (10 and 100 m3): 70 % after two years
• Anaerobic and aerobic bioreactors individually and
together: 44,1% after only several months.
Vietnam- US cooperation
in Danang
Application of bioremediation at pilot scale
(2m3) in Da Nang (6month evaluation) by
VAST and EPA
Vietnam
Dang Thi Cam Ha
Nguyen Ba Huu
Nguyen Nguyen Quang
Nguyen Quang Huy
Dam Thuy Hang
Nguyen Ngoc Anh
Phung Khac Huy Chu
USA
Harry L. Allen
Vance Fong
Acknowledgment – cảm ơn
•
•
•
•
•
•
•
•
Ford foundation- Quỹ Ford
Office 33- Văn phòng 33
US EPA
US Embassy – Sứ quán Hoa Kỳ
Mr. Andrew Herrup
Nguyen Thanh Tu
VAST–Viện Khoa học & Công nghệ Việt Nam
IBT- Viện Công nghệ sinh học
Difficulty in evaluation of
PCDDs/PCDFs concentration or
toxicity
What is a reason?
• Dioxin distributed in the soil matrix is
hetegenous. Probably, “Black small
pieces” came from heavy fraction of DO
(resin and asphentence) contain more
dioxin
How to solve?
Khắc phục như thế nào
• Use GC/MS ( scan method ) look for chemical
composition change or biodegradation products
• Analyze 3-5 samples for each detected soil at
one time
• Use small net of bolt for separation into 2 parts
and analyze parallel
• Chemically analysis will be carried out only each
3 months
• Use the same soil for dioxin analysis in several
laboratories
Da Nang Pilot Study
VAST/EPA Joint Bioremediation Study
Bio -Technologies Tested:
•
Anaerobic Treatment Cells
•
Unamended “Anaerobic” Reference Cell
•
Aerobic (Oxidative) Land Treatment Units (LTU)
•
Unamended Open Reference Cell (Watered to
prevent dehydration, but aerobic and subject to
Natural Attenuation)
System was managed by VAST personnel, who
deserve gratitude for their hard work, diligence,
and care.
Thanks to the VN Ministry of Defense for providing
field support
Product amendment - IBT - Vietnam
Chemical amendment - EPA - US
1
3
2
4
Chemical, physical and biological
parameters for evaluation of
herbicide/dioxin degradation
• Total toxicity and congener toxicity (GC/MS, DR- Calux)
• Concentration of 2,4,5-T, 2,4-D
• Chemical composition change during bioremediation
(GC/MS -Scan)
• Moisture, pH
• Microbial enumeration during treatment (cultivation
methods in specific media + soil extract)
• Evaluation of microbial community structure during
treatment (PCR-DGGE by using 16S rRNA, 18S rRNA
coding genes )
• Detection of some functional genes such as catechol 2,3
dioxygenase gene –C230, rdh gene v.v.
• Laccase producing microbes,
• Dominated dioxin degraders
Overall Summary of Pilot Study Results
Degradation of TCDD Showing Confidence Intervals
TCDD Degradation in Da Nang Pilot Study
All Aerobic and Anaerobic Treatments
70,000
2
Aerobic TCDD = 47,934 -115 ppt./day; R =88%, p=0.0026
TCDD TEQ (ppt.)
60,000
Anaerobic TCDD = 38,184 -45 ppt./day; R 2=25%, p=0.25
50,000
40,000
30,000
20,000
10,000
0
0
30
60
90
120
150
Treatment Duration (Days)
Aerobic TEQ (ppt.)
Anaerobic TEQ (ppt.)
Linear (Anaerobic TEQ (ppt.))
Linear (Aerobic TEQ (ppt.))
180
Conclusions and Recommendations
•
This study builds on other work conducted in Vietnam on bioremediation
of TCDD and Agent Orange in the laboratory and in the field.*
*A rich body of knowledge exists on bioremediation, including published and unpublished research results,
which may be accessed by contacting VAST directly.
•
The bioremediation pilot study has demonstrated that treatment by
bioremediation is a practical alternative for reducing the concentration of
TCDD in Da Nang soil to meet Vietnam’s cleanup goal of 1,000 parts per
trillion (ppt.). It is estimated that the starting concentration of well-mixed
soil and sediment in Da Nang would be approximately 9,900 ppt.
•
Biodegradation was observed in both aerobic and anaerobic treatment
tests. Individual treatment recipes and treatment unit management
schemes yielded different rates.
•
The biodegradation rate may be linear, and if so, the amendment recipes
demonstrated in this study are capable of reducing TCDD at an average
rate of at least 100 parts per trillion (ppt.) per day. This means a stockpile
of excavated soil with average TCDD concentration of 10,000 ppt. could
be detoxified in a matter of months to achieve 1,000 ppt. Anaerobic
bioremediation would take about twice as long.
Conclusions and Recommendations
•
Aerobic biodegradation rate might be first-order, with a fixed percent per
day being degraded. In this case, total remediation time would not exceed 2
years.
•
The pilot study yielded important information relating to the engineering
design factors, including rate, extent and growth conditions, which may be
used for scaling up to full scale bioremediation.
•
Conditions suitable for biodegradation as presented in this study may be
established readily in an active landfill, which may be designed as an
aerobic, anaerobic, or combination system.
•
This study focused on measuring the reduction in dioxin concentration
using chemical analyses in accordance with standard USEPA analytical
methods. Other analytical methods support the chemical results.
•
Monitoring microbial diversity during the course of treatment is effective
for corroborating chemical results. Changes in the microbial community
structure serve as indicators of change in growth conditions within the
treatment unit, which could in turn facilitate dioxin biodegradation.
Conclusions and Recommendations
•
Bioaugmentation with small amounts of treated soil or contaminated
sediment may be effective. However, if suitable growth conditions are
provided, the indigenous microbes in the mixed soil and sediment at Da
Nang appear capable of degrading TCDD without bioaugmentation.
•
Bioremediation is recognized as a “Green Technology” or “white
Biotechnology” which has a very low energy requirement and produces few
emissions. Bioremediation is a permanent solution which produces a soil
which can be returned to beneficial use.
•
Knowledge gained from this project by both Vietnamese and US scientists
will allow for design of customized recipes suitable for addressing dioxin
and other persistent organic pollution problems throughout Vietnam and
elsewhere.
•
Data quality was enhanced by splitting samples among several laboratories.
* The variability in the data was independent of the laboratory.
*Contracted laboratories included SGS Systems in the US, a German dioxin
laboratory, an MOD dioxin laboratory, and DR-CALUX.
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