Detoxification of herbicide/dioxin contaminated soil based on microbial diversity and their gene expression

2ND Meeting of The Agent Orange Working GroupDetoxification of herbicide/dioxin contaminated soil based on microbial diversity and their gene expression Institute of Biotechnology IBT Vi

2ND Meeting of The Agent Orange Working Group

Detoxification of herbicide/dioxin contaminated soil based on microbial diversity and their gene expression

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

• 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 vi sinh vật nuôi cấy được –cultural microbes

-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?

Đa dạng VSV: đa dạng về loài VSV và khả năng trao đổi chất

của VSV 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

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 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

Cây phát sinh chủng loại giữa một số trình tự đại diện mã hóa enzym α- 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 HDN7 HDN8 HDN9 3,67 x

Copy number catechol dioxygenase in

aerobic biotreating cells

Detoxification of herbicide/dioxin at

different scale

1 At the lab Condition

2 At field trail (0.5- 100 m 3 ), Danang hot spot

3 Bioreactors at lab condition (50kg soil)

4 Active landfill at Bien Hoa, 3384 m3

5 VAST and EPA pilot scale (11 cells with 2 m 3 soil)

Biotreatment at pilot scale in

lab condition

Treatment

s

2,3,7,8 TCDD concentrati

on (ppb)

degradatio

n percentage

in comparison

• 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.

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,

2,4,6-trichloro-; Phenol, 2,4,6-trichloro-; Phenol, bis(1-methylpropyl); Phenol, 2,3,4,6- tetrachloro-; Phenol, 2,3,5,6-tetrachloro-;

2,6-Phenol, 2,3,5-trichloro-; 2,6-Phenol, trichloro-

2,3,6-Phenol, 2,4,6-trichloro-; 2,3,6-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;

1,2-Dimethoxy-4,5-dichloro-benzene; Benzene, dichlorodimethoxy- ; Naphthalene, 1,3,7- trichloro-; Benzene, 1,2,4-trichloro-5- ethoxy-

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;

IVB 9-Octadecenamide, (Z)-; 9-Octadecenoic acid,

(E)-; Dodecanoic acid; Dodecanoic acid, methylethyl ester;

1-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; Hexadecenoic acid

Z-7-2.9

Soil composition change before and after treatment

• Result of GC/MS analysis indicates that 51,7% of toxicity of several hundreds µg 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

• Initial toxicity:

268.000 pgTEQ/g

• After two year

treatment: 129.200 pgTEQ/g (51,7 % removed)

Change in composition of 10DNT and

100DNT treatments after 24 and 29 months

Grou

p

Compouds Samples of two year treatment from

100DNT biotreatment Detected compounds Pick

Conclusion (Danang trals)

• Application of in situ bioremediation treatment not only dioxin was

reduced but other polluted components in soils, 2,4-D; 2,4,5-T, 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, T; 2,4-D and other chemicals in contaminated soils;

2,4,5-• 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

After 16 weeks treated in

both aerobic and

facultative bioreactors 12.129 44,1

Average 85,3 pg TEQ/g/day detoxified

“ 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)

PROJECT (Bien Hoa)

Products: DHS1, DHS2 AND

SLOW-D

What have been researching and developing in IBT- VAST concerning to the use of bioremediation for detoxification of herbicide/dioxin and other POP

contaminated soil and sediment?

degradation capability analyzed by HPLC, GC/MS, DR-Calux and other methods;

(Lakes) and their change during bioremediation treatments of

Danang and Bien Hoa former military bases;

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.

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

Application of bioremediation at pilot scale

VAST and EPA Vietnam USA

Dang Thi Cam Ha Harry L Allen

Nguyen Ba Huu Vance Fong

Nguyen Nguyen Quang

Nguyen Quang Huy

Dam Thuy Hang

Nguyen Ngoc Anh

Phung Khac Huy Chu

Vietnam- US cooperation

in Danang

• 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

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

Da Nang Pilot Study VAST/EPA Joint Bioremediation Study

Chemical amendment - EPA - US

Product amendment - IBT - Vietnam

1 2

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

• 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

Conclusions and Recommendations

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.

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.

will allow for design of customized recipes suitable for addressing dioxin and other persistent organic pollution problems throughout Vietnam and

elsewhere.

* 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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