Bioremediation of Nanjing Source from Yangtze River (NSYR) with IKPF Strain Artificial Wetland (ISAW)

Cheng ShupeiState Key Laboratory of Pollution Control and Resource Reuse of China

Nanjing University

NSFC-JST Bilateral Workshop on Environmental Impact Assessments and Protection Technologies for Watershed & Ecosystems Sustainability

9:45-10:10 AM， May 17，2006. Beijing

HighlightBioremediation of NSYR with ISAW

needs Bioinformatics Techniques (BiTs)in this research.

Abbreviations in Highlight BiTs: Bioinformatics TechniquesNSYR: Nanjing Source from Yangtze RiverISAW: IKPF Strain Artificial WetlandIKPF : Inter-Kingdom Protoplast Fusion

1. BiTs should be used to control POPs riskson human health.

2. POPs risks in NSYR concerning cancer frequency reported in the last 5 years

3. BiTs will be used to detect NSYR molecular toxicities to human in this research

4. BiTs will be used for bioremediation of NSYR with ISAW in this research

Contents

Content 1.BiTs should be used to control POPs

risks on human health.

1.1.Chinese Government Put Signature on Stockholm Convention on Persistent Organic Pollutants (POPs) on May 23, 2001

Chinese Government has taken measures to eliminate or reduce the release of POPs into the environment and to protect human health and the environment from POPs.

1.2. There were three topics for China 244th

Science Meeting held on Dec.24, 2004.

Topic 1. Genomics it means thattesting POPs toxicities on genome substance must use BiTs

Topic 2. Proteomics it means thattesting POPs toxicities on proteome expressed must use BiTs

Topic 3. Bioinformatics it means thatassessing POPs risks on human health must use software of BiTs

Content 2. POPs risks in NSYR concerning cancer frequency reported in the

last 5 years

2.1. POPs in NSYR Reported (1) PCBs existed in NSYR was > 0.5 (μg/L)

reported in 2001. PCBs as one of POPs was listed here only.

(2) PCBs value should be ≤0.5 (μg/L)in both [GB 5749-2005] & [EPA816-F-02-013-2002]

(3) Ames & chromosome tests showed that NSYR had risks reported in 1995.

PCBs has a lot of toxicities to human health.including increased risk of cancer; thymus gland problems, immune deficiencies; reproductive or nervous system difficulties.

2.2. Conclusions of NSYR risks in the last 5 years reported

(1) The frequency for POPs detected in NSYR was at the highest level among 22 sections of Yangtze River.

(2) The effects of NSYR risks on human health was at the highest level among 22 cities along with Yangtze River.

(3) Data illustrate that cancer frequency was as high as 0.65% for Nanjing concerning POPs in NSYR .

2.3. Cancer frequency for Nanjing reported

Items China Jiangsu Nanjing

Total population 1.3 billions 70 millions 7 millions

Cancer Patients /y 2 millions 0.15 millions

0.0456 millions

Cancer frequencyPatients/Population

0.15% Globe value=0.16%

0.21% 0.65%=4fold of Globe

Total-lost, USD/y 34 billions 2.5 billions 0.776 billions

Total-lost, RMB/y 272 billions 20 billions 6 billions

(1) 20 thousand USD Lost /(Y• patient), medical treatment + working disability(2) Reproductive, immune, nervous system, children diseases don’t been included.

Content 3. BiTs will be used to test NSYR

molecular toxicities to human in this research.

3.1. Why should we use BiTs to test NSYR toxicities in this research ?

(1) POPs existed in NSYR is as low as the level of μg/L, but POPs may have toxicities to genome & proteome.

So, we must test POPs toxicities to genome & proteome.(2) POPs toxicities to genome & proteome can be detected

by use of BiT only. So, we must use BiTs to test POPs toxicities to genome & proteome.(3) POPs risks on health couldn’t be controlled by use of

a simple standard for COD. So, we must program software to control POPs resks with BiTs.

which were chemical byproducts existing in petrochemical WW & pharmaceutical WW

(such as phthalic acid or ester, and tetrahydrofuran et al).

Definitions of POPs(1) POPs are set of toxic chemicals in the environment for long

period of time & biomagnified moving up through food chain. (2)POPs linked to adverse effects ( A) cancer; (B) nervous system;

(C) reproductive system; ( D) immune system (3) POPs in 3 major categories (A) Insecticides; ( B) PCBs; (C).

certain chemical byproducts, dioxins et al.

3.2. POPs we have researched

3.2(1) BiT we have used to measure POPsmolecular toxicities

(1)Genome toxicities of petrochemical POPs WW tested in 2005 by use of Mouse Genome 430A 2.0 GeneChip on mice liver genes

(2) Reproductive toxicities of pharmaceutical POPs WW testedin 2005 by use of Flow Cytometer on mice sperm DNA

(3) Serological toxicities of pharmaceutical POPs WW testedin 2005 by use of Biochemical Meter to mice serum

(4) Programmed Software to predicate risks & functionsin 2001-2004 programmed for petrochemical WW treated with IKPF strain

3.2.(1)-1, GeneChip we used to test toxicities of POPs WW on genome of mouse liver in 2005

(Mouse Genome 430A 2.0), There were 14000 Gene DNA Probes Fixed in 1cm 2 of Gene Chip Area.

3.2.(1)-2, Scatter Graph of Gene Chip

There were 232 genes for expression enhanced and 74 genes for expression weakened for the mice after treated with PTA WW.

3.2.(2)-1, Photos of sperm morphology from the mice treated with Pharmaceutical POPs WW

Abnormal sperms from Treated Group Normal sperms from Control Group

3.2.(2)-2. Photos of dead cells for mice sperm dyed with double fluorochromes.

The red markers show the dead sperm cells.

Control Group Treated Group

3.2.(3) Serological toxicities of pharmaceutical POPs WW

on mice detected by use of Biochemical Meter to serum in 2005, PTA WW

Items Control Group Treated Group Statistics

LDH, (U/L) 772.25±72.18 687.00±87.88 P < 0.05

GGT, (U/L) 0.88±0.64 0.25±0.44 P < 0.05

CHOL, (mmol/L) 1.98±0.37 2.50±0.51 P < 0.05

URIC, (μmol/L） 113.00±11.34 133.89±18.24 P < 0.05

There were 4 parameters being different from the control mice. It means that pharmaceutical WW had toxicities to liver and kidney.

3.2.(4) Surface of Ebis software we programmedin 2001-2004 to predicate petrochemical WW risks & treatment efficiency

3.3. How to use BiTs to assess NSYR molecular toxicities in this research?

intra-gastric perfusion of rats for 90d-720d

Animal Rats Treatedwill be treated with influent & effluent

of ISAW, solution of POPs chemicals and clean water during

for 90-720dAnimal TreatedInformation

NSYR Sample Takencontinuously will

include the influent &effluent

of ISAW at the pilotof bioremediation

NSYR SampleTaken Information

Analysis of POPsExisting in NSYZ

detected by use of GC/LC-MS,

POPs Existing in NSYR Information

Measuring Molecular Toxicities with BiTs

(1) Genome toxicity: Gene Chip U34

(2) Proteome toxicity: Protein Chip C12

(3) Reproductive risk: RIA Kit+T&E2

(4) Immune toxicity: U34+ C12

(5) Nervous toxicity: NTE+ AChE

Toxicological BiTsInformation on Animals

At last integratingall the information

& programming BiTSoftware to assess NSYR

risks on citizen health during bioremediation

Content 4. BiTs will be used for

bioremediation of NSYR in this research

4.1. Why does bioremediation of NSYR need BiTs?

• First: Measurement & judgment of POPs risks in NSYR on citizen health will need BiTs.

• Second: Measurement & judgment of IKPF strain abilities & ISAW functions will need BiTs.

• Third: Optimization & assessment of feasibility for bioremediation of NSRY will need BiTs.

4.2. IKPF we have researched for 15 years from 1991-2006

(1)1991-1994: Inter-species protoplast fusion (ISPF) /MSG WW2 bacteria (Rhodopseudomonas sphaeroides & Rh. palustris)

(2)1994-1997: Inter-genius protoplast fusion (IGPF)/alcohol WW 2 fungi (Saccharomyces cerevisiae & Candida tropicals)

(3)1997-2000: Inter-kingdom protoplast fusion (IKPF)/food WW1 fungus + 1 bacterium (S. cerevisiae & R. sphaeroides)

(4)2001-2004: Inter-kingdom protoplast fusion (IKPF)/POPs WW2fungi+1 bacterium, Phanerochaete chrysosporium & S. cerevisiae & Bacillus YZ1

(5)2003-2006: Inter-kingdom protoplast fusion (IKPF)/POPs WW2fungi +1bacterium, P. chrysosporium & S. cerevisiae & Bacillus XZ1

4.2(2)-1. The First IKPF Strain Fhhh Photos for treating petrochemical WW

Hybrid Fhhh, Fhh+SC

Parental strain PC Parental strain YZ1 Parental strain SC

Hybrid Fhh, PC+YZ1

SEMScreening Electron Microscope

AMR 1000 USA; EHT=12.00KV,

WD=16mm, Mag=5.00kx, Detector=SE1,

University of Hong Kong2001.8

4.2.(2)-2. Pilot system for Petrochemical POPs WW Treatment with IKPF-Fhhh Strainin 2001-2004, 10m3 of aeration basin, q (SLR)=1.35d-1; μ=0.12h-1; ；f=0.30 h-1

Pilot Study System, 100m3/d Biofilm of Fhhh in Aeration Tank

4.2.(3)-1. The Second IKPF Strain Xhhh Photos for Treatment of Pharmaceutical WW

XZ1, native bacteria PC fungus SC fungus

Xhh form XZ1+PC; Xhhh from Xhh+SC

SEMScreening Electron Microscope

AMR 1000 USA; EHT=12.00KV,

WD=16mm, Mag=5.00kx, Detector=SE1,

University of Hong Kong2003

4.2.(3)-2.Pharmaceutical POPs WW Treatment Plant by Use of IKPF-Xhhh Strain in 2003, 500m3/d

Aeration Tank for POPs WW Treatment Biofilm of Xhhh in aeration tank

4.2(4). Efficiencies of two IKPF strains for treatment of POPs WWs in 2001-2005

(1) Fhhh sludge loading rate was 1.35 d-1 as high as that of 6fold of the native bacterium YZ1 (POPs were phthalic acid or phthalic ester et al.).

(2) Xhhh sludge loading rate was 0.22 d-1 as high as that of 2fold of the native bacterium XZ1(POPs were tetrahydrofuran et al.) .

4.3. How to use BiTs for bioremediation of NSYR in this research?

IKPF-NShhh Strainwill be constructed via

IKPF of 3 parental strains & their genes

IKPF Information

NShhh functionsjudged with BiTsby testing genome

& proteomeBiTs Information

Artificial WetlandISAW will be built

with reed root-zone and IKPF-NShhh strain

Construction Informationof Artificial Wetland

POPs in NSYRwill be analyzed by use of GC/LC-MS

methods POPs in NSYR

Information

Bioremediation of NSYR will be conducted

and genes & process will be optimized

Optimal Information of Artificial Wetland

At lastIntegrating all the

information & programming BiTsoftware to assess& optimize ISAW

for bioremediation of NSYR

4.3(1). New Functional Strain IKPF-NShhhwill be constructed by IKPF of 3 parental strains in this research

Three parental strains will be:(a) The first will be fungus: Phanerochaete chrysosporium

(PC) with high degradability to degrade POPs in NSYZ(b) The second will be a native bacterium (NRpcbs)

with high adaptability isolated from PCBs polluted environment(c) The third will be fungus: Saccharomyces cerevisiae(SC)

with high flocculation to make the effluent more limpid

IKPF-NShhh will be a functional strain with high degradability, adaptability & flocculation from integration of 3 parental genes.

4.3(2). IKPF-NShhh Strain Artificial Wetland (ISAW) will be constructed with Reed-Zone+IKPF-

NShhh+Gravels

1

2

6

4

3

8

5

7

(1) influent of ISAW; (2) Input basin; (3)Bed of reed-zone+ IKPF-NShhh + gravels; (4) Up-flow of ISAW; (5) Output basin; (6) Effluent of ISAW to treatment plant; (7)Sediment of ISAW effluent; (8) Reed plant; (9)Sun light, the energy of ISAW

9

Reed-Zone+IKPF-NShhh+gravel

4.3(3). Some important parameters will be measured in this research

(1) IKPF-NShhh Strain & its BiTsA. genome DNA map; B. proteome AA map; C. MnP; D. SDR value (d-1) in ISAW

(2) Bioremediation of NSYR & its BiTsA. Flow of Pilot ISAW for bioremediation of 10m3/d of NSYZB. Sizes of Pilot ISAW being same to the control without IKPF-NShhh strain

depth of reed-zone bed=1m; depth of surface water=0.7m; length≈18m; width≈10m, area≈180m2; volume of reed-zone bed=180m3; HRT=12dvolume of surface water=120m3; Biomass of IKPF-NShhh existing at reed-zone≥360kg;

C. PCBs Degradation PCBs in influent=0.869(mg/m3); PCBs in effluent≤0.5(mg/m3); SDR of IKPF-NShhh for PCBs≥10-4•0.103(d-1) [referred to SDR=10-4·3.375d-1-10-4·5.4d-1]; VLR of ISAW for PCBs≥10-4•0.205kg/(d•m3); COD, BOD, TOC,TSS will be tested.

D. Optimization of ISAW regulation of metals, flow rate, COD:TN:TP, and IKPF-NShhhBioinformation will be measured.

(3) Reed-Zone Bed Construction & its BiTshas aerobic, anaerobic and simultaneous areas for all microbes to degrade PCBs and has the functions of filtering and adsorbing the water.

The 6 patents we applied for both IKPF and BiTs

处理PTA石化POPs废水-IKPF-Fhhh(1)两真菌与一细菌原生质体融合的特效菌株及其构建方法,

专利申请号: 02138171.0 申请时间： 2002.8.26.(2)特效菌剂的制备与处理化工废水或常规有机废水的方法

专利申请号: 02138171.2; 申请时间： 2002.8.26.处理合成制药POPs废水-IKPF-Xhhh(3)降解合成制药有机废水的特效菌株及其构建方法

专利申请号: 200410041125.5;申请时间：2004.6.30(4)特效菌株处理合成制药废水的生物技术方法

专利申请号: 200410041126.X;申请时间：2004.6.30POPs分子毒性检测-基因芯片-精子毒性(5)一种制药废水的生殖毒性检测与评价方法，

申请号：200510122775.7；申请日期2005/12/2(6)一种石化废水的基因毒性检测方法，

申请号：200510122774.2；申请日期2005/12/2

The 4 sets of software we applied for treatment of WW & predication of WW toxicities

第005607号:NJU-Ebis1废水处理技术预测软件:

第005608号:NJU-Ebis2废水处理技术诊断软件:

第009243号:NJU-Ebis3废水毒性安全控制软件:

第009244号:NJU-Ebis4废水处理工艺调试软件:

Resume, Shupei Cheng• Working experiences 2000-at present, Professor, Nanjing University (NJU)1991-2000: Associate Professor, NJU, China1985-1991: Lecturer, NJU, China1979-1984:Teaching assistant, NJU, China• Exchanges and collaborations1989.9-1990.10: Visiting Scholar, University of Toronto, Canada 1996.4-1996.7: Adjunct Scientist, Washington State University, USA2004.9-2004.9: Visiting Scholar, University of Washington, USA 2000-at present: Collaboration with University of Hong Kong, China• Academic positions Director, Institute of Environment Health & Ecology Security, NJU Director, Biology Section, The State Key Laboratory of PCRR, ChinaDirector, Environment & Agriculture Microbiology of Jiangsu, ChinaVice director, Environmental Microbiology Committee of ChinaVice director, Environmental Biotechnology of Jiangsu, Chian

••Thank You Thank You