1. Molecular Microbial Ecology – Application in EEWS (Energy, Environment, Water, and Sustainability) Quan, Zhe-Xue ( 全 哲 学 ) School of Life Sciences, Fudan University, Shanghai, China E-mail: quanzx@fudan.edu.cn

2. History of Molecular Microbial Ecology

3. Carl Woese – Analysis of 16S rRNA 1)represent a new kingdom “Archaebacteria” 2)A universal and quantitative phylogeny is possible The “Woesian” Revolution

4. Alignment of a highly conserved region of the 16S/18S rRNA Homo sapiens...GTGCCAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTAAAGTTGCTGCAGTTAAAAAG... S. cereviceae...GTGCCAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTAAAGTTGTTGCAGTTAAAAAG... Zea maize...GTGCCAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTTAAGTTGTTGCAGTTAAAAAG... Escherichia coli...GTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCG... Anacystis nidulans...GTGCCAGCAGCCGCGGTAATACGGGAGAGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCG... Thermotoga maritima...GTGCCAGCAGCCGCGGTAATACGTAGGGGGCAAGCGTTACCCGGATTTACTGGGCGTAAAGGG... Methanococcus vannielii...GTGCCAGCAGCCGCGGTAATACCGACGGCCCGAGTGGTAGCCACTCTTATTGGGCCTAAAGCG... Thermococcus celer...GTGGCAGCCGCCGCGGTAATACCGGCGGCCCGAGTGGTGGCCGCTATTATTGGGCCTAAAGCG... Sulfolobus sulfotaricus...GTGTCAGCCGCCGCGGTAATACCAGCTCCGCGAGTGGTCGGGGTGATTACTGGGCCTAAAGCG... E. coli Human Yeast Corn Green algae Thermophile

5. ProkaryotesEukaryotes Macroorganisms Bacteria Archaea Eukarya Not include virus Three domain theory

6. Now: uncultured: ~800,000 cultured: ~200,000

8. “Domain” of Bacteria 1994 - 13 divisions (all cultured) 1997 - 36 divisions 24/12 2003 - 53 divisions 26/27 2004 - 80 divisions 26/54 2008: 30/70

9. Research Field Taxonomy Ecology Bioinformatics Cultured <5% Uncultured >95% Molecular Microbial Ecology Metagenome Microbial Diversity CultivationClassification

10. Molecular analysis

11. Nitrogen Cycle Environmental Technology Removal of organic carbon Removal of nitrogen and phosphate Ecology Carbon cycle (Greenhouse gas) Coupling of Carbon and Nitrogen cycles

12. Ammonium-Oxidizing Microorganisms Aerobic Ammonium-Oxidizing Bacteria Aerobic Ammonium-Oxidizing Archaea Anaerobic Ammonium-Oxidizing (ANAMMOX) Bacteria

13. Diversity of Ammonium-Oxidizing Bacteria and Archaea in Changjiang (Yangtze River) Estuary

14. (PNAS 2005, 102, 14683-14688)

16. Diversity of ammonium-oxidizing archaea (Nature, 2005, 437, 543-546)

17. Chongming Island At the estuary of Yangtze river The 3rd largest island in China Area: >1000 square kilometers, increasing >10 square kilometers per year

19. Diversity of Ammonium-Oxidizing Bacteria in a Granular Sludge Anaerobic ammonium- Oxidizing (ANAMMOX) Reactor

20. ANAMMOX (anaerobic ammonium oxidation) In 1977, the existence of chemolithoautotrophic anammox bacteria was predicted: NH 4 + + NO 2 - → N 2 + 2H 2 O (ΔG= -357 kJ/mol) (Z Allg Mikrobiol, 1977, 17, 491-493) In 1995, it was scientifically confirmed that ANAMMOX is biologically mediated process 15 NH 4 + + 14 NO 3 - → 14,15 N 2 (98%) 5NH 4 + + 3NO 3 - → 4N 2 + 9H 2 O + 2H + NH 4 + + NO 2 - → N 2 + 2H 2 O (AEM, 1995, 61,1246-1251)

21. ANAMMOX in marine ecological system 30-50% of fixed-nitrogen in marine environment would be removed through ANAMMOX process. Black Sea and Golfo Dulce, Costa Rica (Nature, 2002,422, 608-611; 606-608) Benguela upwelling system (PNAS, 2005, 102,6478-6483)

22. ANAMMOX application in wastewater treatment The first full-scale ANAMMOX reactor (2002) at the Dokhaven wastewater treatment plant, Rotterdam, the Netherlands. (http://www.anammox.com/research.html) Normal nitrogen –removal process: NH 4 + + 2O 2 → NO 3 - + H 2 O + 2H + NO 3 - + CH 2 O → N 2 + CO 2 ANAMMOX Process: ( NH 4 + + 1.5O 2 → NO 2 - + H 2 O + 2H + ) NH 4 + + NO 2 - → N 2 + 2H 2 O

23. Reactor operation Influent Recycle Gas Effluent Water bath Artificial Wastewater: NaNO 2 + NH 4 HCO 3 (1:1), KH 2 PO 4 10 mg/l, yeast extract 5 mg/l, and TE. Sludge: river sediment (1400 mg VSS/l) Loading rate: 1-130 days: at 0.3 kg NH 4 + -N/(m 3 · d) Up to 250 days: 0.4-0.8 kg NH 4 + -N/(m 3 · d) (80% removal) 351 days: stable removal 82-86% Sludge sampling: day 377. ANAMMOX reactor

24. Phylogenetic tree based on 16S rRNA gene sequences amplified from the anammox reactor sludge using Planctomycetales-specific primers.

25. The relationships of the different families of anammox bacter ia among the Planctomycetes. (Nature Reviews Microbiology 2008, 6, 320-326 ) Defined as the fifth ANAMMOX genus

26. Anammox bacteria Matched contigs3042 Number of assembled reads269,212 (31.7%) Sum of contig length561.25Kb Matched ORFs in Kuenenia1346 Best match with Kuenenia3023 Best match with KSU-119 Best match with others145 Metagenomic analysis

27. - Microbial population in ANAMMOX reactor - Isolation of novel species from ANAMMOX reactor - Anaerobic ammonium oxidation with sulfate reduction  Cowork with environmental engineers ：

28. Biological treatment of metal containing wastewater

29. Free heavy metals Cyanide-complexed heavy metals High concentration of heavy metals High concentration of cyanide Heavy metal wastewater Biological treatment of heavy metal containing wastewater

30. Treatment of heavy metals with sulfate reduction Sulfide production SO 4 2- + 2CH 2 O + 2H + H 2 S + 2H 2 O + 2CO 2 Metal sulfide precipitation H 2 S + Me 2+ MeS(s) +2H + Bacteria Solid substrates: UASB granule Cow manure

31. Method for recovering heavy metals from the drainage containing heavy metals, 10-0414891, Korea.

32. Acid mine drainage Production (a) FeS 2 ＋ 7/2 O 2 ＋ H 2 O → Fe 2+ ＋ 2 SO 4 2- ＋ 2 H + (b) Fe 2+ ＋ 1/4O 2 ＋ H + → Fe 3+ ＋ 1/2 H 2 O (c) FeS 2 ＋ 14 Fe 3+ ＋ 8 H 2 O → 15 Fe 2+ ＋ 2 SO 4 2- ＋ 16 H + The rate of (b) increase million times by bacteria.

33. SO 4 2- ＋ 2CH 2 O ＋ 2H + → H 2 S ＋ 2H 2 O ＋ 2CO 2 H 2 S ＋ Me 2+ → MeS(s) ＋ 2H + Pilot-scale treatment

34. Anaerobic treatment of cyanide- and metal- containing wastewater Cyanide- and metal- containing wastewater Me 2+, CN -, [Me(CN) 4 ] 2- CN -, [Me(CN) 4 ] 2- MeS CO 2, NH 3 Cyanide degrading SRB Granular sludge (SRB)

35. [ Ni(CN) 4 ] 2- -> Ni 2+ + CO 2 + NH 3 Ni 2+ + S 2- -> NiS

37. Aerobic treatment of metal-complexed cyanide

38. Analysis with DGGE

40. Free heavy metals Cyanide-complexed heavy metals High concentration of heavy metals High concentration of cyanide Heavy metal wastewater Degrade different types cyanide in aerobic condition Precipitate heavy metal with sulfate reduction Degrade cyanide in sulfate reducing condition Biological treatment of metal containing wastewater

41. Monitoring of microorganisms in water

42. Microbial Monitoring for Drinking Water Evaluation of drinking water - Previous: Plate counting of total bacteria and enterobacteria - New: Pathogenic protozoa Cryptosporidium, Giardia - Future: Detection of viruses

43. Detection of viruses: using fecal bacteriophage (host: E. coli, Bacteroides fragilis) using real-time PCR quantification cultivating viruses in cells and detecting with quantum-dot nanocomplexes

44. Change of microbial populations in swimming pools treated with non-chlorine disinfectant

45. Requirement of the company the identity of the organisms found in each sample an idea of the proportions of each (i.e. which are the dominant bacteria/and fungi in each sample) how this dynamic changes over the sampling time during the pool summer if there are distinct differences in the ecology of the two groups of pools that were sampled.

46. Samples 12 swimming pools (6 pools were treated with original chemical and the others were treated with new chemicals) Three sites: pool water, sand filter, pipe line 8 sampling times: (June-Sept. two weeks interval)

47. DNA extraction Liquid nitrogen grinding Enzyme extraction oLysozyme, Lyticase PCR amplification Reconditioning PCR o (18S, most sand_16S, some water_16S (7 sample) ) Nested PCR o (ITS, all water_16S, some sand_16S) PCR primers 16S o27F+1390R o27F+1512R, 519F+1390R 18S o18S-nu0817+18S-nu1536 ITS oNSA3+NLC2, NSI1 +NLB4 Construction of clone libraries

48. Software for sequence analysis

49. Library type Total number of clones sequenced OTU uncertainty value_0(%) OTU uncertainty value_1(%) OTU uncertainty value_2(%) OTU uncertainty value_3(%) OTU uncertainty value_4(%) OTU uncertainty value_10(%) 16S rRNA gene 1100979.01.21.70.53.913.7 ITS986049.817.77.67.17.510.3 18S rRNA gene 35408.811.229.413.127.210.3 Percent of uncertainty level during microbial identification from different type clone libraries (Sample sources: Swimming Pool)

50. Average contents of major types bacteria in different group sand samples. (‘Sand-new-good-743(13)’ means the data is analyzed from the 743 clones from 13 ‘good’ samples treated with new chemical.) The number at X-axis are matching to the order of major bacterial type. Methylobacterium （甲基杆菌属）

51. ChemicalLibrarySand (%)Water(%) Old GO1.83.2 GR1.83.3 MA2.61.1 MU5.27.5 SC18.71.9 WI3.34.6 old average 5.6 3.6 New BR0.86.6 BT12.811.6 RI29.010.4 ST32.37.8 ZJ16.85.8 ZP9.44.8 new average 16.8 7.8 Pipe 30.3 Content of Methylobacterium in each swimming pool

52. Average contents of major types bacteria in different group water samples. (‘Water-new-good-855(14)’ means the data is analyzed from the 855 clones from 14 ‘good’ samples treated with new chemical.) The number at X-axis are matching to the order of major bacterial type. Sphingomonas （鞘氨醇单胞菌）

53. Relationships between turbidity and content of Sphingomonas in the water samples. Y=0.033*X+0.44 R2=0.39

54. Average contents of major types fungi identified by ITS gene in different group sand samples. The number at X-axis are matching to the order of major fungal type with ITS analysis. Candida （念珠菌） Alternaria （链格孢） Epicoccum （附球菌） Cladosporium （枝孢霉）

55. Average contents of major types fungi identified by ITS gene in different group water samples. The number at X-axis are matching to the order of major fungal type with ITS gene analysis. Candida

56. BioEnergy

57. Growth rate of energy consumption per year(%) Growth rate of energy consumption in future 20 years

59. 20042005200620072008 United States223261335457656 Europe5280130154214 Russia00000 India34455 Indonesia0.00.21.52.63.5 Vietnam00000 South Korea0.10.20.91.83.2 China1721283538 Total Biofuels Production (Thousand Barrels Per Day)

60. USA Korea (South) IndiaIndonesiaVietnamChina Urban SO2 concentration (mg/m3) 15.43 (114/141) 52.41 (63)27.55 (93)51.05 (65)64.07 (51)97.07 (28) SO2 emissions per populated area (thousand metric tons/squ) 1680 (38/141)19430 (2)1150 (47)360 (84)260 (92)2680 (22) Urban N2O concentration (mg/m3) 60.57 (45/141)52.86 (65)29.7 (122)34.6 (111)65.5 (30)71.7 (15) NOx emissions per populated area (thousand metric tons/squ) 1.29 (13/141)1.24 (14)0.52 (33)0.18 (94)0.56 (32)0.75 (27) CO2 Emissions (kt) 5788181 (1/195) 455878 (9) 1273175 (4) 295033 (21)76095 (41)4143494(2) CO2 Emissions (kt/1000 people)19.9 (11/196)9.5 (34)1.2 (119)1.4 (114)0.9 (129)3.2 (89) CO2 from fossil fuels 2000 (per $ GDP) (per $100 million) 0.0133 (9/25) 0.0124 (11) 0.0076 (22) 0.0084 (20)-0.0107 (14) CO2 from fossil fuels 2000 (per capita) (per 1 million people) 5.31 (1/25)2.36 (12)0.23 (25)0.29 (24)-0.59 (22) Forest area (sq. km) 3030890 (4/195) 62650 (68) 677010 (10) 884950 (8) 129310 (41) 1972900 (5) Forest area > (% of land area)33 (84/195)63 (20)23 (115)49 (42)41.7 (59)21 (122) Forest area (sq. km/1000 people)10225 (44/195)1297 (135)619 (162)4012 (87)1556(126)1512 (130) Fertiliser consumption (hundred grams/hectare) 1117 (48/141)5117 (8)1040 (52)1546 (34)3416(15)2825 (21) Air Pollution Reated data (ranking)

61. USA South Korea IndiaIndonesiaChina Organic water pollutant (BOD) emissions (Kg/d) 180586131517715198427329656088663 Organic water pollutant (BOD) emissions (Kg/d/worker) 0.13 (47/115)0.12 (49)0.2 (14)0.18 (15)0.14 (60) Water pollution source (% of total BOD emissions) chemical industry14 (9/114)13 (11)9.24 (27)9.17 (13)14.8 (8) food industry42 (31/114)26 (45)53.7 (14)53.7 (12)28.1 (60) metal industry9.6 (13/94)11.3 (9)12.2 (7)2.5 (19)20.4 (5) paper &pulp industry10.6 (35/111)18.9 (16)7.6 (46)8.2 (25)10.9 (43) textile industry5.4 (40/114)13.6 (16)12.8(19)19.4 (8)15.47 (16) Water Polution Related Data (Ranking) Source: www.nationmaster.com

62. Energy Petroleum  Biosurfactant  Application of in-situ microorganisms Coal  Desulfuration Bioleaching Biofuel  Microalgae  Anerobic digestion  Hydrogen-gas production  MFC (microbial Fuel Cells)  Plant: Cellulase

64. ProductionImportsExportsConsumption United States19089460882223047 Europe1071615305611520106 Russia230642059837716746 India110835201460 Indonesia2422011991224 Japan190337703738 South Korea14117901231 China2446138952490 Natural Gas Overview 2007 (Billion Cubic Feet)

68. Metagenomics (Environmental genomics) Example: Ethanol from starch and lignocellulose Metagenomic screening of applicable cellulase Source: rumen (IM), termite (SIBS), biogas fermentation reactor with rice straw (SIBS) Enzyme screening for new source of energy

69. The U.S. Department of Energy (DOE) Office of Science: support sequencing - 485 microbial genomes - 30 microbial communities (metagenomes) Objectives: seek solutions to difficult DOE mission challenges: - alternative sources of energy - cleaning up environmental wastes - understanding biological carbon cycling as it relates to global climate change (sustainability)

70. Identification of novel bacteria

71. Novel Bacteria Genus novel ：  Henriciella marina  Joostella marina Species novel ：  Altererithrobacter dongtanensis  Flavobacterium dongtanense  Pseudomonas caeni  Chryseobacterium caeni  Azonexus caeni  Rhizobium daejeonense Novel bacteria list accepted by ICSB (International committee on systematic bacteriology) （ as first or corresponding author ）

72. Class novel

73. Im WT, Kim KY, Rhee SK, Jung HM, Meng H, Lee ST, & ZX Quan* Description of Fimbriimonadia class nov. of the phylum Armatimonadetes and the diversity and abundance of this class in various environments. Appl Environ Microbiol (submitted)

74. Full genome sequencing

75. Published SCI Papers -First author No.TitlesTimeJournalCitation 1 Henriciella marina gen. nov., sp. nov., a novel member of the family Hyphomonadaceae isolated from the East Sea 2009.4 J Microbiol (IF 1.5) 2 Diversity of ammonium-oxidizing bacteria in a granular sludge anaerobic ammonium- oxidizing (anammox) reactor 2008.11 Environ Microbiol （ IF 4.9 ） 14 3 Joostella marina gen. nov., sp. nov., a novel member of the family Flavobacteriaceae isolated from the East Sea. 2008.6. Int J Syst Evol Microbiol （ IF 2.1 ） 1 4 Chryseobacterium caeni sp. nov., isolated from bioreactor sludge. 2007.1 Int J Syst Evol Microbiol (IF 2.1) 13 5 Azonexus caeni sp. nov., a denitrifying bacterium isolated from the sludge of wastewater treatment plant 2006. 5 Int J Syst Evol Microbiol （ IF 2.1 ） 3 6 Rhizobium daejeonense sp. nov., nickel- complexed cyanide-degrading bacterium 2005. 11 Int J Syst Evol Microbiol （ IF 2.1 ） 12 7 Hydrolyzed molasses as an external carbon source in biological nitrogen removal 2005. 10 Bioresource Technol （ IF 4.3 ） 19

76. Published SCI Papers -Corresponding author No.TitlesTimeJournalCitation 1 Flavobacterium dongtanense sp. nov., isolated from the rhizosphere of reed in wetland 2010.3 Int J Syst Evol Microbiol (IF 2.1) 2 Bacterial diversity of water and sediment in the Changjiang estuary and coastal area of the East China Sea 2009.11 FEMS Microbiol Ecol (IF 3.6) 1 3 Pseudomonas caeni sp. nov., denitrifying bacteria isolated from sludge of an anaerobic ammonium-oxidizing bioreactor 2009.10 Int J Syst Evol Microbiol (IF 2.1) 4 Could nested-PCR be applicable for the study of microbial diversity? 2009.8 World J Microbiol Biotechnol (IF 1.1) 5 The bacterial diversity in an anaerobic ammonium-oxidizing (anammox) reactor community 2009.7 Syst Appl Microbiol (IF 2.6) 2 6 Analyses of Microbial Consortia in the Starter of Fen Liquor 2009.4 Lett Appl Microbiol (IF 1.6)

77. Recent Projects –Project manager 2011.1-2013.12 “Study of carbon- and nitrogen- cycle related active microbial population in soil of tidal flat”, Supported by National Natural Foundation of China. 2010.7-2012.6 “Investigation of pollutant contamination and bioremediation potential on the seashores neighboring on the Yellow Sea in Korea and China” ， Supported by the NSFC-NRF Scientific Cooperation Program 2008.3-2009.4 “Population of microbiology in fermentation of Fen-liquor” Supported as the Project of Scientific and Technological Innovation in Shanxi province, China. 2007.1-2009.12 “Diversity of anaerobic ammonium-oxidizing bacteria and metagenomic research”, Supported by National Natural Foundation of China. 2006.6-2006.12 “Microbial diversity in swimming pools” Supported by one of Chemical Compony in USA 2005.7-2008.3 “Metagenomics of anaerobic nitrogen removal bacteria and isolation of related microorganisms”, Supported by Korea Advanced Institute of Science and Technology

78. Lab members