Molecular Microbial Ecology – Application in EEWS (Energy, Environment, Water, and Sustainability)

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

4. 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... Alignment of a highly conserved region of the 16S/18S rRNA Human Yeast Corn E. coli Green algae Thermophile

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

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

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

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

10. Molecular analysis

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

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: • NH4+ + NO2-→ N2 + 2H2O (ΔG= -357 kJ/mol) • (Z Allg Mikrobiol,1977, 17, 491-493) • In 1995, it was scientifically confirmed that • ANAMMOX is biologically mediated process • 15NH4+ + 14NO3- →14,15N2(98%) • 5NH4+ + 3NO3- → 4N2 + 9H2O + 2H+ • NH4+ + NO2- → N2 + 2H2O • (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 • Normal nitrogen –removal process: NH4+ + 2O2 → NO3- + H2O + 2H+ NO3- + CH2O → N2 + CO2 • ANAMMOX Process: ( NH4+ + 1.5O2 → NO2- + H2O + 2H+) NH4+ + NO2-→N2 + 2H2O The first full-scale ANAMMOX reactor (2002) at the Dokhaven wastewater treatment plant, Rotterdam, the Netherlands. (http://www.anammox.com/research.html)

23. Gas Effluent Water bath Recycle Influent Reactor operation • Artificial Wastewater: • NaNO2 + NH4HCO3 (1:1), KH2PO4 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 NH4+-N/(m3·d) • Up to 250 days: 0.4-0.8 kg NH4+-N/(m3·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. Defined as the fifth ANAMMOX genus The relationships of the different families of anammox bacteria among the Planctomycetes. (Nature Reviews Microbiology 2008, 6, 320-326 )

26. Metagenomic analysis

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

28. Biological treatment of metal containing wastewater

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

30. Treatment of heavy metals with sulfate reduction Sulfide production SO42- + 2CH2O + 2H+ H2S+ 2H2O + 2CO2 Metal sulfide precipitation H2S + Me2+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) FeS2 ＋ 7/2 O2 ＋ H2O • → Fe2+ ＋ 2 SO42-＋ 2 H+ • (b) Fe2+ ＋ 1/4O2 ＋ H+ • → Fe3+ ＋ 1/2 H2O • (c) FeS2 ＋ 14 Fe3+ ＋ 8 H2O • → 15 Fe2+ ＋ 2 SO42-＋16 H+ • The rate of (b) increase million times by bacteria.

33. Pilot-scale treatment • SO42- ＋ 2CH2O ＋ 2H+ • → H2S ＋ 2H2O ＋2CO2 • H2S ＋ Me2+ → MeS(s) ＋ 2H+

34. Anaerobic treatment of cyanide- and metal- containing wastewater Cyanide- and metal- containing wastewater CN-, [Me(CN) 4]2- Me2+, CN-, [Me(CN) 4]2- Granular sludge (SRB) Cyanide degrading SRB MeS CO2, NH3

35. [ Ni(CN)4]2--> Ni2+ + CO2 + NH3 Ni2+ + S2- -> NiS

37. Aerobic treatment of metal-complexed cyanide

38. Analysis with DGGE

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

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 dominantbacteria/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. Construction of clone libraries • DNA extraction • Liquid nitrogen grinding • Enzyme extraction • Lysozyme, Lyticase • PCR amplification • Reconditioning PCR • (18S, most sand_16S, some water_16S (7 sample) ) • Nested PCR • (ITS, all water_16S, some sand_16S) • PCR primers • 16S • 27F+1390R • 27F+1512R, 519F+1390R • 18S • 18S-nu0817+18S-nu1536 • ITS • NSA3+NLC2, NSI1 +NLB4

48. Software for sequence analysis

49. Percent of uncertainty level during microbial identification from different type clone libraries (Sample sources: Swimming Pool)

50. Methylobacterium（甲基杆菌属） 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.