Mini-study of four environmentally relevant contaminant-degrading enrichment cultures. and

Transcription

1 Mini-study of four environmentally relevant contaminant-degrading enrichment cultures KB-1 T3L ACT-3 ORCH4 and Biofilm formation study of the KB-1 and T3L cultures using confocal microscopy Luz A. Puentes Jácome Ph.D. student, University of Toronto, Canada Microbial Diversity 2013

2 Environment Canada:

3 Soil and groundwater contamination Environment Canada:

4 1. Acenaphthene 2. Acrolein 3. Acrylonitrile 4. Benzene 5. Benzidine 6. Carbon tetrachloride 7. Chlorobenzene 8. 1,2,4-trichlorobenzene 9. Hexachlorobenzene 10. 1,2-dichloroethane 11. 1,1,1-trichloreothane 12. Hexachloroethane 13. 1,1-dichloroethane 4. Benzene EPA s priority pollutants 36. 2,6-dinitrotoluene 37. 1,2-diphenylhydrazine 38. Ethylbenzene 39. Fluoranthene chlorophenyl phenyl ether bromophenyl phenyl ether 42. Bis(2-chloroisopropyl) ether 43. Bis(2-chloroethoxy) methane 44. Methylene chloride 45. Methyl chloride 46. Methyl bromide 47. Bromoform 48. Dichlorobromomethane 14. 1,1,1 1,1,2-trichloroethane Trichloroethane 49. REMOVED 15. 1,1,2,2-tetrachloroethane 50. REMOVED 16. Chloroethane 51. Chlorodibromomethane 17. REMOVED 52. Hexachlorobutadiene 18. Bis(2-chloroethyl) ether 53. Hexachlorocyclopentadiene chloroethyl vinyl ethers 54. Isophorone chloronaphthalene 55. Naphthalene 21. 2,4,6-trichlorophenol 56. Nitrobenzene 22. Parachlorometa cresol nitrophenol 23. Chloroform nitrophenol chlorophenol 59. 2,4-dinitrophenol 25. 1,2-dichlorobenzene 60. 4,6-dinitro-o-cresol 26. 1,3-dichlorobenzene 61. N-nitrosodimethylamine 27. 1,4-dichlorobenzene 62. N-nitrosodiphenylamine 28. 3,3-dichlorobenzidine 63. N-nitrosodi-n-propylamine 29. 1,1-dichloroethylene 64. Pentachlorophenol 30. 1,2-trans-dichloroethylene 65. Phenol 31. 2,4-dichlorophenol 66. Bis(2-ethylhexyl) phthalate 32. 1,2-dichloropropane 67. Butyl benzyl phthalate 33. 1,2-dichloropropylene 68. Di-N-Butyl Phthalate 34. 2,4-dimethylphenol 69. Di-n-octyl phthalate 35. 2,4-dinitrotoluene 70. Diethyl Phthalate PCB mixtures! 71. Dimethyl phthalate 72. benzo(a) anthracene 73. Benzo(a)pyrene 74. Benzo(b) fluoranthene 75. Benzo(k) fluoranthene 76. Chrysene 77. Acenaphthylene 78. Anthracene 79. Benzo(ghi) perylene 80. Fluorene 81. Phenanthrene 82. Dibenzo(,h) anthracene 83. Indeno (1,2,3-cd) pyrene 84. Pyrene 85. Tetrachloroethylene 86. Toluene 90. Dieldrin 91. Chlordane 92. 4,4-DDT 93. 4,4-DDE 94. 4,4-DDD 95. Alpha-endosulfan 96. Beta-endosulfan 97. Endosulfan sulfate 98. Endrin 99. Endrin aldehyde 100. Heptachlor 101. Heptachlor epoxide 102. Alpha-BHC 103. Beta-BHC 104. Gamma-BHC 105. Delta-BHC 106. PCB 1242 (Arochlor 1242) 107. PCB 1254 (Arochlor 1254) 108. PCB 1221 (Arochlor 1221) 109. PCB 1232 (Arochlor 1232) 110. PCB 1248 (Arochlor 1248) 111. PCB 1260 (Arochlor 1260) 112. PCB 1016 (Arochlor 1016) 113. Toxaphene 114. Antimony 115. Arsenic 116. Asbestos 117. Beryllium 118. Cadmium 119. Chromium 120. Copper 121. Cyanide, Total 122. Lead 123. Mercury 124. Nickel 125. Selenium 126. Silver 127. Thallium 128. Zinc ,3,7,8-TCDD Toluene 87. Trichloroethylene 88. Vinyl chloride 89. Aldrin 87. Trichloroethene

5 Let the microbes save our planet! KB-1 E- donor: methanol E- acceptor: Trichloroethene Key players: Dehalococcoides, acetogens, methanogens: Methanomicrobiales ACT-3 E- donor: MEL Methanol + Ethanol + Lactate E- acceptor: 1,1,1 trichloroethane Key players: Dehalobacter, Desulfovibrio, Methanosarcinales

6 Let the microbes save our planet! T3L E- donor: toluene E- acceptor: CO2 Methanogenic consortium: Methanosaeta, Methanospirillium, others ORCH4 E- donor: benzene E- acceptor: CO2 Community: Deltaproteobacteria, Firmicutes, methanogens 5 µm µm

7 T3L s Methanosaeta 5 µm

8 T3L s Methanosaeta

9 Mini-project Questions 1. Can we potentially use FISH to monitor the community composition and behaviour in these cultures? Can FISH help us identify our organisms of interest and reveal cell morphology? 2. Can KB-1 and T3L develop biofilms on glass cover slips? 22/07/2013 Microbial Diveristy MBL

10 In the playground with KB-1 Enrich for methanogens: methane production KB-1 -> 20% v/v in 60 ml Enrich for acetogens: acetate production KB-1 -> 20% v/v in 60 ml Microscopy Search for Dehalococcoides by scanning electron microscopy (SEM)

11 Methane production by KB-1 methanogens Moles of methane produced (µmol) Jul 06-Jul 10-Jul 14-Jul 18-Jul

12 KB-1 s methanogens

13 A pleasant surprise: Methanosarcina!!!

14 Confocal images 63X

15 3D view of Methanosarcin aggregate

16 Acetate consumption and production Acetate concentration (mm) Methanogens Acetogens 0 04-Jul Jul Jul Jul Jul-13

17 KB-1 s acetogens

18 Dehalococcoides sp. and PCBs

19 Dehalococcoides Mccartyi sp. Loffler et. al, 2013, Int. J. of Systematic and Evolutionary Microbiology 63 (Pt 2):

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25 1. Can we potentially use FISH to monitor the community composition and behaviour of the TCE, 1,1,1 TCA, toluene, and benzene degrading cultures? FISH: Eub I-III and Archaea 915 probes

26 TCE degrading culture: KB-1 TCE = trichloroethene DAPI

27 TCE degrading culture: KB-1 Eub I III probe

28 TCE degrading culture: KB-1 Archaea 195 probe

29 Toluene degrading culture: T3L

30 Toluene degrading culture: T3L Archaea 195 probe

31 Toluene degrading culture: T3L

32 Benzene degrading culture: ORCH4 DAPI

33 Benzene degrading culture: ORCH4 Eub I III probe

34 Benzene degrading culture: ORCH4

35 The benzene degrading organism is so unknwon!???

36 1,1,1 TCA degrading culture: ACT 3 1,1,1 TCA = 1,1,1 trichloroethane DAPI

37 1,1,1 TCA degrading culture: ACT 3 Eub I III probe

38 1,1,1 DCA degrading culture: ACT 3 DAPI + Eub I III filter piece: 6.67 x 10 7 DAPI stained cells/ml of culture 3.04 x 10 7 EUB I - III hybridized cells/ml of culture Relative abundance: 46% DAPI + Archaea filter piece: 5.37 x 10 7 DAPI stained cells/ml of culture 2.3 x 10 7 Archaea 915 hybridized cells/ml of culture Relative abundance: 43% Eub I III probe

39 What is the cell morphology of Dehalobacter in ACT-3 and the sub-culture WL 1,2 DCA, rods and/or filaments???

40 ACT 3 + Probe Dhb 210

41 ACT 3 + Probe Dhb 442

42 Experiment matrix for probe Dhb 442 Cultures Probe only Probe + Helpers 10% 20% 30% 10% 20% 30% ACT WL 1,2 DCA Negative Control (KB-1) OK OK OK OK OK OK ACT-3 s signal instensity was better with 10% formamide hybridization buffer WL 1,2 DCA s signal intensity was better with 30% formamide concentration + helper oligos

43 ACT 3 + Dhb 442 (10% hybridization buffer) DAPI

44 ACT 3 + Dhb 442 (10% hybridization buffer)

45 ACT 3 + Dhb 442 (10% hybridization buffer) DAPI + Dhb 442 filter piece: 6.73 x 10 7 DAPI stained cells/ml of culture 1.82 x 10 7 Dhb 442 hybridized cells/ml of culture Relative abundance: 27%

46 WL 1,2 DCA + Dhb 442 (30% hybridization buffer) DAPI

47 Wl 1,2 DCA + Dhb 442 (10% hybridization buffer)

48 WL 1,2 DCA + Dhb 442 (10% hybridization buffer) DAPI + Dhb 442 filter piece: 2.2 x 10 7 DAPI stained cells/ml of culture 8.55 x 10 6 hybridized cells/ml of culture Relative abundance: 39%

49 Negative Control (KB-1) 10% Hybridization buffer DAPI

50 Negative Control (KB-1) 10% Hybridization buffer

51 Negative Control (KB-1) 10% Hybridization buffer

52 2. Can KB-1 and T3L develop biofilms on glass cover slips? Laser Scanning Confocal Microscopy

53 T = 5 days, only KB-1 biofilm 22/07/2013 Microbial Diveristy MBL

54 T = 5 days, KB-1 biofilm

55 T = 9 days, KB-1 biofilm

56 Z-stack 3D model KB-1 T = 9 days Maximum biofilm thickness is about 4 µm

57 T = 9 days, T3L biofilm

58 T = 15 days, KB-1 biofilm

59 Z-stack 3D model KB-1, T = 15 days Maximum biofilm thickness is about 6 µm

60 T = 15 days, T3L biofilm

61 Conclusions Luz had so much fun meeting Edlab s cultures in this great environment! FISH could be further explored to monitor Edlab s cultures based on first success trials. Dehalobacter appears to be a thin rod in ACT-3 and filament-forming rods in WL 1,2 DCA; are these different strains? KB-1 developed nice biofilm areas in glass cover slips, future tests should correlate growth and substrate depletion with time.

62 Acknowledgements Microbial Diversity faculty, teaching assistants, and students Central Microscopy Facility at the MBL Zeiss support team Edwards lab, BioZone Dr. Elizabeth A. Edwards Thomas B. Grave and Elizabeth F. Grave Scholarship, Patricia A. Case Endowed Scholarship Family and friends in Canada and Colombia, especially Luz s parents: Oscar and Luz Amparo, and husband, Kristian.