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

Luz A. Puentes Jácome Ph.D. student, University of Toronto, Canada

Microbial Diversity 2013

and

Biofilm formation study of the KB-1 and T3L cultures using confocal microscopy

KB-1 T3L ACT-3 ORCH4

Environment Canada: http://www.ec.gc.ca/eau-water/default.asp?lang=En&n=3F93145A-1

Soil and groundwater contamination

Environment Canada: http://www.ec.gc.ca/eau-water/default.asp?lang=En&n=3F93145A-1

EPA’s priority pollutants 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 14. 1,1,2-trichloroethane 15. 1,1,2,2-tetrachloroethane 16. Chloroethane 17. REMOVED 18. Bis(2-chloroethyl) ether 19. 2-chloroethyl vinyl ethers 20. 2-chloronaphthalene 21. 2,4,6-trichlorophenol 22. Parachlorometa cresol 23. Chloroform 24. 2-chlorophenol 25. 1,2-dichlorobenzene 26. 1,3-dichlorobenzene 27. 1,4-dichlorobenzene 28. 3,3-dichlorobenzidine 29. 1,1-dichloroethylene 30. 1,2-trans-dichloroethylene 31. 2,4-dichlorophenol 32. 1,2-dichloropropane 33. 1,2-dichloropropylene 34. 2,4-dimethylphenol 35. 2,4-dinitrotoluene

36. 2,6-dinitrotoluene 37. 1,2-diphenylhydrazine 38. Ethylbenzene 39. Fluoranthene 40. 4-chlorophenyl phenyl ether 41. 4-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 49. REMOVED 50. REMOVED 51. Chlorodibromomethane 52. Hexachlorobutadiene 53. Hexachlorocyclopentadiene 54. Isophorone 55. Naphthalene 56. Nitrobenzene 57. 2-nitrophenol 58. 4-nitrophenol 59. 2,4-dinitrophenol 60. 4,6-dinitro-o-cresol 61. N-nitrosodimethylamine 62. N-nitrosodiphenylamine 63. N-nitrosodi-n-propylamine 64. Pentachlorophenol 65. Phenol 66. Bis(2-ethylhexyl) phthalate 67. Butyl benzyl phthalate 68. Di-N-Butyl Phthalate 69. Di-n-octyl phthalate 70. Diethyl Phthalate

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 87. Trichloroethylene 88. Vinyl chloride 89. Aldrin 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 129. 2,3,7,8-TCDD 130.

4. Benzene

14. 1,1,1 Trichloroethane

87. Trichloroethene 86. Toluene

106 - 112. PCB mixtures!

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

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 5 µm

5 µm

T3L’s Methanosaeta

T3L’s Methanosaeta

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 2013 9

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)

0

100

200

300

400

500

600

700

800

02-Jul 06-Jul 10-Jul 14-Jul 18-Jul

Mol

es o

f m

etha

ne p

rod

uced

(µm

ol)

Methane production by KB-1 methanogens

KB-1’s methanogens

A pleasant surprise: Methanosarcina!!!

Confocal images 63X

3D view of Methanosarcina aggregate

0

2

4

6

8

10

12

14

04-Jul-13 08-Jul-13 12-Jul-13 16-Jul-13 20-Jul-13

Ace

tate

con

cent

rati

on (

mM

) Acetate consumption and production

Methanogens

Acetogens

KB-1’s acetogens

Dehalococcoides sp. and PCBs

Dehalococcoides Mccartyi sp.

Loffler et. al, 2013, Int. J. of Systematic and Evolutionary Microbiology 63 (Pt 2): 625-35.

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

TCE degrading culture: KB-1

TCE = trichloroethene DAPI

TCE degrading culture: KB-1

Eub I – III probe

TCE degrading culture: KB-1

Archaea 195 probe

Toluene degrading culture: T3L

Toluene degrading culture: T3L

Archaea 195 probe

Toluene degrading culture: T3L

Benzene degrading culture: ORCH4

DAPI

Benzene degrading culture: ORCH4

Eub I – III probe

Benzene degrading culture: ORCH4

“The benzene degrading organism is so unknwon !”

???

1,1,1 TCA degrading culture: ACT 3

1,1,1 TCA = 1,1,1 trichloroethane DAPI

1,1,1 TCA degrading culture: ACT 3

Eub I – III probe

1,1,1 DCA degrading culture: ACT 3

DAPI + Eub I –III filter piece:

• 6.67 x 107 DAPI stained cells/ml of culture

• 3.04 x 107 EUB I - III hybridized cells/ml of culture

• Relative abundance: 46%

DAPI + Archaea filter piece:

• 5.37 x 107 DAPI stained cells/ml of culture

• 2.3 x 107 Archaea 915 hybridized cells/ml of culture

• Relative abundance: 43%

Eub I – III probe

What is the cell morphology of Dehalobacter in ACT-3 and the

sub-culture WL 1,2 DCA, rods and/or filaments???

ACT 3 + Probe Dhb 210

ACT 3 + Probe Dhb 442

Experiment matrix for probe Dhb 442

Cultures Probe only Probe + Helpers

10% 20% 30% 10% 20% 30%

ACT-3 +++ + ++ + + +

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

ACT 3 + Dhb 442 (10% hybridization buffer)

DAPI

ACT 3 + Dhb 442 (10% hybridization buffer)

ACT 3 + Dhb 442 (10% hybridization buffer)

DAPI + Dhb 442 filter piece: • 6.73 x 107 DAPI stained cells/ml of culture • 1.82 x 107 Dhb 442 hybridized cells/ml of culture • Relative abundance: 27%

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

DAPI

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

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

DAPI + Dhb 442 filter piece:

• 2.2 x 107 DAPI stained cells/ml of culture

• 8.55 x 106 hybridized cells/ml of culture

• Relative abundance: 39%

Negative Control (KB-1) 10% Hybridization buffer

DAPI

Negative Control (KB-1) 10% Hybridization buffer

Negative Control (KB-1) 10% Hybridization buffer

2. Can KB-1 and T3L develop biofilms on glass cover slips?

Laser Scanning Confocal Microscopy

T = 5 days, only KB-1 biofilm

22/07/2013 Microbial Diveristy – MBL 2013 53

T = 5 days, KB-1 biofilm

T = 9 days, KB-1 biofilm

Z-stack 3D model KB-1 T = 9 days

Maximum biofilm thickness is about 4 µm

T = 9 days, T3L biofilm

T = 15 days, KB-1 biofilm

Z-stack 3D model KB-1, T = 15 days

Maximum biofilm thickness is about 6 µm

T = 15 days, T3L biofilm

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.

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.