Advanced microbial methods status and prelimenary results

Transcription

1 Advanced microbial methods status and prelimenary results Group composition and stakeholders: GEUS: Jacob Bælum, Carsten Suhr Jacobsen DTU Miljø: Charlotte Scheutz, Mette Broholm 1

2 Outline Overall objective Brief repetition of working document Results Nucleic acid extraction optimization Quantitative real-time PCR optimization TCE degradation experiment Further plans TCE/TCA treatability studies Publications/conferences 2

3 Overall aim Develop tools suitable to detect and quantify activity of specific degraders in clayey sediment Identify key species involved in TCA/TCE dechlorination Develop primers suitable to quantify key species involved in TCE/TCA dechlorination 3

4 Working document / work plan Ongoing activities Optimization of mrna extraction method Sorption of DNA to clayey sediments Respike experiment with VC DNA/rRNA/mRNA extraction optimization TCE degradation experiment Chemical analyses Quantitative microbial analysis (phylogenetic and functional genes) Microbial diversity analysis 4 Activities to be done TCA degradation experiment Respike experiment with 1,1-DCE Chemical analysis Microbial diversity analysis

5 Nucleic acid extraction optimization Background Difficulties in extracting DNA from sub soil environments Extraction yield Extraction reproductivity Objective Why is clayey sub soils often difficult extractable? Optimize extraction yield from clayey sub soils 5

6 Experimental setup DNA sorption experiment 3 H-labelled DNA from E.coli in conc. 1, 1, 1, 1 mg/kg soil Sorption experiment was done in 3ml,1 M Tris and 2 mg soil 9 different soils/sediments were tested Solution G1 in different conc. was added in order to saturate binding sites 6

7 Sorption isoterms ul G1 1 ul G1 5 ul G1 1 ul G1 Burned soil Langmuir fits Conc. on on solid phase (mg/kg) Vasbyvej Clayey Vasbyvej Sandy SjO A Vasbyvej Clayey Vasbyvej Sandy 14 Højbakke gård A SjO SjO B Højbakke Gård Gård A 1 Acidic soil soil 5 5 SjO B Højbakke gård A 1 1 Acidic soil Jyndevad A 14 Jyndevad B 1 Alluvial soil soil Jyndevad A 1 1 Jyndevad B Alluvial soil Conc. Conc. in in liquid liquid phase phase (mg/l) (mg/l) Conc. in liquid phase (mg/l) 7

8 Quantification of Dhc 16s rrna genes 2 ul of KB-1 2 mg of sediment (sortebrovej) 1ml milliq H 2 O with G1 Freeze dried Extracted with Mobio DNA kit Flouresence genes reaction genes reaction genes reaction genes reaction -1-1 KB-1 KB-1+sediment KB-1+sediment+1 ul G1 KB-1+sediment+1 ul G1 KB-1+sediment+25 ul G1 KB-1+sediment+5 ul G # of PCR cycles 8

9 Quantification of Dhc 16s rrna genes 2 ul of KB-1 2 mg of sediment (sortebrovej) 1ml milliq H 2 O with G1 Freeze dried Extracted with Jacob DNA/RNA extraction method Flouresence Flouresence DNA Jbae method prior to ph adjustment genes reaction genes reaction genes pr reaction 1 5 genes reaction genes 1 6 genes pr reaction reaction genes pr reaction 1 6 genes pr reaction KB-1 KB-1 + sediment KB-1+sediment+1 ul G1 KB-1+sediment+25 ul G1 KB-1+sediment+5 ul G # of PCR cycles Cycles 9

10 Quantification of Dhc 16s rrna genes 2 ul of KB-1 2 mg of sediment (sortebrovej) 1ml milliq H 2 O with G1 Freeze dried Extracted with Jacob DNA/RNA extraction method Flouresence Flouresence genes reaction DNA jbae method after ph adjustment 1 4 genes reaction genes pr reaction 1 5 genes reaction genes 1 6 genes pr reaction reaction genes pr reaction 1 6 genes pr reaction KB-1 KB-1+sediment KB-1+sediment+1 ul G1 KB-1+sediment+25 ul G1 KB-1+sediment+5 ul G # of PCR cycles Cycles 1

11 Quantification of Dhc 16s rrna 2 ul of KB-1 2 mg of sediment (sortebrovej) 1ml milliq H 2 O with G1 Freeze dried Extracted with Jacob DNA/RNA extraction method Flouresence Flouresence genes reaction genes reaction genes 1 5 pr reaction genes reaction genes 1 6 genes pr reaction reaction genes pr reaction 1 6 genes pr reaction KB-1 KB-1+sediment KB-1+sediment+1 ul G1 KB-1+sediment+25 ul G1 KB-1+sediment+5 ul G1 rrna fra KB # of PCR cycles Cycles 11

12 Respike experiment with VC Respike with 2 µl VC to bottle 25 and 26 from the TCE degradation experiment µmol bottle Bottle 25 VC Ethene Bottle 27 remains as a negative control Chemical analysis was done on the headspace with GC-FID,5 ml sediment/water was taken at all timepoints µmol bottle Bottle 26 Time (hours) Time (hours) VC Ethene 12

13 Quantification of vcra mrna 2 mg of sediment from VC respike experiment 1ml milliq H 2 O with G1 Freeze dried Extracted with Jacob DNA/RNA extraction method Flouresence Flouresence genes reaction genes 4 genes pr reaction genes 5 genes pr reaction genes reaction genes pr reaction 1 6 genes pr reaction Sediment Sediment+1 ul G1 Sediment+25 ul G1 Sediment+5 ul G1 vcra mrna # of PCR cycles Cycles 13

14 Developement of qpcr assays 1 8 genes reaction genes reaction genes reaction genes reaction genes reaction genes reaction genes reaction -1 Flouresence Flouresence 7 6 tcea Cycles bvca Flouresence Flouresence vcra Cycles Dhb Cycles Cycles 14

15 What remains to be done mrna extraction and measurements of the VC respike experiment mrna extraction optimization 2,4-D degradation scenario with the bacteria Cuprivarius necator 9 different soils 15

16 TCE degradation experiment - Aim To investigate the effects of different concentrations of KB-1 culture towards dechlorination of different concentrations of TCE Speed of the dechlorination Growth/death of Dehalococcoides sp. Microbial community composition To investigate activity of specific degraders through mrna of functional genes To collect samples on which we can test our mrna extraction procedure 16

17 Setup Three different concentrations of TCE Three different concentrations of KB-1 In total 27 airthight glass flasks 1 g sediment from TCE-polluted area at Rugårdsvej 2 ml sterile tap water Addition of lactate ~6 mm The analyses Chlorinated ethenes Fatty acids An-ions Methane DNA and RNA extraction KB-1 TM (celler/ml) TCE (µg/l)

18 Degradation data TCE: mg L -1 TCE: 1 mg L -1 TCE: 1 mg L -1 TCE 1,1 dce trans dce cis dce VC Ethene Ethane µmol bottle-1 3, 2,5 2, 1,5 1,,5 Bottle 1-2, , Bottle ,5 2, 1,5 1,,5, , 2,5 2, 1,5 1,,5 Bottle 4-6, , Bottle ,5 2, 1,5 1,,5, Bottle Bottle KB-1: cells ml -1 KB-1: 1 5 cells ml , 2,5 2, 1,5 1,,5 Bottle 19-21, , 2,5 2, 1,5 1,,5 Bottle 22-24, Time (days) Bottle KB-1: 1 7 cells ml -1

19 Quantification of Dhc and Geob 16s rrna genes µg TCE L -1 TCE 1,1 DCE tdce cdce VC Ethene Ethane Dhc Gbc µmol bottle -1,1,8,6,4,2,,1,8,6,4,2,,1,8,6,4,2, Dhc og Gbc Log cells ml -1 1e+9 1e+8 1e+7 1e+6 1e+5 1e+4 1e+3 1e+9 1e+8 1e+7 1e+6 1e+5 1e+4 1e+3 1e+9 1e+8 1e+7 1e+6 1e+5 1e+4 1e Time, days

20 Quantification of Dhc and Geob 16s rrna genes 1 µg TCE L -1 TCE 1,1 DCE tdce cdce VC Ethene Ethane Dhc Gbc µmol bottle -1 2, 1,5 1,,5, 2, 1,5 1,,5, 2, 1,5 1,,5, Dhc og Gbc Log cells ml -1 1e+9 1e+8 1e+7 1e+6 1e+5 1e+4 1e+3 1e+9 1e+8 1e+7 1e+6 1e+5 1e+4 1e+3 1e+9 1e+8 1e+7 1e+6 1e+5 1e+4 1e Time, days

21 Quantification of Dhc and Geob 16s rrna genes 1. µg TCE L -1 TCE 1,1 DCE tdce cdce VC Ethene Ethane Dhc Gbc µmol bottle Dhc and Gbc. Log cells ml -1 1e+9 1e+8 1e+7 1e+6 1e+5 1e+4 1e+3 1e+9 1e+8 1e+7 1e+6 1e+5 1e+4 1e+3 1e+9 1e+8 1e+7 1e+6 1e+5 1e+4 1e Time, days

22 What remains to be done The experiment has to be fulfilled More DNA and RNA extraction Dhc quantifications based on 16S rrna genes, vcra-, tcea-, and bvca-genes has to be fullfilled Quantification of activity based on vcra-, tcea-, and bvcatranscripts (mrna) in selected microcosms Community analysis on selected microcosms 22

23 Community analysis of TCA degradation experiment Aim To identify bacteria central to degradation of TCA 23 Methods Respike experiment with 1,1-DCA on selected microcosms Community analysis done on an existing treatability study (Charlotte/Mette) Community studies will be limited to the Vasbyvej experiment Community analysis will consist of rrna and DNA analysis with DGGE, cloning, and sequencing brdu incorporation in active microbial populations (still under consideration)

24 Treatability experiments Material Source: Clayey till and groundwater, 2 batch Plume: Sand and groundwater, 14 batch Experimental set-up Abiotic controls: Autoclaved Biotic controls: no amendments Stimulated batches: Lactate added Bioaugmented batches: KB1 (Dehalococcoides) and ACTIII (Dehalobacter) added, degrade TCE to ethene and TCA to CA Added compounds Source: TCA (5 µg/l), TCE (1 µg/l), mix of TCA (5 µg/l) and TCE (1 µg/l) Plume: TCA (1 µg/l) and TCE (1 µg/l), CA (1 µg/l) 24

25 Status What has been done Samples for nucleic acid extraction has been taken and freeze dried What remains to be done Respike experiment (brdu incorporation) Extraction of nucleic acids DGGE Cloning and sequencing 25

26 Publications Bælum, J., Jacobsen, C. S. (29). Improvement of low-biomass soil DNA/RNA extraction yield and quality. Patent submitted June 15. Paulin, M. M., Bælum, J., Nicolaisen, M., Sørensen, J., Jacobsen, C. S. (21). Developement and optimization of a novel technique to extract mrna from difficult samples. In prep for??? Bælum, J., Scheutz, C., Broholm, M., Jensen, C. M., Brochmann, R. P., Jacobsen, C. S. (21). The initial cell density of dechlorinating bacterial culture KB-1 is highly determinative of the degradation of TCE to ethene. In prep. for AEM. Bælum, J., Scheutz, C., Broholm, M., Laier, T., Jacobsen, C. S. (21). Transcription of vcra and bvca genes as a respons to dechlorination of TCE in clayey groundwater sediment. In prep. For ISME journal. Bælum, J., Scheutz, C., Broholm, M., Laier, T., Jacobsen, C. S. (21). Microbial community stucture in TCA degrading microcosms.. In prep for???

27 Conferences In-situ and on-site bioremediation, Baltimore, May 5-8. Diversity of vinylchloride functional genes and quantification of Dehalococcoides sp. DNA in a groundwater aquifer during a field trial (Poster). BAGECO 1, Stockholm, June Dynamics of microbial populations in TCE contaminated groundwater sediment (Poster). Biomicroworld, Lisboa, December 2-4. Extraction of nucleic acids from trichloroethene (TCE) degrading bacteria in extremely clayey groundwater sediments