Characterization of an Adapted Microbial Population to the Bioconversion of Carbon Monoxide Into Butanol Using Next-Generation Sequencing Technology
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Characterization of an adapted microbial population to the bioconversion of carbon monoxide into butanol using next-generation sequencing technology Guillaume Bruant Research officer, Bioengineering group Energy, Mining, Environment - National Research Council Canada Pacific Rim Summit on Industrial Biotechnology and Bioenergy December 8 -11, 2013 Butanol from residue (dry): syngas route biomass → gasification → syngas → catalysis → synfuels (CO, H2, CO2, CH4) (alcohols…) Biocatalysis vs Chemical catalysis potential for higher product specificity may be less problematic when impurities present less energy intensive (low pressure and temperature) Anaerobic undefined mixed culture vs bacterial pure culture mesophilic anaerobic sludge treating agricultural wastes (Lassonde Inc, Rougemont, QC, Canada) PRS 2013 - 2 Experimental design CO Alcohols Serum bottles incubated at Next Generation RDP Pyrosequencing mesophilic temperature Sequencing (NGS) pipeline 35°C for 2 months Ion PGMTM sequencer http://pyro.cme.msu.edu/ sequences filtered CO continuously supplied Monitoring of bacterial and to the gas phase archaeal populations RDP classifier atmosphere of 100% CO, http://rdp.cme.msu.edu/ 1 atm 16S rRNA genes Ion 314TM chip classifier VFAs & alcohol production bootstrap confidence cutoff low level of butanol of 50 % Samples taken after 1 and 2 months total genomic DNA extracted, purified, concentrated PRS 2013 - 3 NGS: bacterial results Bacterial population - Phylum level 100% 80% Other Chloroflexi 60% Synergistetes % of total Firmicutes sequences Proteobacteria 40% Bacteroidetes Spirochaetes 20% Actinobacteria unclassified Bacteria 0% 1 month 2 months Initial sludge Other (<0.5%): TM7, Cyanobacteria/Chloroplast, Thermotogae, Chlorobi, Acidobacteria, Verrucomicrobia, Planctomycetes, OP11, Elusimicrobia, Thermodesulfobacteria, Lentisphaerae and SR1 Phylum of interest: Firmicutes (8.0% to 82.5%) PRS 2013 - 4 NGS: bacterial results Bacterial population - class level 100% Other Anaerolineae (Ch) 80% Synergistia (Sy) Negativicutes (F) Clostridia (F) 60% % of total Gammaproteobacteria (P) identified classes Alphaproteobacteria (P) 40% Betaproteobacteria (P) Deltaproteobacteria (P) 20% Epsilonproteobacteria (P) Flavobacteria (B) Sphingobacteria (B) 0% Bacteroidia (B) Spirochaetes (S) 1 month 2 months Actinobacteria (A) Initial sludge Other (<0.5%): Bacteroidetes incertae sedis (B), Bacilli (C), Erysipelotrichia (C) and Dehalococcoidetes (Ch) A: Actinobacteria, S: Spirochaetes, B: Bacteroidetes, P: Proteobacteria, F: Firmicutes, Sy: Synergistetes and Ch: Chloroflexi Class of interest: Clostridia (6.5% to 82.4%) PRS 2013 - 5 NGS: bacterial results Bacterial population - order level Other Anaerolineales (Ch) Synergistales (Sy) 100% Selenomonadales (F) 90% Clostridiales (F) Methylococcales (P) 80% Rhodospirillales (P) 70% Caulobacterales (P) 60% Rhizobiales (P) % of total 50% Hydrogenophilales (P) identified orders Burkholderiales (P) 40% Desulfuromonadales (P) 30% Desulfovibrionales (P) 20% Syntrophobacterales (P) 10% Campylobacterales (P) 0% Flavobacteriales (B) Sphingobacteriales (B) Bacteroidales (B) 1 month 2 months Spirochaetales (S) Initial sludge Actinomycetales (A) Other: 20 orders < 0.5% A: Actinobacteria, S: Spirochaetes, B: Bacteroidetes, P: Proteobacteria, F: Firmicutes, Sy: Synergistetes and Ch: Chloroflexi Order of interest: Clostridiales (6.4% to 82.3%) PRS 2013 - 6 NGS: bacterial results Order Clostridiales - family level 100% Gracilibacteraceae Peptostreptococcaceae 80% Eubacteriaceae Clostridiales Incertae Sedis III 60% Ruminococcaceae % of total Syntrophomonadaceae identified families Clostridiales Incertae Sedis XI 40% Lachnospiraceae Clostridiaceae 1 20% Peptococcaceae 2 Peptococcaceae 1 0% Clostridiales Incertae Sedis XIII Clostridiales Incertae Sedis XII unclassified Clostridiales 1 month 2 months Initial sludge genera of interest: Clostridium (~0% to 9.0%), Oscillibacter (~0% to 9.9%), Acetobacterium (~0% to 49.8%) PRS 2013 - 7 NGS: bacterial main results Significant decrease of the 3 principal components of the initial bacterial population Bacteroidetes (30.3% to 4.3%) Actinobacteria (23.7% to 9.3%) Proteobacteria (20.9% to 1.4%) Significant increase of the Firmicutes became ultra-dominant (82.5%) 1 class (Clostridia, 82.4%), 1 order (Clostridiales, 82.3%) Emergence of 3 families Clostridiaceae (Clostridium, known solvent producers) Ruminococcaceae (Oscillibacter, valeric acid producer) Eubacteriaceae (Acetobacterium, known acetate producers) PRS 2013 - 8 NGS: archaeal main results Limited impact of CO on archaeal population Archaeal population - class level 100% 2 phyla 90% Euryarchaeota 80% (98.1% to 99.2%) 70% 60% Crenarchaeota % of total 50% Thermoprotei (C) identitfied classes No impact on 40% Methanomicrobia (E) Methanobacteria (E) 30% diversity 20% 2 phyla 10% 3 classes 0% e th s g n th 5 orders d o n lu o s m l 1 m ia 2 it In Methanobacteria increased (13.9% to 45.9%) specifically Methanobacterium (44.6%), hydrogenotrophic methanogen Methanomicrobia decreased (82.8% to 52.7%) emergence of Methanosarcina, methanogen metabolically diverse and nonmethanogenically CO user PRS 2013 - 9 Conclusions and perspectives Ion Torrent Next Generation Sequencing platform powerful technique for microbial population monitoring Impact of CO on microbial population insights on microbial species adapted to bioconversion of CO into butanol importance of Clostridiales (Clostridium and Oscillibacter) Next: bio-augmentation strategies microorganisms identified through NGS optimize and stabilize the process (improve butanol production) PRS 2013 - 10 Acknowledgments Serge Guiot Marie-Josée Lévesque Alain Corriveau Questions? PRS 2013 - 11 .