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