From living cells to stable isotopes: An interdisciplinary approach for unravelling microbial interactions in ammonia‐ overloaded anaerobic digesters Francesc X. Prenafeta‐Boldú ([email protected]) L. Ruiz, J. Vila, B. Fernández, V. Riau, M. Guivernau, M. Viñas GIRO Joint Research Unit IRTA‐UPC, Torre Marimon, Caldes de Montbui, Spain. MELiSSA Workshop Lausanne 8th –9th June 2016 MELiSSA Workshop, Lausanne 8th –9th June 2016 Closing loops in 'Micro Ecological Life Support System Alternative' MELiSSA Workshop, Lausanne 8th –9th June 2016 Closing loops in circular economy MELiSSA Workshop, Lausanne 8th –9th June 2016 Nitrogen fate and toxicity in anaerobic digesters 14 14 CO2/ CH4 >1 + ‐1 Ammonium concentrations of 2 –5 g NH4 ‐N L cause significant inhibition of methanogenesis, depending on T°, and pH conditions (free ammonia is the toxic species) (Angelidaki & Ahring 1994, Sung & Liu 2003) MELiSSA Workshop, Lausanne 8th –9th June 2016 The syntrophic acetate oxydation SAOB are homoacetogenic bacteria that can reverse the Wood‐Ljungdahl pathway, oxidizing acetate to CO2 and H2 , which are further metabolized by hydrogenotrophic archaea (SAOA) SAOB have relatively low growing rates (doubling times above 40 days) SAOB are polyphyletic but mainly linked to the SAOB Clostridia class (phylum Firmicutes) Knowledge on the biodiversity, ecophysiology, and biochemistry of SAOB is limited SAOB are very important for a stable anaerobic SAOA Inhibition by digestion process under high ammonia ammonia and VFA concentrations (Müller et al. 2012) MELiSSA Workshop, Lausanne 8th –9th June 2016 Case study: methanogenic biomass from an agricultural biogas plant Location: Vilasana, Lleida (Spain) Reactor type: CSTR Volume: 1500 m3 (2x) HRT: 65 days TAN: 2 –4 gTAN L‐1 Operation regime: Mesophilic Treatment capacity: 11.000 m3 of pig slurry and 4.500 m3 of organic residues MELiSSA Workshop, Lausanne 8th –9th June 2016 Batch activity assays 40 TAN: 1 g/L Treatment conditions (x3) 35 TAN: 3 g/L Acetic Acid: 3.5 g Ac L‐1 13 30 (*Acetic acid : CH3‐COOH) ‐1 Inoculum: 12.5 gVSS L 25 Ammonia: 1, 3 and 6 gTAN L‐1 4 20 Incubation: 65 days at 37oC TAN: 6 g/L NmL CH 15 10 Monitored parameters 20% total CH4 5 VFA/COD Inoculum CO2/CH4 GC‐TCD and GC‐IRMS 0 NGS MiSeq (Eub/Arch) DNA/cDNA 0 20406080 qPCR 16S rRNA and mcrA (DNA/cDNA) days MELiSSA Workshop, Lausanne 8th –9th June 2016 Compound Stable Isotopic Analysis (CSIA) of biogas: Unlabelled experiments Apparent fractionation factor (αc) (Conrad 2005, Conrad et al. 2009) 15 14 13 13 13 12 11 αc = (δ CO2 + 1000)/ (δ CH4 + 1000) 10 9 8 7 2 6 5 4 HM αc < 1.055 Dominance of acetotrophic methanogenesis 3 2 α > 1.065 Dominance of hydrogenotrophic methanogenesis d13C-CO 1 c 0 TAN 3.5 g/L δ13C-CO2 -1 αc > 1.080 Exclusively hydrogenotrophic methanogenesis -2 TAN 6 g/L -3 -4 -5 AM -6 TAN 1 g/L -7 -8 -20 C2+N1 C2+N2 C2* + N1 C2* + N2 C2* ‐1 Mixture Gas 1gN‐TAN ∙L αc = 1.054 ± 0.017 Acetotrophic (Blank) Inocula -30 C2 Control (Initial N) ‐1 -40 AM 3gN‐TAN ∙L αc = 1.077 ± 0.001 Hydrogenotrophic 4 -50 d13C-CH ‐1 TAN 1 g/L δ13C-CH4 6gN‐TAN ∙L αc = 1.080 ± 0.001 Hydrogenotrophic -60 -70 TAN 3.5 g/L TAN 6 g/L HM -80 C2+N1 C2+N2 C2* + N1 C2* + N2 Gas Mixture Gas Inocula (Blank) C2 ControlC2 (InitialN) MELiSSA Workshop, Lausanne 8th –9th June 2016 Compound Stable Isotopic Analysis (CSIA) of biogas: labelled experiments 13 13 AM CH3‐COOH CH4 + CO2 13 13 13 13 SAO CH3‐COOH + 2H2O CO2 + CO2 +4H2 CH4 / CH4+ CO2 /CO2 SAOB SAOA 120000 100000 80000 60000 4 40000 4 20000 TANAc-*C2-N1 3.52c* g/L TANAc-*C2-N2 3c*6 g/L C-CH d13C-CH 13 δ -20 -30 -40 InoculumInoculum Gas Mixture -50 -60 Ac-C21c N0 Ac-2c -70 C2-N1 C2-N2Ac-3c -80 -50 -40 -30 -20 -10 0 10 20 30 40 50 2000 3000 4000 5000 d13C-CO13 δ C-CO22 MELiSSA Workshop, Lausanne 8th –9th June 2016 Microbial molecular characterization Biomass sampling Raw data (sff) Centrifugiation (4ºC) Biomass Pellet ‐80ºC QIIME/Mothur Simultaneous 1.33.1 DNA + RNA extraction Total DNA + RNA (‐80ºC) SILVA/greengenes alignment RT (PrimeScript) Total DNA + cDNA (‐80ºC) Pick OTUs OTUs distribution NGS (MiSeq) qPCR (Qualitative assessment) (Quantitative assessment) 16S rRNA (Eubacteria) 16S rRNA genes (DNA + cDNA) Diversity indices 16Sr RNA (Archaea) mcrA genes (DNA + cDNA) Taxonomy (RDP) PCA/CA/CCA MELiSSA Workshop, Lausanne 8th –9th June 2016 Ammonia versus transcription level (RT‐qPCR) Molecular targets: The hypervariable V3‐V5 region from eubacterial 16S rRNA genes The functional mcrA gene (methyl coenzyme‐M reductase) specific of methanogenic archaea Total eubacteria (16S rRNA) Methanogenic archaea (mcrA) 5 0.20 A 1 gTAN/L B 1 gTAN/L copies 4 copies 0.15 3.5 gTAN/L 3 3.5 gTAN/L 0.10 2 transcrpts/gene transcrpts/gene 0.05 6 gTAN/L 1 6 gTAN/L rRNA mcrA 16S 0 0.00 0 5 10 15 20 0 5 10 15 20 Time (days) Time (days) MELiSSA Workshop, Lausanne 8th –9th June 2016 NGS of eubacterial 16S rRNA libraries 1 gTAN/L 3.5 gTAN/L 6 gTAN/L DNAt0 DNAt4 cDNAt4 DNAt0 DNAt4 cDNAt4 DNAt0 DNAt4 cDNAt4 Relative abundance (%) Some of the identified ribotypes were somewhat homologous to the SAOB Clostridium ultunense and Tepidanaerobacter acetatoxydans MELiSSA Workshop, Lausanne 8th –9th June 2016 NGS of archaeal 16S rRNA libraries 1 gTAN/L 3.5 gTAN/L 6 gTAN/L DNAt0 DNAt4 cDNAt4 DNAt0 DNAt4 cDNAt4 DNAt0 DNAt4 cDNAt4 Relative abundance (%) MELiSSA Workshop, Lausanne 8th –9th June 2016 Implementation in high‐rate anaerobic digesters Enrichment of SAO biofilm on different support materials (nylon, zeolite, magnetite, steal, graphite, etc.) Process optimization in a continuous lab‐scale anaerobic filter MELiSSA Workshop, Lausanne 8th –9th June 2016 Conclusions 1.A diversified approach (batch methanogenic activity tests, biogas isotopic fractionation, and molecular characterization of microbial communities) demonstrated the occurrence of SAO activity in a full‐scale anaerobic digester. 2.Methanogenic activity switched from predominantly acetotrophic to hydrogenotrophic (linked to SAO activity) at 3 gN‐TAN L‐1 and could be sustained up to 6 gN‐TAN L‐1, but a slight inhibition could already be observed. 3.No significant microbial shift upon ammonia supplementation were apparent for the Eubacteria. Members of the phylum Chloroflexi were predominant, but the most active belonged to the Firmicutes. 4.About 5% ‐ 10% of identified OTUs were related to homoacetogenic bacteria. Some of them were homologous to the SAOB Clostridium ultunense and Tepidanaerobacter acetatoxydans. 5.Concerning the Archaea, representatives of the genera Methanoculleus and Methanosarcina appear to be fundamental hydrogenotrophic syntrophic partners (SAOA). 6.Current efforts are aimed at lab‐scale process implementation and optimization in high‐rate anaerobic digesters based on SAO biofilms. MELiSSA Workshop, Lausanne 8th –9th June 2016 Acknowledgements Special thanks to: Javier Garcia, Laura Tey, Arantxa Matos, Josep Illa, Xavier Flotats Funding: Optimization of the anaerobic digestion and biogas production process of proteins and lipids rich wastes, with ammonia recovery (Ref. RTA2012‐00098‐00‐00).