Use of Microbial Consortia for Conversion of Biomass Pyrolysis Liquids into Value- Added Products Julian Pietrzyk [email protected] Use of Microbial Consortia for Conversion of Biomass Pyrolysis Liquids Outline into Value-Added Products •Pyrolysis •Bio-oil Py-AD •AD •Illumina Sequencing •Mass Spectrometry •Relatedness Julian Pietrzyk [email protected] Use of Microbial Consortia for Conversion of Biomass Pyrolysis Liquids Pyrolysis into Value-Added Products Reactor Heat Heat Heat Cold Cold Receiver Trap 1 Trap 2 Trap 3 Trap 1 Trap 2 N2 • Biomass burned at high temperatures (300°C - 600°C) in the absence of oxygen • Thermal depolymerisation of lignocellulosic biomass • Products are char, bio-oil & syngas Julian Pietrzyk [email protected] Use of Microbial Consortia for Conversion of Biomass Pyrolysis Liquids Bio-oil into Value-Added Products • Product of pyrolysis of biomass • Dark brown organic liquid Organic • High water content (~25 wt %) Aqueous • Extremely high oxygen content catechol, guaiacol, • + 1000s other compounds syringol, isoeugenol, pyrones, vanillin, • Ages instantly furans, acetic acid, formic acid, sugars, • Composition dependant carboxylic acids, phenolics, on feedstock hydroxyketones, • Low pH & biocatalyst inhibitors hydroxyaldehydes Julian Pietrzyk [email protected] Use of Microbial Consortia for Conversion of Biomass Pyrolysis Liquids Anaerobic Digestion into Value-Added Products Hydrolysis • High molecular weight organic polymers split into smaller more bioavailable monomers. • Proteins > Amino acids | Carbohydrates > Monosaccharides | Fats > Fatty acids | + H2 Acidogenesis • Acidogenic fermentation of hydrolysed products to short chain… • volatile acids (propionic, butyric, acetic, formic, lactic) • alcohols (ethanol, methanol) • H2 + CO2 + NH3 + H2S Acetogenesis • Further digestion of acids by acetogens to H2, CO2 and acetic acid. Methanogenesis • Methanogenic archaea convert H2 and acetic acid to CH4, CO2. Julian Pietrzyk [email protected] Use of Microbial Consortia for Conversion of Biomass Pyrolysis Liquids Py-AD into Value-Added Products Vast range of microorganisms capable of bioconversion across a spectrum no single species could accomplish. An ideal platform for the detoxification of complex organic mixtures such as bio- oil. Enables relevant primary energy savings of non- renewable sources without worsening abiotic resources depletion + a strong reduction of GHGs emissions. (Fabbri & Torri, 2016) Julian Pietrzyk [email protected] Use of Microbial Consortia for Conversion of Biomass Pyrolysis Liquids Hohenheim Biogas Yield Test into Value-Added Products silicone layer scale gas compartment opening for sampling plunger substrate glass syringe stopcock • 12 × 100 ml glass syringes • 30 ml Seafield water treatment plant anaerobic digestate • Supplemented with 10 g/l COD bio-oil, dried anaerobic digestate (AD), wood pellets (WP) or seaweed (SW) • Mesophilic (~37°C) for 102 days (adapted Mittweg et al., 2012) Julian Pietrzyk [email protected] Use of Microbial Consortia for Conversion of Biomass Pyrolysis Liquids Biogas into Value-Added Products 250 200 150 DIGESTATE AD BO WP BO 100 SW BO Biogas Generated (ml) Generated Biogas 50 0 0 10 20 30 40 50 60 70 80 90 100 Time (days) Julian Pietrzyk [email protected] Use of Microbial Consortia for Conversion of Biomass Pyrolysis Liquids Illumina sequencing into Value-Added Products Target gene Conserved 16S rRNA Conserved region V4 hypervariable region region 5’ Illumina adapter (adapted) Primer pad Primer linker V4 Forward primer aV4 Reverse primer Adapted V4 Primer linker forward primer Primer pad Golay barcode 100 3’ Illumina adapter 80 Read 1 Read 2 60 40 Index Coverage (%) Coverage 20 0 V3 V4 aV4 Archaea Bacteria Julian Pietrzyk [email protected] Use of Microbial Consortia for Conversion of Biomass Pyrolysis Liquids Illumina sequencing into Value-Added Products Julian Pietrzyk [email protected] Use of Microbial Consortia for Conversion of Biomass Pyrolysis Liquids Illumina sequencing into Value-Added Products 100 Phylum Class Order Family Genus 90 Euryarchaeota Methanobacteria Methanobacteriales Methanobacteriaceae Methanobrevibacter 80 Euryarchaeota Methanomicrobia Methanosarcinales Methanosaetaceae Methanosaeta Euryarchaeota Methanobacteria Methanobacteriales Methanobacteriaceae Methanobacterium 70 Lokiarchaeota uncultured uncultured uncultured uncultured 60 WSA2 WCHA1-57 uncultured uncultured uncultured 50 Euryarchaeota Methanomicrobia Methanosarcinales Methanosarcinaceae Methanosarcina 40 Lokiarchaeota uncultured uncultured uncultured uncultured Relative Abundance (%) Abundance Relative 30 Lokiarchaeota uncultured uncultured uncultured uncultured Euryarchaeota Methanomicrobia Methanomicrobiales Methanomicrobiaceae Methanoculleus 20 Marine Benthic Group Euryarchaeota Thermoplasmata Thermoplasmatales uncultured D and DHVEG-1 10 0 d0 DIG2 d0 d0 DIG3 d0 d0 DIG1 d0 Archaeal top 10 d102 AD1 d102 AD2 d102 AD3 d102 d102 MP1 d102 d102 MP2 d102 MP3 d102 d102 DIG1 d102 DIG2 d102 DIG3 d102 d102 SWP1 d102 SWP2 d102 SWP3 d102 Julian Pietrzyk [email protected] Use of Microbial Consortia for Conversion of Biomass Pyrolysis Liquids Illumina sequencing into Value-Added Products 100 Phylum Class Order Family Genus 90 Thermotogae Thermotogae Petrotogales Petrotogaceae Defluviitoga Cloacimonetes W5 uncultured uncultured uncultured 80 Bacteroidetes Sphingobacteriia Sphingobacteriales Lentimicrobiaceae uncultured 70 Bacteroidetes Bacteroidia Bacteroidales Porphyromonadaceae Proteiniphilum 60 Firmicutes Clostridia D8A-2 uncultured uncultured 50 Firmicutes Clostridia Thermoanaerobacterales Thermoanaerobacteraceae Gelria 40 Bacteroidetes Bacteroidia Bacteroidales Porphyromonadaceae uncultured Relative Abundance (%) Abundance Relative 30 Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae Pseudomonas Firmicutes Clostridia Clostridiales Caldicoprobacteraceae Caldicoprobacter 20 Firmicutes BSA1B-03 uncultured uncultured uncultured 10 0 d0 DIG1 d0 DIG2 d0 DIG3 d0 d102 AD1 d102 AD2 d102 AD3 d102 d102 MP2 d102 MP3 d102 d102 MP1 d102 Bacterial top 10 d102 DIG1 d102 DIG2 d102 DIG3 d102 d102 SWP1 d102 SWP2 d102 SWP3 d102 Julian Pietrzyk [email protected] Use of Microbial Consortia for Conversion of Biomass Pyrolysis Liquids Illumina sequencing into Value-Added Products 1.2 1 Archaea Bacteria d102 AD 0.6 d102 AD 0.5 d102 SW d0 DIG d102 SW 0 0 d102 WP d102 DIG NMDS2 NMDS2 d0 DIG -0.6 -0.5 d102 WP d102 DIG -1.2 -1 -2 -1 0 1 2 -2 -1.5 -1 -0.5 0 0.5 1 NMDS 1 NMDS 1 Julian Pietrzyk [email protected] Use of Microbial Consortia for Conversion of Biomass Pyrolysis Liquids ESI FT-ICR MS DCNC CJIℏ⊙Щ M CNIOinto Value RW∞-Added Products 1.6 3.0 7 1.4 2.0 10 1.2 × 1.0 8 1.0 10 × 0.0 0.8 Intensity 299.05 299.10 299.15 0.6 m/z 0.4 Intensity 0.2 0.0 200 250 300 350 400 450 500 m/z 2 protein lipid carbohydrate 1 H/C lignin condensed Van Krevelen diagram hydrocarbon 0 0.0 0.5 1.0 O/C Julian Pietrzyk [email protected] Use of Microbial Consortia for Conversion of Biomass Pyrolysis Liquids ESI FT-ICR MS into Value-Added Products d0 Digestate d0 AD/BO d0 SW/BO d0 WP/BO d102 Digestate d102 AD/BO d102 SW/BO d102 WP/BO Julian Pietrzyk [email protected] Use of Microbial Consortia for Conversion of Biomass Pyrolysis Liquids ESI FT-ICR MS into Value-Added Products 1.5 d102 DIG1 1 d102 DIG2 0.5 d102 DIG3 d102 AD1 d102 WP1 d102 AD2 0 d102 WP2 NMDS2 d102 AD3 -0.5 d102 WP3 d102 SW3 d102 SW2 -1 d102 SW1 -1.5 -2 -1 0 1 2 NMDS 1 Julian Pietrzyk [email protected] Use of Microbial Consortia for Conversion of Biomass Pyrolysis Liquids Relatedness into Value-Added Products Julian Pietrzyk [email protected] Use of Microbial Consortia for Conversion of Biomass Pyrolysis Liquids Relatedness into Value-Added Products 40 d102 DIG2 30 d102 DIG3 20 d102 DIG1 d102 AD1 10 0 d102 WP3 d102 AD3 d102 SW2 -10 Defluviitoga Abundance Abundance Defluviitoga (24.4% of total variation) total of (24.4% d102 WP2 d102 AD2 d102 SW1 d102 SW3 -20 d102 WP1 -30 -40 -30 -20 -10 0 10 20 30 40 Cloacimonetes Abundance (38.2% of total variation) Julian Pietrzyk [email protected] Use of Microbial Consortia for Conversion of Biomass Pyrolysis Liquids Relatedness into Value-Added Products Analysis: • Distance-based linear model (DistLM). • Multivariate chemical data matrix using the abundance profiles of Candidatus. Cloacimonetes and the Defluviitoga as the predictor variables. • The predictor variables are additionally plotted as vectors (annotated arrows). • The abundance profiles of these two microorganisms are cumulatively able to explain 62.61% of the chemical variation observed. • Candidatus Cloacimonetes phylum abundance correlates with the chemical pattern separating reactor conditions – propionate degradation? • Defluviitoga suggests that increases in its abundance are related to the chemistry observed for digestate-only control reactors – specific inhibition by bio-oil? Julian Pietrzyk [email protected] Use of Microbial Consortia for Conversion of Biomass Pyrolysis Liquids Importance into Value-Added Products • Patterns in reactor chemistry can be correlated to fluxes in microbial community • Understanding the microbial players involved at each stage of AD • Longitudinal studies: continuous sampling of both the chemical and biological species involved to identify process bottlenecks • Strategies to overcome inhibition Julian Pietrzyk [email protected] Use of Microbial Consortia for Conversion of Biomass Pyrolysis Liquids What next? into Value-Added Products Biochar supplementation aids AD by the adsorption of Biochar inhibitory compounds and via the adherence of microbial cells in biofilms. • High surface area, biofilm formation • Biofilms show increased resistance to environmental stresses • Partially conductive to the flow of electrons, Scanning electron microscope image capable of supporting direct interspecies electron of a biochar surface. From Schulz, H. transfer (DIET) & Glaser, B. (2012). Julian Pietrzyk [email protected] Use of Microbial Consortia for Conversion of Biomass Pyrolysis Liquids Thank you! into Value-Added Products Julian Pietrzyk [email protected].