Use of Microbial Consortia for Conversion of Biomass Pyrolysis Liquids into Value- Added Products Julian Pietrzyk julian.pietrzyk@ed.ac.uk
Use of Microbial Consortia for Outline Conversion of Biomass Pyrolysis Liquids into Value-Added Products • Pyrolysis • Bio-oil Py-AD • AD • Illumina Sequencing • Mass Spectrometry • Relatedness Julian Pietrzyk julian.pietrzyk@ed.ac.uk
Use of Microbial Consortia for Pyrolysis Conversion of Biomass Pyrolysis Liquids into Value-Added Products Reactor Heat Heat Heat Cold Cold Receiver Trap 1 Trap 2 Trap 3 Trap 1 Trap 2 N 2 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 julian.pietrzyk@ed.ac.uk
Use of Microbial Consortia for Bio-oil Conversion of Biomass Pyrolysis Liquids into Value-Added Products Product of pyrolysis of biomass • Dark brown organic liquid • Organic Aqueous High water content (~25 wt %) • Extremely high oxygen content • catechol, guaiacol, + 1000s other compounds • syringol, isoeugenol, Ages instantly • pyrones, vanillin, furans, acetic acid, Composition dependant formic acid, sugars, • carboxylic acids, phenolics, on feedstock hydroxyketones, hydroxyaldehydes Low pH & biocatalyst inhibitors • Julian Pietrzyk julian.pietrzyk@ed.ac.uk
Use of Microbial Consortia for Anaerobic Digestion Conversion of Biomass Pyrolysis Liquids into Value-Added Products Hydrolysis High molecular weight organic polymers • split into smaller more bioavailable monomers. • Proteins > Amino acids | Carbohydrates > Monosaccharides | Fats > Fatty acids | + H 2 Acidogenesis Acidogenic fermentation of hydrolysed • products to short chain… • volatile acids (propionic, butyric, acetic, formic, lactic) alcohols (ethanol, methanol) • H 2 + CO 2 + NH 3 + H 2 S • Acetogenesis Further digestion of acids by acetogens to • H 2 , CO 2 and acetic acid. Methanogenesis Methanogenic archaea convert H 2 and • acetic acid to CH 4 , CO 2 . Julian Pietrzyk julian.pietrzyk@ed.ac.uk
Use of Microbial Consortia for Py-AD Conversion of Biomass Pyrolysis Liquids 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 julian.pietrzyk@ed.ac.uk
Use of Microbial Consortia for Hohenheim Biogas Yield Test Conversion of Biomass Pyrolysis Liquids into Value-Added Products silicone layer scale gas compartment opening for sampling stopcock plunger substrate glass syringe • 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 julian.pietrzyk@ed.ac.uk
Use of Microbial Consortia for Biogas Conversion of Biomass Pyrolysis Liquids into Value-Added Products 250 200 Biogas Generated (ml) 150 DIGESTATE AD BO WP BO 100 SW BO 50 0 0 10 20 30 40 50 60 70 80 90 100 Time (days) Julian Pietrzyk julian.pietrzyk@ed.ac.uk
Use of Microbial Consortia for Illumina sequencing Conversion of Biomass Pyrolysis Liquids 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 Adapted V4 Reverse primer forward primer Primer linker Primer pad 100 Golay barcode 3’ Illumina adapter 80 Coverage (%) Read 1 Read 2 60 40 Index 20 0 V3 V4 aV4 Archaea Bacteria Julian Pietrzyk julian.pietrzyk@ed.ac.uk
Use of Microbial Consortia for Illumina sequencing Conversion of Biomass Pyrolysis Liquids into Value-Added Products Julian Pietrzyk julian.pietrzyk@ed.ac.uk
Use of Microbial Consortia for Illumina sequencing Conversion of Biomass Pyrolysis Liquids into Value-Added Products 100 Phylum Class Order Family Genus 90 Euryarchaeota Methanobacteria Methanobacteriales Methanobacteriaceae Methanobrevibacter Euryarchaeota Methanomicrobia Methanosarcinales Methanosaetaceae Methanosaeta 80 Euryarchaeota Methanobacteria Methanobacteriales Methanobacteriaceae Methanobacterium Relative Abundance (%) 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 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 DIG1 d0 DIG2 d0 DIG3 d102 DIG1 d102 DIG2 d102 DIG3 d102 AD1 d102 AD2 d102 AD3 d102 MP1 d102 MP2 d102 MP3 d102 SWP1 d102 SWP2 d102 SWP3 Archaeal top 10 Julian Pietrzyk julian.pietrzyk@ed.ac.uk
Use of Microbial Consortia for Illumina sequencing Conversion of Biomass Pyrolysis Liquids into Value-Added Products 100 Phylum Class Order Family Genus Thermotogae Thermotogae Petrotogales Petrotogaceae Defluviitoga 90 Cloacimonetes W5 uncultured uncultured uncultured 80 Bacteroidetes Sphingobacteriia Sphingobacteriales Lentimicrobiaceae uncultured Relative Abundance (%) 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 Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae Pseudomonas 30 Firmicutes Clostridia Clostridiales Caldicoprobacteraceae Caldicoprobacter 20 Firmicutes BSA1B-03 uncultured uncultured uncultured 10 0 d0 DIG1 d0 DIG2 d0 DIG3 d102 DIG1 d102 DIG2 d102 DIG3 d102 AD1 d102 AD2 d102 AD3 d102 MP1 d102 MP2 d102 MP3 d102 SWP1 d102 SWP2 d102 SWP3 Bacterial top 10 Julian Pietrzyk julian.pietrzyk@ed.ac.uk
Use of Microbial Consortia for Illumina sequencing Conversion of Biomass Pyrolysis Liquids into Value-Added Products 1.2 1 Bacteria Archaea d102 AD 0.6 0.5 d102 AD d102 SW d0 DIG NMDS 2 NMDS 2 d102 SW 0 0 d102 DIG d102 WP 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 julian.pietrzyk@ed.ac.uk
Use of Microbial Consortia for ESI FT-ICR MS DCNC CJI ℏ⊙ Щ M CNIO RW∞ ESI FT-ICR MS Conversion of Biomass Pyrolysis Liquids into Value-Added Products 3.0 1.6 Intensity × 10 7 1.4 2.0 1.2 1.0 Intensity × 10 8 1.0 0.0 0.8 299.05 299.10 299.15 0.6 m/z 0.4 0.2 0.0 200 250 300 350 400 450 500 m/z 2 protein lipid carbohydrate H/C 1 lignin condensed Van Krevelen diagram hydrocarbon 0 0.0 0.5 1.0 O/C Julian Pietrzyk julian.pietrzyk@ed.ac.uk
Use of Microbial Consortia for ESI FT-ICR MS Conversion of Biomass Pyrolysis Liquids into Value-Added Products d0 WP/BO d0 AD/BO d0 Digestate d0 SW/BO d102 Digestate d102 AD/BO d102 WP/BO d102 SW/BO Julian Pietrzyk julian.pietrzyk@ed.ac.uk
Use of Microbial Consortia for ESI FT-ICR MS Conversion of Biomass Pyrolysis Liquids into Value-Added Products 1.5 d102 DIG1 d102 DIG2 1 d102 DIG3 0.5 d102 AD1 NMDS 2 d102 AD2 d102 WP1 0 d102 WP2 d102 AD3 d102 WP3 -0.5 d102 SW3 d102 SW2 d102 SW1 -1 -1.5 -2 -1 0 1 2 NMDS 1 Julian Pietrzyk julian.pietrzyk@ed.ac.uk
Use of Microbial Consortia for Relatedness Conversion of Biomass Pyrolysis Liquids into Value-Added Products Julian Pietrzyk julian.pietrzyk@ed.ac.uk
Use of Microbial Consortia for Relatedness Conversion of Biomass Pyrolysis Liquids into Value-Added Products 40 d102 DIG2 30 d102 DIG3 d102 DIG1 20 Defluviitoga Abundance (24.4% of total variation) d102 AD1 10 0 d102 WP3 d102 AD3 d102 SW2 -10 d102 AD2 d102 WP2 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 julian.pietrzyk@ed.ac.uk
Use of Microbial Consortia for Relatedness Conversion of Biomass Pyrolysis Liquids 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 julian.pietrzyk@ed.ac.uk
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