Development of a moving ‐ bed carrier for stimulating direct interspecies electron transfer for improving anaerobic digestion 6 th International Conference on Sustainable Solid Waste Management June 13 ‐ 16, 2018 The Cultural Center former Ursuline School, Naxos Island, Greece HEE ‐ DEUNG PARK SCHOOL OF CIVIL, ENVIRONMENTAL AND ARCHITECTURAL ENGINEERING KOREA UNIVERSITY
Importance of Methanogenesis CH 4 waste (Source: http://msutoday.msu.edu) (Source: http://unlcms.unl.edu) • Methanogenesis refers to methane formation by methanogenic archaea under anaerobic condition • Methanogenesis plays roles in global carbon cycle and waste treatment (bioenergy production) by decomposing organic matters 2
Principles of Methanogenesis Organics CH 4 Diffusive e ‐ carriers (e.g. H 2 and formate) Fermenting Methanogenic Bacteria Archaea CO 2 CO 2 Fermenting Methanogenic Bacteria Archaea • Methanogenesis can be explained as electron transfer deposited in organics to methane by fermenting bacteria and methanogenic archaea (i.e. interspecies electron transfer, IET) • IET occurs via diffusive electron carriers (e.g. H 2 and formate) 3
Direct Interspecies Electron Transfer (DIET) for Methanogenesis CH 4 Organics e ‐ e ‐ e ‐ Diffusive e ‐ carriers (e.g. H 2 and formate) Organics Methanogenic Oxidizing CO 2 Archaea CO 2 Bacteria • DIET removes some steps associated with hydrogen production and consumption, which lead to more energy efficient compared with IET via diffusive electron carrier (Lovley 2011, Energy Environ Sci 4) • Electrical conductance is more efficient than molecular diffusion of electron carriers 4
How is DIET Possible? Conductive pili a e ‐ e ‐ Organics Oxidizing Methanogenic Bacteria Archaea e ‐ e ‐ transport proteins b e ‐ Methanogenic Organics Oxidizing Archaea Bacteria e ‐ Conductive material c e ‐ e ‐ Organics Oxidizing Methanogenic e ‐ Bacteria Archaea e ‐ Modification of Lovley 2017, Annual Rev. Microbiol. 5
Granular Activated Carbon can Facilitate DIET in Methanogenesis • Excellent adsorbent • High surface area • Good electricity conduit • GAC supplementation improved stability and performance in anaerobic digestion due to adsorbing toxic chemicals and attaching microbes (Akta ş and Çeçen 2007, Int Biodet Biodeg 59; Liu et al. 2012, Energy Environ Sci 5) • GAC facilitated DIET in methanogenesis (Kato et al., 2012, Environ Microbiol 14; Liu et al. 2012, Energy Environ Sci 5) 6
Research Questions • Is it possible to generate a condition of DIET by supplementing GAC in anaerobic reactors for wastes treatments? • What microbes can be enriched in the reactors? • What are the potential benefits of DIET in anaerobic digestion? 7
Experimental Approach Reactor performance (control vs. GAC reactors) ‐ COD reduction ‐ Methane production rates Control GAC ‐ Relative contribution (suspended vs. GAC biomass) Microbial community analyses (16S rRNA gene) ‐ Bacterial populations ‐ Archaeal populations ‐ Network analysis Continuous flow and batch Reactors fed with acetate Bioelectrochemical analyses ‐ Anodic current generation ‐ Cathodic current generation 8
GAC Supplementation Produced More Methane 50 Control reactor 35.7 Methane production rate GAC reactor 40 mL ‐ CH 4 /d 30 20.1 20 10 0 0.25 0.18 Control GAC reactor 15 20 25 30 35 40 45 Control reactor reactor Operational day 0.20 Effleunt soluble COD GAC reactor 0.12 0.15 gCOD/L 0.10 0.05 0.00 15 20 25 30 35 40 45 Control GAC reactor reactor Operational day 9
GAC Biomass Showed Higher Methane Production GAC+Bulk Bulk GAC 3.0 3.5 GAC+Bulk GAC+Bulk Cumm. CH 4 production (mL) 3.0 2.5 Bulk Specific CH 4 production Bulk 2.5 GAC 2.0 (mL/mg VSS) GAC 2.0 1.5 1.5 1.0 1.0 0.5 0.5 0.0 0.0 0 10 20 30 40 0 10 20 30 40 Time (hr) Time (hr) 10
Bacterial Community Shift 100 Bacteroidetes 90 Actinobacteria 80 Betaproteobacteria Gammaproteobacteria 70 Deltaproteobacteria Fraction (%) 60 Chloroflexi 50 Synergistetes 40 Firmicutes Minor groups 30 20 10 0 days 3 20 40 3 20 40 20 40 Attached biomass Suspended biomass Control reactor GAC reactor 11
GAC Enriched Geobacter, Thauera, and Gordonia 35 OTU7 61 75 OTU30 Geobacter 57 30 OTU99 99 OTU82 Thauera 100 Geobacter sulfurreducens 25 Geobacter grbiciae 99 Gordonia Thauera terpenica Fraction (%) OTU104 20 100 OTU3 89 98 OTU6 92 15 50 OTU43 OTU42 OTU9 10 75 89 100 OTU97 OTU113 90 5 Gordonia cholesterolivorans 97 53 OTU52 Thermotogae 0 days Aquificae 3 20 40 3 20 40 20 40 Archaea Suspended biomass Attached biomass 0.05 Control reactor GAC reactor 12
Archaeal Community Shift 100 Methanoregulaceae 90 80 Methanospirillaceae 70 Fraction (%) Methanomassiliicoccaceae 60 Methanosarcinaceae 50 40 Others 30 20 10 0 3 20 40 3 20 40 20 40 days Attached biomass Suspended biomass Control reactor GAC reactor 13
GAC Enriched Methanospirillum and Methanolinea OTU2 95 OTU9 57 OTU12 OTU10 25 Methanolinea mesophila 80 Methanospirillum OTU14 Methanosarcina OTU11 20 OTU8 62 58 Methanolinea OTU1 57 65 OTU13 Fraction (%) 100 Methanoculleus receptaculi 15 100 OTU21 Methanospirillum hungatei OTU5 10 100 OTU3 100 OTU7 Methanosarcina thermophila 100 5 OTU4 68 Methanosaeta concilii 100 OTU18 0 60 Methanobacterium ferruginis 100 days 3 20 40 3 20 40 20 40 OTU17 Methanomassiliicoccus luminyensis 62 Suspended biomass Attached biomass Uncultured archaeon clone 100 89 OTU6 Control reactor GAC reactor Escherichia coli 14 0.05
Network Analysis demonstrates a Non ‐ random Co ‐ occurrence Control reactor GAC reactor 97% cutoff 122 OTUs Rho > 0.6, P < 0.05 15
Cyclic Voltammogram Suggests DIET by GAC Biomass CH 3 COO ‐ + 2H 2 O → 2CO 2 + 7H + + 8e ‐ 2,5 2,5 2,0 2,0 Potentiostat 1,5 1,5 RE Current (mA) Current (mA) WE 1,0 1,0 CE 0,5 0,5 0,0 0,0 ‐ 0,5 ‐ 0,5 W/ GAC biomass ‐ 1,0 ‐ 1,0 Acetate W/O GAC biomass ‐ 1,5 ‐ 1,5 GAC biomass CO 2 + 8H + + 8e ‐ → CH 4 ‐ + 2H 2 O ‐ 2,0 ‐ 2,0 ‐ 2,5 ‐ 2,5 three ‐ electrode cell ‐ 1 ‐ 1 ‐ 0,5 ‐ 0,5 0 0 0,5 0,5 1 1 Potentail (V) Potentail (V) • GAC biomass generated anodic current at ‐ 0.36 V and cathodic current at ‐ 0.32 V, respectively 16
More Research Questions • Can we hold conductive materials for DIET in a bioreactor without loss of them? • Can substrates other than acetate also stimulate DIET? 17
Development of a Moving ‐ Bed Carrier 17 mm • Working Volume: 700 mL Carbon or cotton cloth: 10 cm 2 * 50EA • Substrate: Acetate or Glucose (1 gCOD/L) • • Feeding rate: 35 mL/d Temperature: 35 � • • Phase 1 (3 weeks): Acetate Phase 2 (3 weeks): Glucose • • Phase 3 (3 weeks): Acetate • Phase 4 (3 weeks): Glucose 18
Carriers with Carbon Cloth Stimulated More Methane Production P1 ‐ Acetate P2 ‐ Glucose P3 ‐ Acetate P4 ‐ Glucose 200 Cotton cloth CH4 production rate Carbon cloth 150 (ml/day) 100 50 0 Cotton cloth Carbon cloth Total organic acid 8 (gCOD/L) 6 4 2 0 0 20 40 60 80 Operation time (day) 19
Known DIET Microorganisms were not Identified Cotton Cloth Carbon Cloth Seed P1 P2 P3 Seed P1 P2 P3 100 Actinobacteria Actinobacteria Aminicenantes Aminicenantes Armatimonadetes Armatimonadetes Bacterial Fraction (%) 80 Bacteroidetes Bacteroidetes Chloroflexi Chloroflexi Cloacimonetes Cloacimonetes Firmicutes 60 Firmicutes Planctomycetes Planctomycetes Proteobacteria Proteobacteria Spirochaetae Synergistetes Spirochaetae 40 Thermotogae Synergistetes Verrucomicrobia Thermotogae WS6 Verrucomicrobia 20 Unclassified WS6 Others Unclassified Others 0 100 Methanobacteriales Methanobacteriales Methanomicrobiales Methanomicrobiales Methanosarcinales Methanosarcinales Archaeal Fraction (%) 80 Thermoplasmatales Thermoplasmatales WSA2 WSA2 Woesearchaeota Woesearchaeota Others Others 60 40 20 20 0
Summary and Significance or Carbon Cloth • Supplementation of GAC in anaerobic reactors enhanced methane production (1.8 folds), mostly due to the biomass attached on GAC • GAC facilitated DIET between exoelctrogens (e.g. Geobacter) and methanogens (e.g. Methanospirillum) • DIET via carbon cloth was effective only when acetate was provided as the substrate 21
Acknowledgements Hyun ‐ Jin Kang Jung ‐ Yeol Lee, Ph.D. ‐ Design a moving ‐ bed carrier ‐ Experimental design ‐ Reactor operation ‐ Reactor operation and analyses ‐ Bioelectrochemical analyses Sang ‐ Hoon Lee, Ph.D. Jeong ‐ Hoon Park, Ph.D. ‐ Microbial community analyses ‐ Microbial community analyses This work was financially supported by National Research Foundation of Korea (2018R1A2B2002110). 22
GAC Biomass Comprised a Minor Fraction of Total Biomass 8.3% Specific CH 4 production rate 2.0 1.65 ml ‐ CH 4 /gVSS/d 1.5 Suspended 1.25 biomass 1.0 GAC 0.67 biomass 0.44 0.5 91.7% 0.0 Control GAC Suspended GAC reactor reactor biomass biomass • GAC biomass was 3.8 ‐ fold higher than suspended biomass in terms of specific methane production rate 23
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