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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

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SLIDE 1

Development of a moving‐bed carrier for stimulating direct interspecies electron transfer for improving anaerobic digestion

6th 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

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SLIDE 2

Importance of Methanogenesis

  • 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

(Source: http://msutoday.msu.edu)

2

waste CH4

(Source: http://unlcms.unl.edu)

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SLIDE 3

Principles of Methanogenesis

3

  • Methanogenesis can be explained as electron transfer deposited in
  • rganics to methane by fermenting bacteria and methanogenic archaea

(i.e. interspecies electron transfer, IET)

  • IET occurs via diffusive electron carriers (e.g. H2 and formate)

Fermenting Bacteria Methanogenic Archaea

Organics CO2 CO2 CH4

Fermenting Bacteria Methanogenic Archaea

Diffusive e‐ carriers (e.g. H2 and formate)

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SLIDE 4

Direct Interspecies Electron Transfer (DIET) for Methanogenesis

Organics CO2 CO2 CH4 Diffusive e‐ carriers (e.g. H2 and formate) e‐ e‐ e‐

  • 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

Organics Oxidizing Bacteria Methanogenic Archaea

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SLIDE 5

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Organics Oxidizing Bacteria Methanogenic Archaea

Conductive pili

Organics Oxidizing Bacteria Methanogenic Archaea

e‐ transport proteins e‐ e‐ e‐ e‐ e‐

Organics Oxidizing Bacteria Methanogenic Archaea

e‐ e‐ e‐

How is DIET Possible?

a b c

Conductive material Modification of Lovley 2017, Annual Rev. Microbiol. e‐

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SLIDE 6

Granular Activated Carbon can Facilitate DIET in Methanogenesis

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  • 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 (Aktaş 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)

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SLIDE 7

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

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SLIDE 8

Experimental Approach

Control GAC Continuous flow and batch Reactors fed with acetate

Reactor performance (control vs. GAC reactors)

‐ COD reduction ‐ Methane production rates ‐ Relative contribution (suspended vs. GAC biomass)

Microbial community analyses (16S rRNA gene)

‐ Bacterial populations ‐ Archaeal populations ‐ Network analysis

Bioelectrochemical analyses

‐ Anodic current generation ‐ Cathodic current generation

8

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SLIDE 9

GAC Supplementation Produced More Methane

10 20 30 40 50 15 20 25 30 35 40 45 Methane production rate mL‐CH4/d Operational day

Control reactor GAC reactor

20.1 35.7 Control reactor GAC reactor 0.00 0.05 0.10 0.15 0.20 0.25 15 20 25 30 35 40 45 Effleunt soluble COD gCOD/L Operational day

Control reactor GAC reactor

0.18 0.12 Control reactor GAC reactor 9

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SLIDE 10

GAC Biomass Showed Higher Methane Production

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GAC+Bulk Bulk GAC

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 10 20 30 40

  • Cumm. CH4 production (mL)

Time (hr) GAC+Bulk Bulk GAC

0.0 0.5 1.0 1.5 2.0 2.5 3.0 10 20 30 40

Specific CH4 production (mL/mg VSS) Time (hr) GAC+Bulk Bulk GAC

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SLIDE 11

Bacterial Community Shift

Control reactor GAC reactor

10 20 30 40 50 60 70 80 90 100

3 20 40 3 20 40 20 40

Fraction (%)

Bacteroidetes Actinobacteria Betaproteobacteria Gammaproteobacteria Deltaproteobacteria Chloroflexi Synergistetes Firmicutes Minor groups

Attached biomass Suspended biomass

days

11

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SLIDE 12

GAC Enriched Geobacter, Thauera, and Gordonia

5 10 15 20 25 30 35 3 20 40 3 20 40 20 40

Fraction (%)

Geobacter Thauera Gordonia

Control reactor GAC reactor

Attached biomass Suspended biomass

days

OTU7 OTU30 OTU99 OTU82 Geobacter sulfurreducens Geobacter grbiciae Thauera terpenica OTU104 OTU3 OTU6 OTU43 OTU42 OTU9 OTU97 OTU113 Gordonia cholesterolivorans OTU52 Thermotogae Aquificae Archaea

61 75 57 99 100 50 92 89 100 89 99 98 100 75 90 97 53

0.05 12

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SLIDE 13

Archaeal Community Shift

10 20 30 40 50 60 70 80 90 100

3 20 40 3 20 40 20 40

Fraction (%)

Methanoregulaceae Methanospirillaceae Methanomassiliicoccaceae Methanosarcinaceae Others

Control reactor GAC reactor

Attached biomass Suspended biomass

days

13

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SLIDE 14

5 10 15 20 25 3 20 40 3 20 40 20 40

Fraction (%)

Methanospirillum Methanosarcina Methanolinea

GAC Enriched Methanospirillum and Methanolinea

Control reactor GAC reactor

Attached biomass Suspended biomass

days

OTU2 OTU9 OTU12 OTU10 Methanolinea mesophila OTU14 OTU11 OTU8 OTU1 OTU13 Methanoculleus receptaculi OTU21 Methanospirillum hungatei OTU5 OTU3 OTU7 Methanosarcina thermophila OTU4 Methanosaeta concilii OTU18 Methanobacterium ferruginis OTU17 Methanomassiliicoccus luminyensis Uncultured archaeon clone OTU6 Escherichia coli

89 100 100 100 100 68 62 60 100 100 100 100 62 95 57 80 65 57 58

0.05

14

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SLIDE 15

Network Analysis demonstrates a Non‐random Co‐occurrence

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Control reactor GAC reactor

97% cutoff 122 OTUs Rho > 0.6, P < 0.05

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SLIDE 16

Cyclic Voltammogram Suggests DIET by GAC Biomass

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  • GAC biomass generated anodic current at ‐ 0.36 V and cathodic current

at ‐ 0.32 V, respectively

Potentiostat

CE WE RE

three‐electrode cell Acetate GAC biomass

‐2,5 ‐2,0 ‐1,5 ‐1,0 ‐0,5 0,0 0,5 1,0 1,5 2,0 2,5 ‐1 ‐0,5 0,5 1

Current (mA) Potentail (V)

‐2,5 ‐2,0 ‐1,5 ‐1,0 ‐0,5 0,0 0,5 1,0 1,5 2,0 2,5 ‐1 ‐0,5 0,5 1

Current (mA) Potentail (V)

W/O GAC biomass W/ GAC biomass

CH3COO‐ + 2H2O → 2CO2 + 7H+ + 8e‐ CO2 + 8H+ + 8e‐→ CH4

‐ + 2H2O

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SLIDE 17

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?

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SLIDE 18

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Development of a Moving‐Bed Carrier

  • Working Volume: 700 mL
  • Carbon or cotton cloth: 10 cm2 * 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

17 mm

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SLIDE 19

CH4 production rate (ml/day)

50 100 150 200

Cotton cloth Carbon cloth

Operation time (day)

20 40 60 80

Total organic acid (gCOD/L)

2 4 6 8

Cotton cloth Carbon cloth

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Carriers with Carbon Cloth Stimulated More Methane Production

P1 ‐ Acetate P2 ‐ Glucose P3 ‐ Acetate P4 ‐ Glucose

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SLIDE 20

Archaeal Fraction (%)

20 40 60 80 100 Methanobacteriales Methanomicrobiales Methanosarcinales Thermoplasmatales WSA2 Woesearchaeota Others

Known DIET Microorganisms were not Identified

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Bacterial Fraction (%)

20 40 60 80 100 Actinobacteria Aminicenantes Armatimonadetes Bacteroidetes Chloroflexi Cloacimonetes Firmicutes Planctomycetes Proteobacteria Spirochaetae Synergistetes Thermotogae Verrucomicrobia WS6 Unclassified Others

Cotton Cloth Carbon Cloth Seed P1 P2 P3 Seed P1 P2 P3

Actinobacteria Aminicenantes Armatimonadetes Bacteroidetes Chloroflexi Cloacimonetes Firmicutes Planctomycetes Proteobacteria Spirochaetae Synergistetes Thermotogae Verrucomicrobia WS6 Unclassified Others Methanobacteriales Methanomicrobiales Methanosarcinales Thermoplasmatales WSA2 Woesearchaeota Others

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SLIDE 21

Summary and Significance

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  • 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

  • r

Carbon Cloth

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SLIDE 22

Acknowledgements

22

Jung‐Yeol Lee, Ph.D. ‐ Experimental design ‐ Reactor operation and analyses ‐ Bioelectrochemical analyses Sang‐Hoon Lee, Ph.D. ‐ Microbial community analyses

This work was financially supported by National Research Foundation of Korea (2018R1A2B2002110).

Jeong‐Hoon Park, Ph.D. ‐ Microbial community analyses Hyun‐Jin Kang ‐ Design a moving‐bed carrier ‐ Reactor operation

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SLIDE 23

GAC Biomass Comprised a Minor Fraction of Total Biomass

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Suspended biomass GAC biomass

8.3% 91.7%

0.67 1.25 0.44 1.65 0.0 0.5 1.0 1.5 2.0

Control reactor GAC reactor Suspended biomass GAC biomass Specific CH4 production rate ml‐CH4/gVSS/d

  • GAC biomass was 3.8‐fold higher than suspended biomass in terms of

specific methane production rate

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SLIDE 24

More Research Questions

  • Does a voltage application to the conductive

materials accelerate the DIET between two groups

  • f microorganisms to produce methane?
  • Is DIET via conductive materials also effective to

mitigate sensitiveness to operational and environmental conditions in methanogenesis?

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SLIDE 25

Operational time (day)

2 4 6 8 10 12 14 16

Cumulative CH4 (mL)

10 20 30 40 Voltage applied cell No voltage applied cell Estimated

Voltage Application was not Helpful for DIET

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  • A voltage (0.35 V) applied cell showed a 168% higher methane

production rate

  • Methanogens generating DIET were observe only in the no voltage

applied cell

OTU1 Methanosaeta harundinacea OTU3 OTU4 Methanosaeta concilii strain Methanosarcina mazei OTU5 Methanosarcina thermophila Methanospirillum hungatei Methanobacterium palustre OTU2 Methanobacterium subterraneum 95 100 66 100 100 100 99 100 53 0.02

Voltage application No voltage application

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SLIDE 26

DIET Moderated the Effects of Several Adverse Operational Conditions

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  • GAC supplementation moderated the harsh conditions, such as low pH

(pH = 5.0), high organic loading (12 ml Ac/L), and high ammonia (0.25 g NH3‐N/L)

Time (d) 20 40 60 Time (d) 20 40 60 Time (d) 20 40 60 Cumulative CH4 (mL) 20 40 60 80 100 120 140

Time (d) 20 40

pH = 7.0 4 ml Ac/L pH = 5.0 4 ml Ac/L pH = 7.0 12 ml Ac/L pH = 8.0 2 ml Ac/L 0.25 g NH3‐N/L w/o GAC w/ GAC