From living cells to stable isotopes

Francesc X. Prenafeta‐Boldú ([email protected])

L. Ruiz, J. Vila, B. Fernández, V. Riau, M. Guivernau, M. Viñas

From living cells to stable isotopes:

An interdisciplinary approach forunravelling microbial interactions in ammonia‐

overloaded anaerobic digesters

GIRO Joint Research Unit IRTA‐UPC, Torre Marimon, Caldes de Montbui, Spain.

MELiSSA WorkshopLausanne 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


14CO2/14CH4 >1

Ammonium concentrations of 2 – 5 g NH4+‐N L‐1 cause

significant inhibition of methanogenesis, depending on T°, and pH conditions (free ammonia is the toxic species)(Angelidaki & Ahring 1994, Sung & Liu 2003)

Nitrogen fate and toxicity in anaerobic digesters


The syntrophic acetate oxydation

SAOB

SAOAInhibition byammonia and VFA

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 Clostridia class (phylum Firmicutes)

Knowledge on the biodiversity, ecophysiology, and biochemistry of SAOB is limited

SAOB are very important for a stable anaerobic digestion process under high ammoniaconcentrations(Müller et al. 2012)


Location: Vilasana, Lleida (Spain)Reactor type: CSTRVolume: 1500 m3 (2x)HRT: 65 daysTAN: 2 – 4 gTAN L‐1Operation regime: MesophilicTreatment capacity: 11.000 m3 of pig slurry and 4.500 m3 of organic residues

Case study: methanogenic biomass from an agricultural biogas plant


Batch activity assays

0

5

10

15

20

25

30

35

40

0 20 40 60 80

NmLCH

4

days

Inoculum

TAN: 6 g/L

TAN: 1 g/LTAN: 3 g/L

20% total CH4

Treatment conditions (x3)Acetic Acid: 3.5 g Ac L‐1(*Acetic acid : 13CH3‐COOH)Inoculum: 12.5 gVSS L‐1Ammonia: 1, 3 and 6 gTAN L‐1Incubation: 65 days at 37oC

Monitored parametersVFA/CODCO2/CH4 GC‐TCD and GC‐IRMSNGS MiSeq (Eub/Arch) DNA/cDNAqPCR 16S rRNA and mcrA (DNA/cDNA)


Compound Stable Isotopic Analysis (CSIA) of biogas: Unlabelled experiments

Apparent fractionation factor (αc) (Conrad 2005, Conrad et al. 2009)

αc = (δ13CO2 + 1000)/ (δ13CH4 + 1000)

αc < 1.055 Dominance of acetotrophic methanogenesisαc > 1.065 Dominance of hydrogenotrophic methanogenesisαc > 1.080 Exclusively hydrogenotrophic methanogenesis

Gas

Mix

ture

Inoc

ula

(Bla

nk)

C2

Con

trol (

Initi

al N

)

C2+

N1

C2+

N2

C2*

+ N

1

C2*

+ N

2

d13C

-CO

2

-8-7-6-5-4-3-2-10123456789

101112131415

Gas

Mix

ture

Inoc

ula

(Bla

nk)

C2

Con

trol (

Initi

al N

)

C2+

N1

C2+

N2

C2*

+ N

1

C2*

+ N

2

d13C

-CH

4

-80

-70

-60

-50

-40

-30

-20

TAN 6 g/LTAN 3.5 g/L

TAN 3.5 g/LTAN 6 g/L

TAN 1 g/L

TAN 1 g/LAM

HM

δ13C-CO2

AM

HM

δ13C-CH4

1gN‐TAN ∙L‐1 αc = 1.054 ± 0.017

3gN‐TAN ∙L‐1 αc = 1.077 ± 0.001

6gN‐TAN ∙L‐1 αc = 1.080 ± 0.001

Acetotrophic

Hydrogenotrophic

Hydrogenotrophic


Compound Stable Isotopic Analysis (CSIA) of biogas: labelled experiments

13CH3‐COOH 13CH4 + CO2AM

SAO 13CH3‐COOH + 2H2O 13CO2 + CO2 +4H213CH4 / CH4+13CO2 / CO2

SAOB SAOA

d13C-CO2

-50 -40 -30 -20 -10 0 10 20 30 40 50 2000 3000 4000 5000

d13C

-CH

4

-80

-70

-60

-50

-40

-30

-20

20000

40000

60000

80000

100000

120000

Gas Mixture

C2-N2C2-N1

C2 N0

Inoculum

*C2-N1 *C2-N2Ac-2c* Ac-3c*

Inoculum

Ac-2cAc-3c

δ13C-CO2

δ13C-CH4

Ac-1c

TAN 3.5 g/L TAN 6 g/L


Microbial molecular characterization

Total DNA + RNA (‐80ºC)

Biomass sampling

Biomass Pellet ‐80ºC

Simultaneous DNA + RNA extraction

Total DNA + cDNA (‐80ºC)

RT (PrimeScript)

qPCR(Quantitative assessment)

16S rRNA genes (DNA + cDNA)mcrA genes (DNA + cDNA)

NGS (MiSeq) (Qualitative assessment)16S rRNA (Eubacteria)16Sr RNA (Archaea)

Centrifugiation (4ºC)Raw data (sff)

QIIME/Mothur1.33.1

SILVA/greengenesalignment

Pick OTUs

OTUs distribution

Diversity indicesTaxonomy (RDP)PCA/CA/CCA


0

1

2

3

4

5

0 5 10 15 20

16S rRNAtran

scrpts/gen

e copies

Time (days)

A

0.00

0.05

0.10

0.15

0.20

0 5 10 15 20

mcrAtran

scrpts/gen

e copies

Time (days)

B 1 gTAN/L3.5 gTAN/L

3.5 gTAN/L

6 gTAN/L 6 gTAN/L

1 gTAN/L

Total eubacteria (16S rRNA) Methanogenic archaea (mcrA)

Molecular targets:The hypervariable V3‐V5 region from eubacterial 16S rRNA genes The functionalmcrA gene (methyl coenzyme‐M reductase) specific of methanogenic archaea

Ammonia versus transcription level (RT‐qPCR)


NGS of eubacterial 16S rRNA librariesRe

lativeabun

dance(%

)

DNAt0 DNAt4 cDNAt4 DNAt0 DNAt4 cDNAt4 DNAt0 DNAt4 cDNAt41 gTAN/L 3.5 gTAN/L 6 gTAN/L


Some of the identified ribotypes were somewhat homologous to the SAOB Clostridium ultunense and Tepidanaerobacter acetatoxydans

NGS of archaeal 16S rRNA librariesRe

lativeabun

dance(%

)

DNAt0 DNAt4 cDNAt4 DNAt0 DNAt4 cDNAt4 DNAt0 DNAt4 cDNAt41 gTAN/L 3.5 gTAN/L 6 gTAN/L


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

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 werehomologous to the SAOB Clostridium ultunense and Tepidanaerobacter acetatoxydans.

5.Concerning the Archaea, representatives of the genera Methanoculleus andMethanosarcinaappear 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.

Conclusions


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)

Documents

From living cells to stable isotopes