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Francesc X. Prenafeta‐Boldú (francesc.prenafeta@irta.cat)
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
MELiSSA Workshop, Lausanne 8th – 9th June 2016
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
MELiSSA Workshop, Lausanne 8th – 9th June 2016
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)
MELiSSA Workshop, Lausanne 8th – 9th June 2016
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
MELiSSA Workshop, Lausanne 8th – 9th June 2016
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)
MELiSSA Workshop, Lausanne 8th – 9th June 2016
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
MELiSSA Workshop, Lausanne 8th – 9th June 2016
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
MELiSSA Workshop, Lausanne 8th – 9th June 2016
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
MELiSSA Workshop, Lausanne 8th – 9th June 2016
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)
MELiSSA Workshop, Lausanne 8th – 9th June 2016
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
MELiSSA Workshop, Lausanne 8th – 9th June 2016
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
MELiSSA Workshop, Lausanne 8th – 9th June 2016
Implementation in high‐rate anaerobic digesters
MELiSSA Workshop, Lausanne 8th – 9th June 2016
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
MELiSSA Workshop, Lausanne 8th – 9th June 2016
Acknowledgements
MELiSSA Workshop, Lausanne 8th – 9th June 2016
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)
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