experimental access of microbial processes in constructed
TRANSCRIPT
Uwe KappelmeyerDepartment of Environmental Biotechnology - UBTEWaTT Group
Experimental Access of Microbial Processes in Constructed Wetlands Plant-Rhizosphere-Reactors
Scaling Issue in Wetland Research
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Microscale Macroscale
Scaling Issue in Wetland Research
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Microscale Macroscale
Aerobic conditionsrhizospheric effects
Anaerobic conditionsS2- + metals (e.g. FeS)
black precipitation
EWaTT - Wetland Infrastructure
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Planted fixed bed reactor (PFR) –a constructed wetland model
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Constructed wetlandon lab-scale (10 L)
Continous monitoring ofprocess parameters
- pH- Oxygen- Redox potential
Continous inflow anddegradationof toluene for 6 years
Toluene concentration measured in the in-and outflow
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27/4 7/5 17/5 27/5 6/6 16/6 26/6 6/7 16/7 26/7 5/8
cTolulene [mg/L]
PFR-1 inflow
PFR-1 outflow
PFR-2 inflow
PFR-2 outflow
2011
c Tol
ulen
e[m
g/L]
date
date
09/01/16 10/01/16 11/01/16 12/01/16 01/01/17
c Tolu
lene
[mg/
L]
0
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60PFR-1 outflowPFR-1 inflow
Toluene Concentration during Peak Experiment
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• 8 hours gas samples collection on Tenax TA
• Thermo-desorption GC/MS system
• Toluene emission rate fluctuated between 0.1-0.6 mg/h
• 5.1 mg/h Toluene inflow rate
Bacterial community composition of PFRToluene Experiment
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0 20 40 60 80 100
Acidobacteria ActinobacteriaBacteroidetes ChlamydiaeFirmicutes ProteobacteriaTM7 VerrucomicrobiaOther Phyla Unclassified Bacteria
0 20 40 60 80 100
PFR-1
PFR-2
Bradyrhizobiaceae Burkholderiaceae ChitinophagaceaeClostridiaceae 1 Comamonadaceae HolophagaceaeRhodocyclaceae Rhodospirillaceae SphingobacteriaceaeSporichthyaceae Xanthomonadaceae Other FamiliesUnclassified Bacteria
Martínez-Lavanchy et al. 2015. Appl. Environ. Microbiol. 81:6241-6252.
Degradation pathways for toluene
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Functional Gens in PFRBSSA
RHDO
RHMO
CYP153
ALKB
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5
ALKB_YP_691842.1 Alcanivorax borkumensis SK2
ALKB_AAB70825.1 xylene monooxygenase Pseudomonas putida
ALKB_YP_790621.1 Pseudomonas aeruginosa UCBPP-PA14
ALKB_CAH56098.1 putative alkane hydroxylase uncultured bacterium
ALKB_ABB96072.1 putative alkane monooxygenase uncultured bacterium
ALKB_ABB96087.1 putative alkane monooxygenase uncultured bacterium
ALKB_ABB96096.1 putative alkane monooxygenase uncultured bacterium
ALKB_NP_771234.1 delta 9 acyl-lipid fatty acid desaturase B. japonicum USDA 110
ALKB_YP_552229.1 alkane 1-monooxygenase Polaromonas sp. JS666
ALKB_YP_921354.1 alkane 1-monooxygenase Nocardioides sp. JS614
ALKB_NP_251264.1 alkane-1-monooxygenase Pseudomonas aeruginosa PAO1
CYP153_BAE47487.1 cytochrome P450 alkane hydroxylase uncultured bacterium
RHMO_YP_001110001.1 methane/phenol/toluene hydroxylase Burkholderia vietnamiensis G4
RHMO_AF349675_4 TomA3 Burkholderia cepacia
RHDO_NP_251202.1 anthranilate dioxygenase Pseudomonas aeruginosa PAO1
RHDO_BAB21463.1 chlorobenzoate 1,2-dioxygenase Burkholderia sp. NK8
RHDO_YP_587013.1 benzoate 1,2-dioxygenase Ralstonia metallidurans CH34
RHDO_NP_887278.1 dioxygenase system Bordetella bronchiseptica RB50
BSSA_BAD42366.1 putative benzylsuccinate synthase Magnetospirillum sp. TS-6
Normilzed IntensityPage 10
Catabolic activity according to oxygen and redox fluctuations ?
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Activity of different microbes or different metabolism?
https://femkedegrijs.com/wintertijd-problemen-oplossen/
Isotope Fractionation ∆δ13C
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Isotope fractionation
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Time [h]0 10 20 30 40 50 60 70 80 90
Toluene [mg/L]
0
10
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6013C-toluene
Time [h]0 10 20 30 40 50
Tolu
ene
[mg/
L]
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Num
ber o
f lab
elle
d pe
ptid
es
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13C-toluene Labelled peptides
Lünsmann V et al., Environ Microbiol. 2016;18(4):1176-86
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0 10 20 30 40 50 60 70Time (h)
PFR-1
PFR-2
PFR-1 model
PFR-2 model
Period 1 Period 2
13C - Labeling Isotope Fractionation
Workflow Protein-SIP
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tryptic in-gel digest
Desalting
LC-MS
Time [h]
0 10 20 30 40 50 60
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SIP Analysis
Protein extraction
Pathways and keyplayers
Method
Illumina EFTU Clone Library Metaproteomics
Relative abundance
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Betaproteobacteria Alphaproteobacteria Unclassified Acidobacteria Gammaproteobacteria Sphingobacteria Actinobacteria Acidobacteria Deltaproteobacteria Others Unclassified
Community structure
Metaproteomics
Herbst et al. 2016. Proteomics. 16:783-798. Jehmlich et al. 2016. Curr Opin Biotechnol. 41:26-33.
The toluene degrading bacteria in the PFR show a diurnal regulation of PHA metabolic activity
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Lünsmann V et al., Environ Microbiol. 2016;18(4):1176-86
Day/Night Cycle
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day night
o Within the PFR, the oxygen concentration varied between 0 and 1 mg/l,but never reached saturation (8 mg/l).
o Toluene was exclusively degraded via one aerobic pathway.
o Diurnal redox shifts enable separated carbon storage and energymetabolism
o Thus, a general principle for rhizospheres could be described thatoffers process management and bioaugmentation options for all kindsof organic compounds treated in rhizosphere processes.
General Observation
Martínez-Lavanchy et al. 2015. Appl. Environ. Microbiol. 81:6241-6252.Lünsmann et al. 2016. Environ. Microbiol. 18:1176-1186.Lünsmann et al. 2016. Appl Environ Microbiol. 82:4126-4132.Meyer-Cifuentes et al. 2017. PLoS One12:e0174750.
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Acknowledgment
Paula Martínez-Lavanchy, Vanessa LünsmannIngrid Meyer-CifuentesHermann Heipieper, Jochen A. Müller, Nico Jehmlich