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Prerequisites for reliable VFA quantification from anaerobic digestion systems
Wagner A.O., Markt R., Puempel T., Illmer P., Insam H., Ebner, C.
3rd International Conference on Monitoring & Process Control of Anaerobic Digestion Plants (CMP)
Leipzig, March 2017 1/22
Why Anaerobic digestion: • Locally available resources (regional) • Energy generation from bio-“wastes” (closed cycles) • Digestion of non-fossile resources (renewable) • Energy can be conserved in a chemical form:
• Storage • Availability • Mobility
Introduction
2/22
VFA – volatile fatty acids: • six or fewer C-atoms: C1 – C6
• distillable under atmospheric pressure
Volatile fatty acids
Formic acid – C1
Valeric acid – C5
Acetic acid - C2
Propionic acid – C3
Butyric acid – C4
According to APHO, 1992 Capronic acid – C6 4/22
Recovery/Quantification of VFA
Separation of solid and liquid phase: • Filtration: gravity, vacuum • Centrifugation • Dialysis
Extraction: • using an organic solvent • using an acid: eg. formic acid (10 – 30%)*
Qualification and Quantification: • Titration • TLC (DC), IC • Electrophoresis • GC • HPLC
Sample characteristics!!!
*patented?? 5/22
Aims and setup
Evaluation1 of: 1. separation techniques on VFA recovery 2. decomposition of VFA under original reactor temperature
conditions and at +4 °C 3. preservation and precipitation/coagulation agents on VFA
recovery 4. sample storage at +4 and -20 °C
6/22 1: published in: Engineering in Life Science 2017, 17, 132-139. DOI: 10.1002/elsc.201600095
Material and Methods
spike
sludge sludge + VFAs
Addition of Phenoxy acetic acid as inert tracer
1. Separation techniques 2. Sampling (4 vs 38 °C) 3. Preservation 4. Storage
Sludge: from full-scale WWTP Zirl, Austria (mesophilic)
Spiking:
7/22
Material and Methods
Detection : • HPLC: Phenomenex Fast Fruit Column • Detection: UV@220 nm • Using an external standard (Sigma, Germany):
C1 – C7; 5 mM each
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0 42.5 45.0 47.5 50.0 52.5 55.0 57.5 min-2.5
0.0
2.5
5.0
7.5
10.0
12.5
15.0
17.5
20.0
22.5
25.0
mV
0
50
100
150
200
250
300
350
bar
A.Press.(Status)Detector A:220nm
/8.1
12
Fo/1
4.79
4
Ac/
16.0
38
Pr/1
7.63
7
i-Bu/
18.6
95B
u/19
.332
i-Val
/20.
511
Val
/21.
995
/22.
920
/24.
099
/25.
660
/32.
809
8/22
Material and Methods
1. Separation: • Gravity filtration (MN 615) • Centrifugation: 15 min@15 000 x g • Dialysis tube: Visking #44114 - 24h on ice • Vacuum filtration: Rapid-Flow (Thermo)
9/22
Results – 1. Extraction/Separation
fluted filters centrifugation dialysis bottle topsample preparation
6
8
10
12
14
16
18
20
tota
l VFA
[mM
]
Mean Mean±2*SD Mean±0,95 Conf. Interval
a
a
a
b
• Total spike: 16.5 mmol kg-1
• Maximum recovery with centrifugation yielding 94.9% of theoretically added sum
• Filtration methods work surprisingly good
• High losses by application of dialysis
Σ 16.5 mmol kg-1
10/22
Results – 1. Extraction/Separation
fluted filters centrifugation dialysis bottle topsample preparation
-1
0
1
2
3
4
5
6
mm
ol k
g-1
Mean; Box: Mean±2*SD; Whisker: Mean±0,95 Conf. Interval
formate butyrate
a
b
AA
B
A
c
b
• Dialysis showed a poor recovery of butyrate and failed to recover formate
• Nondiffusible volume fraction of larger solids particles and/or nondiffusible microbial biomass
11/22
Material and Methods
2. Decomposition: • Spiking of sludge with 5.0 mmol kg-1 Fo – Bu • Sampling in 30 min intervals for 2.5 hours • Incubation of sludge at 4 °C and 38 °C, respectively
spiking
sludge sludge + VFAs
+ 4° C
+38 °C
immediate cooling!!!
keep reactor temperature 12/22
time [min]
0 20 40 60 80 100 120 140 160
VFA
[mm
ol k
g-1]
-1
0
1
2
3
4
5
6
formateacetatepropionatebutyrate
time [min]
0 20 40 60 80 100 120 140 160
formateacetatepropionatebutyrate
Results – 2. Decomposition
4 °C 38 °C
13/22
Results – 2. Decomposition
38 °C 4 °C
Min Formate Acetate Propionate Butyrate Formate Acetate Propionate Butyrate
30 2.7 97.0 99.7 92.5 94.5 98.9 98.9 97.8
60 0.2 95.2 98.0 82.5 91.3 99.1 98.9 97.4
150 0.4 91.2 88.7 66.9 75.1 99.9 91.8 93.4
Loss [mmol kg-1 h-1]* ** 0.172 0.118 0.626 0.372 0.114 0.123
*: for linear regression with R2 > 0.5. **: completely lost
Recovery [%] from initial concentrations of spiked VFA and loss of VFA h-1 at 4 °C and 38 °C, respectively.
14/22
Material and Methods
3. Preservation: • Deep freezing (-20 °C)
• Aliquoted 1 g samples
• Chemical: • ZnCl2 [7.5 mM] • Cu: CuCl2-3Cu(OH)2 [50 mM], CuSO4 [1 mM],
CuCl2 [50 mM], commercial kit [??]
spiking aliquots
• Addition of preservation chemicals
• Deep freezing
5 days storage at room
temperature
15/22
Results – 3. Preservation
time [days]
0 1 2 3 4 5 6
formateacetatepropionatebutyratephenoxy-acetic acid
time [days]
0 1 2 3 4 5 6
VFA
[mm
ol k
g-1]
0
1
2
3
4
5
formate CuCl2acetate CuCl2propionate CuCl2butyrate CuCl2formate kitacetate kitpropionate kitbutyrate kitPhenoxy-acetic acid CuCl2Phenoxy-acetic acid kit
CuCl2
comm. kit
Cu2(OH)3Cl
16/22
Results – 3. Preservation
day Cu2(OH)3Cl Commercial kit CuCl2 -20° C
1 101 60.8 95.1 nd
3 102 7.51 95.5 nd
5 69.7 nd 95.8 92.7
Recovery [%] of spiked VFAs.
• ZnCl2 and CuSO4
inappropriate in the applied concentrations (reducing the extractable VFA concentration at t=0)
• Commercial kit failed to preserve the sample
• Cu2(OH)3Cl: onset of microbial acitivity after 3 days
• CuCl2: >95% recovery after 5 days, but loss of
approx. 5% within 24 h (lag phase Cu toxicity?) • Deep freezing: >92% recovery for all spiked VFA,
approx. 98% recovery for Ac, Pr, Bu
17/22
15 min @ 15 000 g
Storage at
Material and Methods
4. Sample storage: • Spiking of sludge: cooling (4° C) • Extraction by centrifugation 15 min@15 000 x g • Filtration (0.2 µm RC) into HPLC glass vials • Storage of extracted samples at +4 °C and -20 °C
spiking
+ 4 °C
-20 °C Filtered
(0.2 µm RC) 18/22
time [days]
0 5 10 15 20 25 30 35
VFA
[mm
ol k
g-1]
0
2
4
6
8formate acetate propionate butyrate phenoxyacetic acid
time [days]
0 5 10 15 20 25 30 35
formateacetate propionate butyrate phenoxyacetic acid
Results – 4. Storage
-20 °C +4 °C
Storage of extracted, filtered samples at -20 °C and +4 °C
19/22
Conclusions and future prospects
Current projects and further prospects:
• Biological pre-treatment strategies • Inhibition of AD processes
Conclusions: 1. “The simpler the better” centrifugation for “extraction” 2. “The faster the better” immediate cooling after sampling 3. “Chemistry can help” CuCl2 or deep freezing at -20 °C 4. “The cooler the better” storage of extr. samples at -20 °C
… many thanks to …
Julian Sybille
Gerlinde
Kati
Chris
Sieglinde Alex
Nadine
Mira
Michael Vera
Paul Anna Nina
… and the team of Ecophysiology WG