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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

Prerequisites for reliable VFA quantification from ... · Prerequisites for reliable VFA quantification from anaerobic digestion systems Wagner A.O., Markt R., Puempel T., Illmer

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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

Anaerobic digestion – a complex process

3/22 Ruetz, 2016

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