SUPPLEMENTARY DATA

Appendix I. Simplified methodology procedure

Apparatus

- Determination of the specific methanogenic activity

1. 120 or 160 mL serum glass bottle, gas-tight septum and its corresponding screw cap or aluminium

crimp (per sample).

2. Precision balance capable of weighting 310 g with an accuracy of ±0.01 g.

3. 1 mL micropipette.

4. Temperature-controlled incubator set at 35 ± 1 °C.

5. Measurement biogas/methane production device:

- Electronic or liquid manometer

- Liquid displacement system

- Low friction gas syringe

6. Gas chromatograph for biogas production analysis, if required.

- Determination of inoculum total and volatile solids

7. Analytical balance with an accuracy of ±0.001 g.

8. 25 mL crucibles.

9. Drying oven set at 103 ± 2 °C

10. Muffle furnace set at 550 °C.

11. Desiccator with silica gel.

Reagents and materials

1. Deionised water.

2. Sodium acetate stock solution (200 g acetate L-1). Dissolve 69.47 g of sodium acetate anhydrous into

a 0.25 L volumetric flask. Mix well with a magnetic stirrer.

3. Inoculum dilution. Determine the VS concentration of the as-received inoculum and dilute it to reach

a volatile solid concentration of 10 g VS L-1 approximately. It is recommended that anaerobic

1
 granules be diluted with treated process effluent after removing debris using centrifugation (10 min

at 2500 g).

4. Inoculum conditioning. The inoculum should be stored at a 35 °C temperature-control incubator for

20 – 30 hours before starting the experiment.

5. Determine the VS concentration and the inhibitor concentration of the diluted inoculum, since this is

the value to be used for the specific methanogenic activity calculations.

Procedure

1. Add, by volume addition or weighting, the required amount of chemical needed to reach the desired

concentration of inhibitor.

2. Weight 99.0 ± 0.1 g of diluted inoculum (10 g VS L-1) in a serum bottle.

3. Add 1 mL of the sodium acetate stock solution.

4. Swirl and measure the pH of the bottle content.

5. Flush the headspace of the bottle with inert gas (e.g. 99.99% N2, 80/20% N2/CO2, 99.99% He).

6. Seal the bottles with a rubber stopper and fix it with a screw cap or aluminium crimp.

7. Place the serum bottles at the temperature-controlled incubator set at mesophilic conditions.

8. Measure the methane production at approximately 0.5, 1.0 and 1.5 days, after the beginning of the

assay. Although the suggested timing of sample events is applicable to most digester samples,

measurement timing can be shortened (0.25, 0.5 and 1 day) for very active samples (e.g. anaerobic

granules) or extended (1, 2 and 3 days) for poorly active samples (e.g. pond samples after

desludging).

9. Plot the accumulated specific methane production in COD-basis (g COD g-1 VSinoculum) in the course

of time. Remember that 350 mL of methane at standard conditions (0 °C, 1 bar) is 1 gram of COD.

10. Determine the slope and uncertainty of the linear zone.

2
Table I. Details of some methodologies used to quantify compounds inhibition/toxicity

Inhibitor Inoculum conditioning Carbon source & Medium Experimental Ref.

concentration addition time (days)

Pre-treatment co- none 1.5 g L-1 acetate & 0.53 yes 5 (Owen et al., 1979)

products g L-1 propionate

Phenolic compounds degassed with N2 0.56 g L-1 acetate & yes 35 (O’Connor and

0.19 g L-1 propionate Young, 1989)

NH3 none 2 g L-1 HAc no 8.5 (Soto et al., 1991)

Na Inoculum centrifugation, 2 g L-1 HAc, 0.5 g L-1 yes 2.5 – 16.5 (Soto et al., 1993)

removal of supernatant & re- HPr & 0.5 g L-1 HBu or

suspension with medium 2 g L-1 HAc

N-substituted aromatics 3 days exposure to the target 2.5 g COD L-1 of yes 0.25 – 1/3 (Donlon et al.,

compound acetate 1995)

Surfactants, pesticides Filtered (1 mm mesh) & 0.3% yeast extract yes 7 days (Madsen and

& phenolic compounds adjusted to 0.7 g TS L-1 Rasmussen, 1996)

Warfare compounds 24 h incubation in medium & 1 g COD L-1 of acetate no n.d. (Sklyar and

24 h exposure to the target Mosolova, 1999)

compound

Warfare compounds Centrifugation & washed with 1 g COD L-1 of acetate yes n.d. (Sklyar and

medium after 24 h exposure Mosolova, 1999)

to the target compound

Na none 1 g COD L-1 of glucose no 1.5 (Kim et al., 2000)

Heavy metals none 10 mL of fresh whey no n.d. (Zayed and Winter,

solution 2000)

Linear alkylbenzene none 0.5 g L-1 acetate or 0.5 g yes 5 - 10 (Gavala and

sulfonates L-1 propionate Ahring, 2002)

Oleate Washed with tap water, 24 h 1 g COD L-1 of acetate yes 1/3 (Hwu and Lettinga,

incubation in medium & 1997)

overnigh exposure to the

target compound

3
 A

Fig. I Cumulative methane production profiles of the experiments carried out to select the best carbon

source and its dose on DSS inoculum. (A) Experiments carried out with acetic acid: (♦) 1, (●) 2, (■) 3, and

(▲) 4 g Ac L-1. (B) Experiments carried out with sodium acetate: (◊) 1, (○) 2, (□) 3, and (∆) 4 g Ac L-1.

4
Fig. II Cumulative methane production profiles of the experiment carried out with 2 g Ac L-1 and three

undiluted inoculums: (♦) digested sludge from a conventional WWTP, (○) digested sludge from a WWTP

equipped with a thermal hydrolysis process, and (×) digested manure from a piggery lagoon.

5
Fig. III Cumulative methane production profiles of the experiments carried out to select the best inoculum

to substrate ratio on DSS inoculum: (♦) 10.5, (○) 5.2, (□) 3.4, (+) 2.5, (×) 1.7, (▲) 1.2, (∆) 0.9, (●) 0.7, and

(◊) 0.4 g VSinoculum g-1 Ac, respectively.

6
A

7
 C

Fig. IV Cumulative methane production profiles of the experiments carried out to select the simplified

sampling scheme on DSS inoculum. (A) Reference. (B) Addition of 5 g NH4+ L-1. (C) Addition of 10 g

NH4+ L-1. (○) 3 sampling events; (□) 2 sampling events at 1.0 and 1.5 days; (∆) 2 sampling events at 0.5

and 1.5 days; (×) 1 sampling event.