Clausen et al. (2017) Test of aerobic TCE degradation by willows (S. viminalis) and willows inoculated with three root colonizing strains of B. cepacia
Supporting information to: Clausen et al. (2017) Test of aerobic TCE degradation by willows (Salix viminalis) and willows inoculated with three TCE-cometabolizing strains of Burkholderia cepacia
The preliminary experiment
A preliminary study was conducted for consistency and to get experience with the test setup before running the final test. The preliminary test was conducted as described for the final test but with smaller willows (shoot size of approx. 20 cm for the preliminary study vs. 30 cm for the final test) and at slightly lower temperature (18 ± 3 oC). The initial TCE concentration was 40 mg/L in solution and TCE was not refilled. The absolute transpiration (weight loss), TCE in solution and chloride in solution for the four treatment groups; Control with Cl (ISO 8692 nutrient solution – with Cl, no TCE), Control no Cl (Cl free ISO 8692 nutrient solution, no TCE), 40 mg TCE/L (Cl free ISO 8692 nutrient solution, initial conc. 40 mg TCE/L) and a group of dead willow sticks (Cl free ISO 8692 nutrient solution, initial conc. 40 mg TCE/L), were measured every 2-3 days. Chloride in the plant tissue at test termination was not measured due to incomplete extraction method development. Average transpiration of the treatment groups is presented in SI Fig. 1. It is observed that the transpiration of the trees is lower than in the final experiment, which is explained by the smaller willows and lower temperature. The general picture is very consistent and supports the results obtained in the final study.
SI Fig. 1 Absolute transpiration of willows from the preliminary study. “Control with Cl” is the control group grown in ISO 8962 nutrient solution, no TCE, n=3. “Control no Cl” is the control group grown in Cl free ISO 8962 nutrient solution, n=4. “Dead” is a test group with dead trees initially exposed to 40 mg TCE/L in Cl free ISO 8962 nutrient solution, n=4. “40 mg TCE/L” is the test group grown in 40 mg TCE/L in Cl free ISO 8962 nutrient solution, n=4. Error bars indicate standard deviation.
The behavior of TCE in solution can be observed, in SI Fig. 2, for the two TCE exposed treatments (40 mg TCE/L and Dead). TCE is rapidly lost from solution both in flasks with and without living trees. TCE is almost completely lost after two weeks.
SI Fig. 2 Measured concentrations of TCE in solution (mg TCE/L) in the preliminary experiment for the treatment groups; 40 mg/L (Initial TCE concentration) and dead (n=4).
Chloride in solution was never detected above the limit of quantification, LOQ = 0.16 mg Cl/L, SI Fig. 3.
SI Fig. 3 Measured chloride concentration in solution (mg Cl/L) for the preliminary study. a) the control group grown without chloride (n=4), b) the exposed trees (initial TCE concentration of 40 mg/L) grown in chloride free nutrient solution (n=4) and, c) the dead willow sticks grown in chloride free nutrient solution with initially 40 mg TCE/L (n=4). Cl in solution of control with Cl was not measured for the preliminary test.
Transpiration of the microbial study
The average transpiration of the treatment groups of the microbial tests are presented in SI Fig. 4. The control no bac. perform markedly better than the other test groups at test start (P < 0.05, two-tailed t-test) but after approximately one week all test groups performed equally, transpiration of approximately 10 g/d, with no statistically significant difference (P > 0.05, two-tailed t-test).
SI Fig. 4 Absolute transpiration of willows from the bacterial study. “Control no bac.” is the control group grown in Cl free ISO 8962 nutrient solution, initial TCE concentration 25 mg/L, n=6. “301C” is the test trees inoculated with B. cepacia strain 301C grown in Cl free ISO 8962 nutrient solution, initial TCE concentration 25 mg/L, n=6. “PR1-31” is a test group inoculated with B. cepacia strain PR1-31 grown in Cl free ISO 8962 nutrient solution, initial TCE concentration 25 mg/L, n=6. “pTOM” is the test group inoculated with B. cepacia strain pTOM grown in Cl free ISO 8962 nutrient solution, initial TCE concentration 25 mg/L, n=6. “Control no Cl” is the control group grown in Cl free ISO 8962 nutrient solution n=4 with no TCE. Error bars indicate standard deviation.
The removal capacity of the bacterial strains was tested in a batch test set-up. Ten mL bacterial suspension containing 8 mg/L of B. cepacia protein (one of the three strains), 400 mg toluene/L and 5 mg TCE/L were added to a 20 mL GC-MS vial flushed with O2 and capped. Destructive sampling was done day 0, 2, 4 and 50 and samples were analyzed by head space GC-MS and ion-chromatography. Three replicates were done for each sampling day. Measured concentrations of TCE in solution for the three B. cepacia strains and the control (no microbes added) are presented in SI Fig. 5. The result was surprising as co-metabolic degradation of TCE, under described conditions was in-significant. The day 50 samples (not presented) showed no further degradation, which was also confirmed by the chloride and toluene measurements (not presented).
SI Fig. 5 Measured concentrations of TCE in solution (mg/L) for the test of microbial TCE degradation potential. Error bars represent the 95% confidence intervals.
Plant weight
The weights of the individual trees were measured weekly. The results are presented in SI Table 1 and confirm that trees grown in chloride free ISO 8692 nutrient solution suffer and lose weight.
SI Table 1 Tree weight (g) at test start, after 1 and 2 weeks of growth and at test termination.Treatment / Rep. No. / Day 0 / Day 7 / Day 14 / Day 21 / Day 23
Control with Cl / 1 / 38.1 / 41.3 / 42.4 / - / 42.6
2 / 37.8 / 39.8 / 40.9 / - / 41.5
3 / 62.6 / 65.5 / 66.8 / - / 68.8
4 / 49.7 / 53.0 / 54.2 / - / 54.8
5 / 41.4 / 43.9 / 44.3 / - / 44.7
Control no Cl / 1 / 40.2 / 38.9 / 38.8 / - / 38.2
2 / 42.4 / 42.0 / 41.7 / - / 40.0
3 / 42.7 / 41.5 / 40.8 / - / 39.5
4 / 33.0 / 33.1 / 33.1 / - / 33.3
5 / 46.5 / 47.3 / 47.2 / - / 45.1
Treatment 1 / 1 / 42.6 / 41.4 / 45.3 / - / 41.5
2 / 50.6 / 50.3 / 49.8 / - / 47.9
3 / 53.3 / 51.9 / 52.0 / - / 50.9
4 / 38.0 / 38.6 / 39.1 / - / 38
5 / 44.3 / 43.7 / 44.0 / - / 43.5
Treatment 2 / 1 / 63.3 / 62.6 / 62.2 / - / 61.3
2 / 29.0 / 27.9 / 28.3 / - / 28.7
3 / 35.4 / 33.9 / 34.2 / - / 34
4 / 39.9 / 40.1 / 40.6 / - / 40.1
5 / 29.0 / 29 / 29.1 / - / 28.6
Dead / 1 / - / - / - / - / 34.7
2 / - / - / - / - / 41.4
3 / - / - / - / - / 29.5
4 / - / - / - / - / 28.7
Control no bac. / 1 / 45.4 / 45.9 / 45.5 / 44.7 / -
2 / 43.5 / 43 / 42.6 / 41.8 / -
3 / 43 / 43.1 / 42.3 / 41.1 / -
4 / 39.7 / 39.8 / 39 / 37.1 / -
5 / 39.3 / 39.5 / 39 / 37.1 / -
6 / 31.3 / 31.7 / 31.6 / 29.8 / -
301C / 1 / 34.2 / 33.5 / 33.5 / 33.4 / -
2 / 41.9 / 41.4 / 41.4 / 41 / -
3 / 35.1 / 34.1 / 33.9 / 33.7 / -
4 / 58.6 / 58.9 / 58.5 / 56.4 / -
5 / 30.2 / 29.8 / 29.6 / 29.3 / -
6 / 37.6 / 38 / 37.4 / 37.2 / -
PR1-31 / 1 / 34.4 / 34.1 / 34 / 33.8 / -
2 / 39 / 38.8 / 37.7 / 36.4 / -
3 / 47.7 / 47.8 / 47 / 46 / -
4 / 26.8 / 26.5 / 26 / 25.4 / -
5 / 40.1 / 39.8 / 28.8 / 37.8 / -
6 / 40.3 / 40.2 / 39.1 / 37.7 / -
pTOM / 1 / 37.2 / 36.7 / 36 / 34.4 / -
2 / 33.8 / 33.8 / 33.2 / 32.4 / -
3 / 47.9 / 48 / 47.2 / 45.7 / -
4 / 34.3 / 34.5 / 33.6 / 33.1 / -
5 / 37.3 / 37.3 / 35.9 / 34.7 / -
6 / 26.7 / 26.5 / 26.2 / 25.5 / -
Chloride mass in plants
The average chloride mass measured in the plant tissue (roots, leaves and stems) for the final experiment is presented in SI Table 2. It is observed that the willows contain approx. 2 mg of chloride. Elevated level of chloride is only observed for control with Cl.
SI Table 2 Average Cl mass in plant roots, leaves and stems (mg) for the final experiment. 95% C.I. are given in brackets.Roots / Leaves / Stems
Control no Cl / 0.0 (±0.0) / 1.1 (±0.8) / 0.7 (±0.2)
Control with Cl / 0.4 (±0.1) / 2.7 (±1.1) / 1.4 (±0.3)
Treatment 1 / 0.0 (±0.0) / 0.8 (±0.4) / 1.1 (±0.7)
Treatment 2 / 0.0 (±0.0) / 0.6 (±0.1) / 0.7 (±0.3)
Dead / - / - / 0.8 (±0.2)
Control no bac. / 0.0 (±0.0) / 0.6 (±0.1) / 1.9 (±1.0)
301C / 0.0 (±0.0) / 0.5 (±0.2) / 1.1 (±0.7)
PR1-31 / 0.0 (±0.0) / 0.4 (±0.1) / 1.4 (±1.1)
pTOM / 0.0 (±0.0) / 0.4 (±0.1) / 2.0 (±2.8)
TCE metabolites in plant tissue
The plants of the microbial study were sent for analysis of TCE metabolites DCAA, TCEtOH and TCAA. The results are presented in SI Table 3 and 4 for leaf and stem analysis, respectively. Only traces of the metabolites are observed in the plants, with DCAA being the most common. Traces of DCAA were even measured in control tree not exposed to TCE. TCEtOH was not observed in the leaves.
SI Table 3. Analytical results of TCE metabolites in plant leaves (µg/g fresh weight) at test termination. LOD is given in brackets.
Control no bac. 1 / 0.14 (0.03) / <0.13 (0.13) / 0.26 (0.03)
Control no bac. 2 / 0.25 (0.02) / <0.10 (0.10) / <0.02 (0.02)
Control no bac. 3 / 0.28 (0.02) / <0.10 (0.10) / 0.19 (0.02)
Control no bac. 4 / 0.22 (0.02) / <0.08 (0.08) / 0.12 (0.02)
Control no bac. 5 / 0.29 (0.02) / <0.10 (0.10) / 0.18 (0.02)
301C. 1 / 0.27 (0.03) / <0.13 (0.13) / 0.21 (0.03)
301C. 2 / 0.31 (0.02) / <2.00 (2.00) / <0.02 (0.02)
301C. 3 / 0.30 (0.02) / <0.10 (0.10) / <0.02 (0.02)
301C. 4 / 0.27 (0.03) / <2.50 (2.50) / 0.25 (0.03)
301C. 5 / 0.32 (0.03) / <0.13 (0.13) / 0.20 (0.03)
PR1-31. 1 / 0.20 (0.03) / <0.13 (0.13) / 0.16 (0.03)
PR1-31. 2 / 0.18 (0.03) / <0.13 (0.13) / 0.23 (0.03)
PR1-31. 3 / 0.31 (0.03) / <0.13 (0.13) / 0.54 (0.03)
PR1-31. 4 / 0.34 (0.03) / <2.50 (2.50) / <0.03 (0.03)
PR1-31. 5 / 0.27 (0.02) / <1.67 (1.67) / <0.02 (0.02)
pTOM. 1 / 0.07 (0.02) / <2.00 (2.00) / <0.02 (0.02)
pTOM. 2 / 0.32 (0.02) / <2.00 (2.00) / <0.02 (0.02)
pTOM. 3 / 0.50 (0.03) / <2.50 (2.50) / <0.03 (0.03)
pTOM. 4 / 0.21 (0.02) / <0.10 (0.10) / 0.20 (0.02)
pTOM. 5 / 0.33 (0.02) / <2.00 (2.00) / <0.02 (0.02)
Control no Cl. 1 / 0.08 (0.02) / <0.10 (0.10) / <0.02 (0.02)
Control no Cl. 2 / 0.20 (0.02) / <0.10 (0.10) / <0.02 (0.02)
Control no Cl. 3 / 0.13 (0.02) / <0.08 (0.08) / 0.05 (0.02)
SI Table 4. Analytical results of TCE metabolites in plant stems (µg/g fresh weight) at test termination. LOD is given in brackets.
Group and rep. no. / DCAA / TCEtOH / TCAAControl no bac. 1 / 0.05 (0.01) / 0.08 (0.03) / 0.01 (0.01)
Control no bac. 2 / 0.05 (0.01) / 0.09 (0.03) / 0.01 (0.01)
Control no bac. 3 / 0.06 (0.01) / 0.13 (0.04) / <0.01 (0.01)
Control no bac. 4 / 0.06 (0.01) / 0.13 (0.05) / 0.02 (0.01)
Control no bac. 5 / 0.08 (0.01) / 0.14 (0.05) / 0.02 (0.01)
301C. 1 / 0.11 (0.01) / <0.04 (0.04) / <0.01 (0.01)
301C. 2 / 0.06 (0.01) / <0.04 (0.04) / <0.01 (0.01)
301C. 3 / 0.07 (0.01) / <0.56 (0.56) / <0.01 (0.01)
301C. 4 / 0.09 (0.01) / 0.17 (0.05) / 0.01 (0.01)
301C. 5 / 0.13 (0.01) / <0.83 (0.83) / 0.01 (0.01)
PR1-31. 1 / 0.08 (0.01) / <0.04 (0.04) / <0.01 (0.01)
PR1-31. 2 / 0.06 (0.01) / 0.06 (0.03) / 0.02 (0.01)
PR1-31. 3 / 0.11 (0.01) / 0.07 (0.05) / 0.03 (0.01)
PR1-31. 4 / 0.14 (0.01) / <1.11 (1.11) / <0.01 (0.01)
PR1-31. 5 / 0.13 (0.01) / <0.77 (0.77) / <0.01 (0.01)
pTOM. 1 / 0.13 (0.01) / <1.00 (1.00) / <0.01 (0.01)
pTOM. 2 / 0.09 (0.01) / <0.83 (0.83) / <0.01 (0.01)
pTOM. 3 / 0.12 (0.01) / <1.00 (1.00) / <0.01 (0.01)
pTOM. 4 / 0.10 (0.01) / <0.05 (0.05) / <0.01 (0.01)
pTOM. 5 / 0.08 (0.01) / <0.04 (0.04) / <0.01 (0.01)
Control no Cl. 1 / 0.07 (0.01) / <0.77 (0.77) / <0.01 (0.01)
Control no Cl. 2 / 0.06 (0.01) / <0.04 (0.04) / <0.01 (0.01)
Control no Cl. 3 / 0.09 (0.01) / 0.20 (0.04) / <0.01 (0.01)
The amount of TCAA, DCAA and TCEtOH detected in the plants correspond, on molar basis, to approximately 0.3-0.7‰ of the TCE added to the test systems, SI Table 5.
SI Table 5. Promille (‰, molar) of added TCE measured as metabolites in plant tissue at test termination, with corresponding 95% CI for each test group.Group / (‰) / 95% C.I.
Control no bacteria / 0.28 / 0.05
301C / 0.46 / 0.19
PR1-31 / 0.48 / 0.25
pTOM / 0.70 / 0.38
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