Supporting Information for
Long-term Simulation of In Situ Biostimulation of Polycyclic

Aromatic Hydrocarbon-Contaminated Soil

Stephen D. Richardson[*], Maiysha D. Jones, David R. Singleton and Michael D. Aitken
Department of Environmental Sciences and Engineering
Gillings School of Global Public Health
University of North Carolina at Chapel Hill

Number of pages: 9

Number of Figures: 6

Number of Tables: 3

Prepared for Biodegradation

Column Design

Figure S1. Schematic of the column system. SS, stainless steel.

PAH Mass Removal in the Control Column

Table S1. Percent PAH mass removed by dissolution, sampling, and biodegradation in the control column after 534 da.

Percent of PAH Mass Removed
Analyte b / Total / Dissolution / Sampling / Biodegradation c
NAP / 35.3 / ± / 47.4 / 25.5 / ± / 11.2 / 12.6 / ± / 5.6 / NA
ACE / 61.1 / ± / 26.9 / 34.1 / ± / 7.7 / 5.2 / ± / 1.2 / 21.8 / ± / 28.0
FLU / 47.2 / ± / 23.6 / 22.8 / ± / 4.7 / 6.9 / ± / 1.5 / 17.5 / ± / 24.1
PHN / 33.5 / ± / 26.0 / 6.5 / ± / 1.6 / 8.5 / ± / 2.1 / 18.6 / ± / 26.2
ANT / 37.5 / ± / 23.9 / 5.0 / ± / 1.1 / 8.6 / ± / 1.9 / 23.9 / ± / 24.0
FLA / 31.5 / ± / 20.9 / 1.6 / ± / 0.3 / 10.4 / ± / 2.1 / 19.6 / ± / 21.0
PYR / 27.2 / ± / 20.3 / 1.2 / ± / 0.2 / 10.6 / ± / 2.1 / 15.4 / ± / 20.5
BaA / 37.7 / ± / 23.9 / d / 11.7 / ± / 2.6 / 26.0 / ± / 24.0
CHR / 45.3 / ± / 28.3 / d / 13.9 / ± / 3.6 / 31.4 / ± / 28.6
BbF / 27.3 / ± / 26.5 / d / 14.5 / ± / 3.8 / 12.8 / ± / 26.8
BkF / 27.0 / ± / 28.7 / d / 14.2 / ± / 4.0 / 12.8 / ± / 28.9
BaP / 28.8 / ± / 32.8 / d / 14.6 / ± / 4.6 / 14.2 / ± / 33.1
BgP / 13.0 / ± / 31.1 / d / 19.7 / ± / 6.0 / NA
Total / 34.1 / ± / 23.7 / 6.2 / ± / 1.4 / 10.0 / ± / 2.3 / 17.9 / ± / 23.9
3-ring e / 36.5 / ± / 25.8 / 9.4 / ± / 2.2 / 8.2 / ± / 2.0 / 18.9 / ± / 26.0
4-ring e / 32.6 / ± / 21.4 / d / 11.2 / ± / 2.3 / 21.4 / ± / 21.5
5-ring e / 29.1 / ± / 30.5 / d / 14.3 / ± / 4.2 / 14.8 / ± / 30.8

aError associated with the percent removal values was the result of sample variability of the column soil and propagation of error in calculating total mass and percent removal of each PAH in the columns.

b See Table 1 for definitions of abbreviations.

c Calculated by subtracting removal bydissolution and removal by samplingfrom the total removal and accounting for error propagation; NA = not applicable (calculated value was negative).

d Value was below detection.

e Represents the sum of the mass removed for PAHs with the same number of rings (Table 1); NAP and BgP are not included in these values.

Time-Series Graphs of Soil PAH Concentrations

Figure S2. Time courses for PAH concentrations from soil samples collected from surface soil of the biostimulated (●) and control (▲) columns. Each point represents the mean value of duplicate or triplicate samples and the error bars are the range or standard deviation, respectively. Horizontallines represent the mean and standard deviation of the PAH in the column soil (Table 1).

Figure S3. Time courses for PAH concentrations from soil samples collected from Port A of the biostimulated (●) and control (▲) columns. Each point represents the mean value of duplicate or triplicate samples and the error bars are the range or standard deviation, respectively. Horizontallines represent the mean and standard deviation of the PAH in the column soil (Table 1).

Figure S4. Time courses for PAH concentrations from soil samples collected from Port B of the biostimulated (●) and control (▲) columns. Each point represents the mean value of duplicate or triplicate samples and the error bars are the range or standard deviation, respectively. Horizontallines represent the mean and standard deviation of the PAH in the column soil (Table 1).

Figure S5. Time courses for PAH concentrations from soil samples collected from Port C of the biostimulated (●) and control (▲) columns. Each point represents the mean value of duplicate or triplicate samples and the error bars are the range or standard deviation, respectively. Horizontallines represent the mean and standard deviation of the PAH in the column soil (Table 1).The unexpected decrease in PAH concentrations in the control column at t = 380 d was attributed to soil heterogeneities.

Aqueous-Phase PAH Concentrations

Figure S6. Aqueous-phase concentrations of naphthalene (a), acenaphthene (b), fluorene (c), phenanthrene (d), anthracene (e), fluoranthene (f), and pyrene (g) over time in the biostimulated (○) and control (▲) columns. Time 0 (depicted with the vertical dashed line) refers to the time that biostimulation commenced. Data shown before t = 0 were from samples collected during the equilibration phase. Concentrations of BaA, CHR, BbF, BkF, BaP, DBA, and BgP were below detection.

Spearman Rank Analyses

Table S2. Spearman rank coefficients (rs) representing significant correlations between DO concentration (mg/L) and PG2 abundance (16S rRNA genes/g) in the biostimulated and control columns.

Spearman rank coefficients (rs) a
Biostimulated / Control
Surface / Port A / Port B / Port C / Surface / Port A / Port B / Port C
0.70 / 0.49 / 0.88 / 0.78 / -0.12 / -0.20 / NA / NA

a Only rs values representing correlations with p < 0.15 are reported (p <0.05 in bold and underlined; 0.05< p <0.1 in bold; 0.1< p <0.15 in italics).

NA = not applicable; Spearman rank correlations could not be calculated because oxygen was not detected at Ports B and C of the control column.

Table S3. Spearman rank coefficients (rs) representing significant correlations between PAH concentrations (mg/kg) and PG2 abundance (16S rRNA genes/g) in the biostimulated and control columns.

Spearman rank coefficients (rs) with PG2 abundance a
Biostimulated b / Control b
PAH c / Port B / Port C / Port A / Port B / Port C
ACE / -0.77 / -0.83
FLU / -0.83 / -0.77 / -0.71 / -0.94
PHN / -0.83 / -0.83
ANT / -0.94 / -0.71 / -0.94
FLA / -1.00 / -0.83
PYR / -0.94 / -0.71
BaA / -0.94 / -0.94
CHR / -0.94 / -0.77 / -0.94
BbF / -0.83
BkF / -0.71
BaP / -0.81
BgP / -0.90 / 0.71 / -0.89

a Only rs values representing correlations with p < 0.15 are reported (p <0.05 in bold and underlined; 0.05< p <0.1 in bold; 0.1< p <0.15 in italics); rs values with p > 0.15 are omitted for clarity.

b No significant correlations were identified in the surface soil and at Port A of the biostimulated column and the surface soil of the control column.

c Spearman rank coefficients for NAP and DBA were not significant at all sample locations and are, therefore, omitted from the table; see Table 1 for definitions of abbreviations.

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[*] Corresponding author: phone 919-873-1060; e-mail: