Table S1. Sulphur balance over the BCRs and estimates of the masses of FeS formed within the BCRs over their period of operation.

Transcription

1 Water 16, 8, 14 S1 of S7 Supplementary Material: Decline in Performance of Biochemical Reactors for Sulphate Removal from Mine-Influenced Water is Accompanied by Changes in Organic Matter Characteristics and Microbial Population Composition Parissa Mirjafari and Susan A. Baldwin Table S1. Sulphur balance over the BCRs and estimates of the masses of FeS formed within the BCRs over their period of operation. Column Amount of Sulphate Entering Amount of Sulphate Leaving Amount of Sulphate Removed Amount of S Formed as Soluble Sulphide Estimated FeS Formed (g) BCR_W BCR_W BCR_W BCR_H BCR_H BCR_H BCR_W1 BCR_H1 BCR_H BCR_W BCR_W3 BCR_H3 P P Port number P4 P6 Port number P4 P6 P8 P Sulphate Concentration (mg/l) Sulphate Concentration (mg/l) Figure S1. Sulphate concentrations measured in the pore water from Ports 8, 6, 4 and for the early; and late sacrificed bioreactors. Port 8 was located cm above the influent and Port was the effluent leaving the bioreactor matrix. BCR_W indicates a biochemical reactor containing the rich mixture and BCR_H a biochemical reactor containing the -rich mixture.

2 Water 16, 8, 14 S of S7 Percent dry weight Neutral detergent soluble Neutral detergent fibre Ash t= Percent dry weight Neutral detergent soluble Neutral detergent fibre Ash t= Figure S. Composition of the solid matrices in the -rich; and -rich bioreactors. NDF/NDS stands for the ratio of neutral detergent fibre to neutral detergent soluble fractions. Error bars represent standard deviations of three replicate sub-samples and three technical replicates for each section of organic material. X-axis labels refer to the sections analyzed: P8 is the section from the influent to Port 8, P78 is the section from Port 8 to Port 7 and so on Fraction of NDF Other Lignin Hemicellulose Cellulose..1 t= Figure S3. Cont.

3 Water 16, 8, 14 S3 of S Fraction of NDF Other Lignin Hemicellulose Cellulose..1 t= Figure S3. Composition of the neutral detergent fibre fraction in the -rich; and -rich bioreactors. Other refers to components of the NDF not accounted for as cellulose, hemicellulose or lignin. Error bars represent standard deviations of three replicate sub-samples and three technical replicates for each section of organic material. X-axis labels refer to the sections analyzed: P8 is the section from the influent to Port 8, P78 is the section from Port 8 to Port 7 and so on. Figure S4. Shannon diversity index; and diversity index based on phylogeny as a function of sequencing depth as determined using rarefaction of the acquired reads. Curves are summarized according to time of sampling with orange representing the time zero samples, blue the early sacrificed samples and red the late sacrificed samples. Overlapping error bars suggest that diversity does not change over time.

4 Water 16, 8, 14 S4 of S7 Figure S5. Shannon diversity index; and diversity index based on phylogeny as a function of sequencing depth as determined using rarefaction of the acquired reads. Curves are summarized according to content with blue representing the inocula, orange the -rich samples and red the -rich samples. Overlapping error bars suggest that diversity is not different between the samples according to their matrix. Figure S6. Principle co-ordinate plot of UniFrac distance matrix comparison of microbial communities in the cultures (CDB, SRB and manure), inocula and samples taken at time zero and from the earlyand late-sacrificed BCRs. The un-weighted UniFrac matrix was used. Colors depict the type of sample, and size of bubble depicts the time at which the sample was taken.

5 Water 16, 8, 14 S5 of S7.rich3.t.zero. Inoc..rich3.rich.t.zero..rich1.t.zero Inoc..rich1 Inoc..rich Manure CDB3..rich CDB1..rich CDB..rich inoc..rich inoc..rich3.rich1.t.zero..rich.t.zero.rich3.t.zero SRB3 SRB SRB1.rich.early.P34 CDB1..rich.rich.early.P45.rich.early.P56.rich.early.P67.rich.early.P8.rich1.early.P34.rich1.early.P45.rich1.early.P56.rich1.early.P67.rich3.early.P78.rich3.early.P8.rich3.early.P67.rich3.early.P34.rich3.early.P45.rich3.early.P56.rich1.late.P56.rich1.late.P67.rich1.late.P45.rich1.late.P8.rich1.late.P34.rich.late.P78.rich.late.P8.rich.late.P45.rich.late.P56.rich.late.P67.rich3.late.P56.rich3.late.P67.rich3.late.P45.rich3.late.P78.rich3.late.P8.rich1.early.P8.rich1.early.P78 CDB..rich OTU_19 Pseudomonadaceae OTU_63 Chloroplast OTU_3 Enterobacteriaceae OTU_769 Enterobacteriaceae OTU_6 Enterobacteriaceae OTU_14 Erysipelotrichaceae OTU Lachnospiraceae OTU_3 Enterococcaceae OTU_65 Porphyromonadaceae OTU_6 Erysipelotrichaceae OTU_54 Family_XI_Incertae_Sedis OTU_3 Desulfobulbaceae OTU_46 Desulfovibrionaceae OTU_17 Ruminococcaceae OTU_5 Christensenellaceae OTU_9 Bacteroidaceae OTU_199 Lachnospiraceae OTU_35 Bacteroidaceae OTU_1111 S4.7 OTU_11 Syntrophomonadaceae OTU_44 Ruminococcaceae OTU_16 Ruminococcaceae OTU_4 Lachnospiraceae OTU_5 PL.11B8_wastewater.sludge_group OTU_7 WCHB1.6 OTU Rs.E47_termite_group OTU_13 AUTHM97 OTU_3 Treponema OTU_49 Christensenellaceae OTU_7 Ruminococcaceae OTU_1 Terrestrial_Miscellaneous_Gp.TMEG. OTU_31 RF3 OTU_18 RF3 OTU_4 Methanosarcinaceae OTU_17 Spirochaeta OTU Methanocorpusculaceae OTU_36 Ruminococcaceae OTU_1 Porphyromonadaceae OTU_43 Family_XIII_Incertae_Sedis OTU_33 Methanoregulaceae OTU_15 LNR_A.18 OTU_53 Treponema OTU_1 Methanobacteriaceae OTU_347 Ruminococcaceae sacrifice.time none zero early late matrix manure -rich -rich culture Figure S7. Heatmap of the relative abundances of 9% sequence similarity OTUs in the bioreactors. Samples from the bioreactors are presented as columns and were clustered according to Euclidean distances using hierarchical clustering (hclust in the R programming environment). OTUs are arranged in rows clustered according to Pearson s correlation. Colour intensity represents read counts on a log to the base scale. -rich -rich early late DCA NDF.NDS Lignin.hemi DOC Figure S8. Detrended correspondence analysis (DCA) of the Bray-Curtis dissimilarity comparison of microbial communities in the BCR samples (only BCR samples are shown on this diagram). Samples containing higher concentrations of dissolved organic carbon (indicated in the direction of the blue arrow labeled DOC). DCA1

6 Water 16, 8, 14 S6 of S7 SRB 1_g Desulfosporosinus meridiei 11_g Desulfotomaculum 1_g Desulfurispora 9_g Desulfovibrio mexicanus OTU_Genus_level 8_g Syntrophus 7_g Desulfovibrio mexicanus 6_g Desulfobulbus 5_o GW 8 log(read_count, base = ) _g Desulfovibrio mexicanus 3_g Desulfovibrio aminophilus _g Desulfovibrio mexicanus 1_g Syntrophus SRB Culture Figure S9. Read counts for sulphate-reducing bacteria related OTUs (94% similarity cut-off) found in the SRB cultures used to prepare the BCR inocula.

7 Water 16, 8, 14 S7 of S7 tzero early late 1_g Desulfosporosinus meridiei 11_g Desulfotomaculum 1_g Desulfurispora OTU_Genus_level 9_g Desulfovibrio mexicanus 8_g Syntrophus 7_g Desulfovibrio mexicanus 6_g Desulfobulbus 5_o GW 8 4_g Desulfovibrio mexicanus Matrix log(read_count, base = ) 4 6 3_g Desulfovibrio aminophilus _g Desulfovibrio mexicanus 1_g Syntrophus Matrix Figure S1. Read counts for sulphate-reducing bacteria related OTUs (94% similarity cut-off) found in the SRB cultures used to prepare the BCR inocula.