Biogas-based denitrification in a biotrickling filter: Influence of nitrate concentration and hydrogen sulfide
Juan C. López,a,b Estefanía Porca,b Gavin Collins,b Rebeca Pérez,a Alberto Rodríguez-Alija,a Raúl Muñoz,a Guillermo Quijano*,a,c
aDepartment of Chemical Engineering and Environmental Technology, University of Valladolid, Dr. Mergelina, s/n, 47011, Valladolid, Spain.
bMicrobial Communities Laboratory, School of Natural Sciences and Ryan Institute, National University of Ireland Galway, University Road, Galway, H91 TK33, Ireland.
cAgrarian Engineering School, University of Valladolid – Soria Campus, C/Universidad s/n, 42004 Soria, Spain.
*Corresponding author: , Tel. +34 975129404, Fax: +34 975129401.
Supporting Information containing 9 pages, with 4 figures and 1 table.
MATERIAL AND METHODS
Fluorescence in situ hybridization (FISH)
Biomass samples (250 uL) were fixed in 4% (w/v) paraformaldehyde for 3 h and then washed three times with phosphate-buffered saline (PBS). Aliquots of 10 µL of biofilm samples were deposited on the wells of gelatin-coated, acid-washed, glass microscope slides and dehydrated by passing through a 50%, 80% and 96% (v/v) ethanol series. Hybridisation with 30% formamide (v/v) and the oligonucleotide probes was at 46ºC for 2 h. After hybridization, and once the slides were washed and dried, the specimens were counter-stained for 5 min at room temperature with the DNA stain DAPI to quantify the total number of cells. For quantitative FISH analysis, 15 images were randomly acquired from inside each well on the slides using a Leica DM4000B microscope (Leica Microsystems, Wetzlar, Germany). The relative biovolumes of total archaea, total bacteria, DAMO bacteria and DAMO archaea from the total DAPI-stained biomass were calculated using DAIME software and split into individual colour channels before image segmentation (Daims et al. 2006).
End-point PCR of Bacterial 16S rRNA, Archaeal 16S rRNA, narG, nirK, nosZ, pmoA and soxB genes
Purified DNA from the samples was used to amplify the target Bacterial 16S rRNA, Archaeal 16S rRNA, narG, nirK, nosZ, pmoA and soxB genes by end-point PCR. The PCR mixture (25 µL) contained 0.5 mL of each primer (10 mM), 5 µL of 5× ready-to-use MyTaq reaction buffer (Bioline, UK), 0.5 µL of MyTaq DNA Polymerase, 1 µL purified DNA and PCR H2O up to a final volume of 25 µL. PCR was performed in a Super Cycler Trinity (Kyratec, Australia) following thermo-cycling programs for Bacterial and Archaeal 16S rRNA according to Yu et al. (2005). End-point PCR amplifications of narG, nirK, and nosZ genes were performed according to Philippot et al. (2002), Braker et al. (2000), and Henry et al. (2006), respectively. Additionally, the detection of the genes pmoA (encodes for the particulate methane monooxygenase; present in aerobic methane-oxidizing bacteria) and soxB (encodes for the thiosulfate-oxidising Sox enzyme complex; present in hydrogen sulfide-oxidizing bacteria) in the purified samples was assessed according to previous studies (Holmes et al. 1995; Thomas et al. 2014). The success of each amplification reaction was determined by electrophoresis in a 2% (w/v) agarose gel in 5× TAE buffer.
Real-time PCR analysis
Table SI. Primers and thermocycling conditions used during qPCR analysis
Target gene / Primer and sequence (5’ – 3’) / Amplicon size (bp) / qPCR thermocycling program / ReferenceBacterial 16S rRNAa / BAC338F: ACTCCTACGGGAGGCAG
BAC805R:GACTACCAGG GTATCTAATCC / 468 / 94°C for 10 min; 45 cycles of: 94ºC for 10 s, 60ºC for 30 s (acquisition step) (±5ºC/s ramp) / Yu et al. (2005)
Archaeal 16S rRNAa / ARC787F: ATTAGATACCCSBGTAGTCC
ARC1059R: GCCATGCACCWCCTCT / 273 / 94°C for 10 min; 45 cycles of: 94ºC for 10 s, 60ºC for 30 s (acquisition step) (±5ºC/s ramp) / Yu et al. (2005)
narG / narG 1960F: TAYGTSGGCCARGARAA
narG 2659R: TTYTCRTACCABGTBGC / 699 / 95°C for 10 min; 40 cycles of: 95ºC for 45 s, 66ºC for 45 s, 72ºC for 78 s, 80ºC for 18 s (acquisition step); 72°C for 5 min / This study
nirK / nirK 1F: GG(A/C)ATGGT(G/T)CC(C/G)TGGCA
nirK 5R: GCCTCGATCAG(A/G)TT(A/G)TGG / 514 / 95°C for 5 min; 45 cycles of: 95ºC for 30 s, 68ºC for 40 s, 72ºC for 30 s, 83ºC for 15 s (acquisition step) / This study
nosZ / nosz F: CGYTGTTCMTCGACAGCCG
nosz 1R: CAKRTGCAKSGCRTGGCAGAA / 699 / 95ºC for 15 min; 6 cycles of: 95ºC for 30 s, 67ºC for 30 s (-1ºC/cycle), 72ºC for 30 s, 85ºC for 15 s (acquisition data step); 40 cycles of: 95ºC for 30 s, 60ºC for 30 s, 72ºC for 30 s / This study
aTaqMan probes were set according to Yu et al. (2005).
RESULTS
Long-term reactor performance
Figure S1. Time course of the N2 production rates during BTF operation.
Figure S2. Influence of nitrate concentration on nitrous oxide production rate during Stage 1 of BTF operation.
Figure S3. Time course of NO2- concentration during BTF operation.
Figure S4. DNA fragments on gel electrophoresis of a) pmoA (510 bp) and b) soxB (1020 bp) in the samples under study (lane 1, HypperLadderTM 50bp; lane 2, sample A; lane 3, sample B; lane 4, sample C; lane 5, negative template control (NTC)). The arrows indicate the migration place of both fragments.
REFERENCES
Braker G, Zhou J, Wu L, Devol AH, Tiedje JM. 2000. Nitrite reductase genes (nirK and nirS) as functional markers to investigate diversity of denitrifying bacteria in pacific northwest marine sediment communities. Appl Environ Microb 66:2096-2104.
Daims H, Lücker S, Wagner M. 2006. Daime, a novel image analysis program for microbial ecology and biofilm research. Environ Microbiol 8:200-213.
Henry S, Bru D, Stres B, Hallet S, Phillipot L. 2006. Quantitative detection of the nosZ gene, encoding nitrous oxide reductase, and comparison of the abundances of 16S rRNA, narG, nirK, and nosZ Genes in Soils. Appl Environ Microb 72:5181-5189.
Holmes AJ, Costello A, Lidstrom ME, Murrell JC. 1995. Evidence that particulate methane monooxygenase and ammonia monooxygenase may be evolutionarily related. FEMS Microbiol Lett 132:203-208.
Philippot L, Piutti S, Martin-Laurent F, Hallet S, Germon JC. 2002. Molecular analysis of the nitrate-reducing community from unplanted and maize-planted soils. Appl Environ Microb 68:6121-6128.
Thomas F, Giblin AE, Cardon ZG, Sievert SM. 2014. Rhizosphere heterogeneity shapes abundance and activity of sulfur-oxidizing bacteria in vegetated salt marsh sediments. Front Microbiol 5:309.
Yu Y, Lee C, Kim J, Hwang S. 2005. Group-specific primer and probe sets to detect methanogenic communities using quantitative real-time polymerase chain reaction. Biotechnol Bioeng 89:670-679.