Supplementary materials:

Effects of aeration and internal recycle flow on nitrous oxide emission

from a Modified Ludzak–Ettinger process fed with glycerol

Kang Song1,2, Toshikazu Suenaga1, Willie F. Harper Jr.3, Tomoyuki Hori4, Shohei Riya1,Masaaki Hosomi1, Akihiko Terada1*

  1. Department of Chemical Engineering, Tokyo University of Agriculture & Technology, Tokyo 184-8588, Japan
  2. Research Fellow of Japan Society for the Promotion of Science, 5-3-1 Kojimachi, Chiyoda-ku, Tokyo 102-0083, Japan
  3. Department of Systems Engineering and Management, Air Force Institute of Technology, Wright-Patterson Air Force Base, OH 45433, United States
  4. National Institute of Advanced Industrial Science and Technology, Tokyo 305-8568, Japan

* Corresponding author:

Tel: +81-42-388-7069 / Fax: +81-42-388-7731

Figure legendsfor supplementary materials

Fig. S1 Time courses of dissolved N2O concentration in theanoxic and aerobic tanks on day 146.

Fig.S2Representative organic carbon and nitrogen concentrations in the MLE under different IRFs conditions. The samples were taken at the final date of the IRF condition (prior to changeover).

Fig. S3Time courses of the quantities of functional genes encoding (a) amoA, (b) nirK, (c) nirS, (d) nosZ clade I, and (e) nosZ clade II.

Fig. S4 Correlation of N2O emission rate from the MLE system versus the relative gene abundance nosZ/(nirK + nirS).

Fig. S5Correlation of N2O emission rate from the MLE system versus relative abundance of the genus Rhodobacter.

Fig. S6Relative abundances of AOB and NOB during the MLE operation.

Fig. S1Time courses of dissolved N2O concentration in theanoxic and aerobic tanks on day146.

Fig.S2Representative organic carbon and nitrogen concentrations in the MLE under different IRF conditions. The samples were taken at the final date of eachIRF condition (prior to changeover).

Fig.S3Time courses of the quantities of functional genes encoding (a) amoA, (b) nirK, (c) nirS, (d) nosZ clade I,and (e) nosZ clade II.

Fig. S4Correlation of N2O emission rate from the MLE system versus therelative gene abundance nosZ/(nirK + nirS).

Fig. S5Correlation of N2O emission rate from the MLE system versusrelative abundance of the genus Rhodobacter.

Fig.S6Relative abundances of AOB and NOB during the MLE operation

Table S1Primers used inreal-time quantitative PCR

Primer / Purpose / Target gene / Sequence (5’-3’) / Reference
amoA1F / qPCR / amoA / GGGGTTTCTACTGGTGGT / (Rotthauwe et al. 1997)
amoA 2R / qPCR / amoA / CCCCTCKGSAAAGCCTTCTTC
nirk 876-F / qPCR / nirK / ATYGGCGGVCAYGGCGA / (Henry et al. 2004)
nirK 1040-R / qPCR / nirK / GCCTCGATCAGRTTRTGGTT
nirS 4QF / qPCR / nirS / GTSAACGYSAAGGARACSGG / (Throback et al. 2004)
nirS 6QR / qPCR / nirS / GASTTCGGRTGSGTCTTSAYGAA
nosZ1F / qPCR / nosZ / WCSYTGTTCMTCGACAGCCAG / (Henry et al. 2006)
nosZ1R / qPCR / nosZ / ATGTCGATCARCTGVKCRTTYTC
nosZ Clade II-F / qPCR / nosZ / CTI GGICCI YTK CAY AC / (Jones et al. 2013)
nosZ Clade II-R / qPCR / nosZ / GCI GARCAR AAI TCB GTR C

Table S2 Diversity indices of microbial community structure

Glycerol / 0 / 98 / 106 / 113 / 131 / 148 / 154 / 167 / 194
Read number / 32196 / 31336 / 35856 / 25540 / 38560 / 37108 / 46564 / 35033 / 36846
OTUs number (<97%) / 1123 / 1002 / 1128 / 1197 / 1136 / 1345 / 1350 / 993 / 1122
Shannon–Wiener’s H’ / 41.06 / 35.74 / 18.84 / 25.20 / 20.03 / 26.11 / 19.55 / 30.43 / 22.99
Evenness J / 5.85 / 5.17 / 2.68 / 3.56 / 2.85 / 3.62 / 2.71 / 4.41 / 3.27

Additional References

Henry S, Baudoin E, López-Gutiérrez JC, Martin-Laurent F, Brauman A, Philippot L (2004) Quantification of denitrifying bacteria in soils by nirK gene targeted real-time PCR. Journal of microbiological methods 59:327-335

Henry S, Bru D, Stres B, Hallet S, Philippot 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 Microbiol 72:5181-9

Jones CM, Graf DR, Bru D, Philippot L, Hallin S (2013) The unaccounted yet abundant nitrous oxide-reducing microbial community: a potential nitrous oxide sink. The ISME journal 7:417-26

Rotthauwe J-H, Witzel K-P, Liesack W (1997) The ammonia monooxygenase structural gene amoA as a functional marker: molecular fine-scale analysis of natural ammonia-oxidizing populations. Applied and environmental microbiology 63:4704-4712

Throback IN, Enwall K, Jarvis A, Hallin S (2004) Reassessing PCR primers targeting nirS, nirK and nosZ genes for community surveys of denitrifying bacteria with DGGE. FEMS microbiology ecology 49:401-17

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