Advanced biofilm analysis in streams receiving organic deicer runoff

Autoři: Michelle A. Nott aff001;  Heather E. Driscoll aff002;  Minoru Takeda aff003;  Mahesh Vangala aff004;  Steven R. Corsi aff001;  Scott W. Tighe aff005
Působiště autorů: Upper Midwest Water Science Center, U.S. Geological Survey, Middleton, Wisconsin, United States of America aff001;  Vermont Genetics Network, Department of Biology, Norwich University, Northfield, Vermont, United States of America aff002;  Graduate School of Engineering, Yokohama National University, Hodogaya, Yokohama, Japan aff003;  Vermont Genetics Network, University of Vermont, Burlington, Vermont, United States of America aff004;  Advanced Genome Technologies Core, University of Vermont, Burlington, Vermont, United States of America aff005
Vyšlo v časopise: PLoS ONE 15(1)
Kategorie: Research Article


Prolific heterotrophic biofilm growth is a common occurrence in airport receiving streams containing deicers and anti-icers, which are composed of low-molecular weight organic compounds. This study investigated biofilm spatiotemporal patterns and responses to concurrent and antecedent (i.e., preceding biofilm sampling) environmental conditions at stream sites upstream and downstream from Milwaukee Mitchell International Airport in Milwaukee, Wisconsin, during two deicing seasons (2009–2010; 2010–2011). Biofilm abundance and community composition were investigated along spatial and temporal gradients using field surveys and microarray analyses, respectively. Given the recognized role of Sphaerotilus in organically enriched environments, additional analyses were pursued to specifically characterize its abundance: a consensus sthA sequence was determined via comparison of whole metagenome sequences with a previously identified sthA sequence, the primers developed for this gene were used to characterize relative Sphaerotilus abundance using quantitative real-time PCR, and a Sphaerotilus strain was isolated to validate the determined sthA sequence. Results indicated that biofilm abundance was stimulated by elevated antecedent chemical oxygen demand concentrations, a surrogate for deicer concentrations, with minimal biofilm volumes observed when antecedent chemical oxygen demand concentrations remained below 48 mg/L. Biofilms were composed of diverse communities (including sheathed bacterium Thiothrix) whose composition appeared to shift in relation to antecedent temperature and chemical oxygen demand. The relative abundance of sthA correlated most strongly with heterotrophic biofilm volume (positive) and dissolved oxygen (negative), indicating that Sphaerotilus was likely a consistent biofilm member and thrived under low oxygen conditions. Additional investigations identified the isolate as a new strain of Sphaerotilus montanus (strain KMKE) able to use deicer components as carbon sources and found that stream dissolved oxygen concentrations related inversely to biofilm volume as well as to antecedent temperature and chemical oxygen demand. The airport setting provides insight into potential consequences of widescale adoption of organic deicers for roadway deicing.

Klíčová slova:

Airports – Bacterial biofilms – Biofilms – Chemical oxygen demand – Microarrays – Sequence alignment – Sequence analysis – Sequence databases


1. Corsi SR, Graczyk DJ, Geis SW, Booth NL, Richards KD. A fresh look at road salt: Aquatic toxicity and water-quality impacts on local, regional, and national scales. Environ Sci Technol. 2010;44: 7376–7382. doi: 10.1021/es101333u 20806974

2. Findlay SEG, Kelly VR. Emerging indirect and long-term road salt effects on ecosystems. Ann N Y Acad Sci. 2011;1223: 58–68. doi: 10.1111/j.1749-6632.2010.05942.x 21449965

3. Cañedo-Argüelles M, Kefford BJ, Piscart C, Prat N, Schäfer RB, Schulz C-J. Salinisation of rivers: An urgent ecological issue. Environ Pollut. 2013;173: 157–167. doi: 10.1016/j.envpol.2012.10.011 23202646

4. Corsi SR, De Cicco LA, Lutz MA, Hirsch RM. River chloride trends in snow-affected urban watersheds: increasing concentrations outpace urban growth rate and are common among all seasons. Sci Total Environ. 2015;508: 488–497. doi: 10.1016/j.scitotenv.2014.12.012 25514764

5. Muthumani A, Fay L, Shi X, Bergner D. Understanding the Effectiveness of Non-Chloride Liquid Agricultural By-Products and Solid Complex Chloride/Mineral Products. St. Paul, MN: Minnesota Department of Transportation Research Services & Library; 2015 Nov p. 160. Report No.: CR13-02.

6. Ferguson L, Corsi SR, Geis SW, Anderson G, Joback K, Gold H, et al. Aircraft Deicing and Airfield Anti-Icing Formulations: Aquatic Toxicity and Biochemical Oxygen Demand. 2008 Nov pp. 1–103.

7. Corsi SR, Mericas D, Bowman GT. Oxygen Demand of Aircraft and Airfield Pavement Deicers and Alternative Freezing Point Depressants. Water Air Soil Pollut. 2012;223: 2447–2461. doi: 10.1007/s11270-011-1036-x

8. Henze M, Comeau Y. Wastewater Characterization. 1st ed. Biological Wastewater Treatment: Principles, Modelling and Design. 1st ed. London, England: IWA Publishing; 2008. pp. 33–52.

9. U.S. Environmental Protection Agency O. Technical Development Document for the Final Effluent Limitations Guidelines and New Source Performance Standards for the Airport Deicing Category. Washington, D.C.; 2012 Apr p. 193. Report No.: EPA-821-R-12-005.

10. U.S. Environmental Protection Agency O. Environmental Impact and Benefit Assessment for the Final Effluent Limitation Guidelines and Standards for the Airport Deicing Category. Washington, D.C.; 2012 Apr p. 98. Report No.: EPA-821-R-12-003.

11. Corsi SR, Booth NL, Hall DW. Aircraft and runway deicers at General Mitchell International Airport, Milwaukee, Wisconsin, USA. 1. Biochemical oxygen demand and dissolved oxygen in receiving streams. Environ Toxicol Chem SETAC. 2001;20: 1474–1482.

12. Corsi SR, Geis SW, Bowman G, Failey GG, Rutter TD. Aquatic toxicity of airfield-pavement deicer materials and implications for airport runoff. Environ Sci Technol. 2009;43: 40–46. doi: 10.1021/es8017732 19209582

13. Pillard DA. Assessment of Benthic Macroinvertebrate and Fish Communities in a Stream Receiving Storm Water Runoff from a Large Airport. J Freshw Ecol. 1996;11: 51–59. doi: 10.1080/02705060.1996.9663493

14. Dondero NC. The Sphaerotilus-Leptothrix Group. Annu Rev Microbiol. 1975;29: 407–428. doi: 10.1146/annurev.mi.29.100175.002203 1180519

15. Dworkin M, Falkow S, Rosenberg E, Schleifer K-H, Stackebrandt E, editors. The Prokaryotes Volume 3: Archaea. Bacteria: Firmicutes, Actinomycetes. 2006. Available:

16. Stokes JL. Studies on the Filamentous Sheathed Iron Bacterium Sphaerotilus natans. J Bacteriol. 1954; 278–291. 13142992

17. Curtis EJC. Sewage fungus: Its nature and effects. Water Res. 1969;3: 289–311. doi: 10.1016/0043-1354(69)90084-0

18. DeMartini FE. Slime Growths in Sewers. Sew Works J. 1934;6: 950–955.

19. Gray NF. Heterotrophic Slimes in Flowing Waters. Biol Rev. 1985;60: 499–548. doi: 10.1111/j.1469-185X.1985.tb00621.x

20. Pellegrin V, Juretschko S, Wagner M, Cottenceau G. Morphological and Biochemical Properties of a Sphaerotilus sp. Isolated From Paper Mill Slimes. Appl Environ Microbiol. 1999;65: 156–162. 9872774

21. Quinn JM, Gilliland BW. The Manawatu River Cleanup—Has it worked? Trans Inst Prof Eng N Z. 1988;16: 22–26.

22. Ramothokang TR, Drysdale GD, Bux F. Isolation and cultivation of filamentous bacteria implicated in activated sludge bulking. Water A. 2003;29: 405–410.

23. Dworkin M, Falkow S, Rosenberg E, Schleifer K-H, Stackebrandt E, editors. The Prokaryotes Volume 5: Proteobacteria: Alpha and Beta Subclasses. 2006. Available:

24. Mulder EG. Iron Bacteria, particularly those of the Sphaerotilus-Leptothrix Group, and Industrial Problems. J Appl Microbiol. 1964;27: 151–173. doi: 10.1111/j.1365-2672.1964.tb04824.x

25. Battin TJ, Besemer K, Bengtsson MM, Romani AM, Packmann AI. The ecology and biogeochemistry of stream biofilms. Nat Rev Microbiol. 2016;14: 251–263. doi: 10.1038/nrmicro.2016.15 26972916

26. Hirsch A. Biological evaluation of organic pollution of New Zealand streams. N Z J Sci. 1958;1: 500–553.

27. Hynes HBN. The biology of polluted waters. Liverp Engl Liverp Univ PRESS 1960. 1960 [cited 9 Jul 2012]. Available: = gs&collection = ENV&recid = 6905004

28. Smith LL, Kramer RH. Survival of Walleye Eggs in Relation to Wood Fibers and Sphaerotilus natans in the Rainy River, Minnesota. Trans Am Fish Soc. 1963;92: 220–234. doi: 10.1577/1548-8659(1963)92[220:SOWEIR]2.0.CO;2

29. U.S. Environmental Protection Agency. Methods for the determination of inorganic substances in environmental samples. Cincinnati, OH: US EPA, Office of Research and Development; 1993 Aug. Report No.: EPA/600/R-93/100.

30. American Society for Testing and Materials. Annual Book of ASTM Standards, Section 11 (Water and Environmental Technology). Method D-1252-95. Philadelphia, PA: American Society for Testing and Materials; 1995.

31. Rantz SE, others. Measurement and computation of streamflow. Washington, D.C.; 1982 p. 631. Report No.: 2175. Available:

32. U.S. Environmental Protection Agency. Nonhalogenated Organics by Gas Chromatography. 2007 Feb p. 36. Report No.: USEPA Method 8015C. Available:

33. Nott MA, Driscoll HE, Takeda M, Vangala M, Corsi SR, Tighe SW. Data and regression models describing biofilms and water quality in streams surrounding Milwaukee Mitchell International Airport, Milwaukee, Wisconsin (2009–2014). U.S. Geological Survey; 2019. Available:

34. Stevenson RJ, Bahls LL. Periphyton protocols. 2nd ed. Rapid Bioassessment Protocols for Use in Streams and Wadeable Rivers: Periphyton, Benthic Macroinvertebrates, and Fish, Second Edition. 2nd ed. Washington, D.C.: U.S. Environmental Protection Agency; Office of Water; 1999. pp. 6–1 through 6–22.

35. Stevenson RJ, Rollins SL. Chapter 34—Ecological Assessments with Benthic Algae. Methods in Stream Ecology (Second Edition). San Diego: Academic Press; 2007. pp. 785–803. Available:

36. Fitzpatrick FA, Waite IR, D’Arconte PJ, Meador MR, Maupin MA, Gurtz ME. Revised Methods for Characterizing Stream Habitat in the National Water-Quality Assessment Program. 1998. Report No.: 98–4052. Available:

37. R Core Team. R: A Language and Environment for Statistical Computing. Vienna, Austria: R Foundation for Statistical Computing; 2015. Available:

38. Kellogg CA, Piceno YM, Tom LM, DeSantis TZ, Gray MA, Andersen GL. Comparing Bacterial Community Composition of Healthy and Dark Spot-Affected Siderastrea siderea in Florida and the Caribbean. PLOS ONE. 2014;9: e108767. doi: 10.1371/journal.pone.0108767 25289937

39. Yang C, Powell CA, Duan Y, Shatters R, Fang J, Zhang M. Deciphering the Bacterial Microbiome in Huanglongbing-Affected Citrus Treated with Thermotherapy and Sulfonamide Antibiotics. PLOS ONE. 2016;11: e0155472. doi: 10.1371/journal.pone.0155472 27171468

40. Piceno YM, Pecora-Black G, Kramer S, Roy M, Reid FC, Dubinsky EA, et al. Bacterial community structure transformed after thermophilically composting human waste in Haiti. PLOS ONE. 2017;12: e0177626. doi: 10.1371/journal.pone.0177626 28570610

41. Berendsen RL, Vismans G, Yu K, Song Y, Jonge R de, Burgman WP, et al. Disease-induced assemblage of a plant-beneficial bacterial consortium. ISME J. 2018;12: 1496–1507. doi: 10.1038/s41396-018-0093-1 29520025

42. Mapelli F, Marasco R, Fusi M, Scaglia B, Tsiamis G, Rolli E, et al. The stage of soil development modulates rhizosphere effect along a High Arctic desert chronosequence. ISME J. 2018;12: 1188–1198. doi: 10.1038/s41396-017-0026-4 29335640

43. Probst AJ, Lum PY, John B, Dubinsky EA, Piceno YM, Tom LM. Microarray of 16S rRNA gene probes for quantifying population differences across microbiome samples. Microarrays: Current Technology, Innovations and Applications. Norfolk, UK: Horizon Scientific Press and Caister Academic Press; 2014. pp. 99–119.

44. DeSantis TZ, Hugenholtz P, Larsen N, Rojas M, Brodie EL, Keller K, et al. Greengenes, a Chimera-Checked 16S rRNA Gene Database and Workbench Compatible with ARB. Appl Environ Microbiol. 2006;72: 5069–5072. doi: 10.1128/AEM.03006-05 16820507

45. McDonald D, Price MN, Goodrich J, Nawrocki EP, DeSantis TZ, Probst A, et al. An improved Greengenes taxonomy with explicit ranks for ecological and evolutionary analyses of bacteria and archaea. ISME J. 2012;6: 610–618. doi: 10.1038/ismej.2011.139 22134646

46. Howe E, Holton K, Nair S, Schlauch D, Sinha R, Quackenbush J. MeV: MultiExperiment Viewer. In: Ochs MF, Casagrande JT, Davuluri RV, editors. Biomedical Informatics for Cancer Research. Boston, MA: Springer US; 2010. pp. 267–277. doi: 10.1007/978-1-4419-5714-6_15

47. Eisen MB, Spellman PT, Brown PO, Botstein D. Cluster analysis and display of genome-wide expression patterns. Proc Natl Acad Sci. 1998;95: 14863–14868. doi: 10.1073/pnas.95.25.14863 9843981

48. Sokal RR, Michener CD. A statistical method for evaluating systematic relationships. Univ Kans Sci Bull. 1958;38: 1409–1438.

49. Hamady M, Lozupone C, Knight R. Fast UniFrac: facilitating high-throughput phylogenetic analyses of microbial communities including analysis of pyrosequencing and PhyloChip data. ISME J. 2010;4: 17–27. doi: 10.1038/ismej.2009.97 19710709

50. Minot SS, Krumm N, Greenfield NB. One Codex: A Sensitive and Accurate Data Platform for Genomic Microbial Identification. bioRxiv. 2015; 23. doi: 10.1101/027607

51. Mendenhall W, Beaver RJ, Beaver BM. Introduction to Probability and Statistics. 10th ed. Pacific Grove, California, USA: Duxbury Press; 1999.

52. Mao D-P, Zhou Q, Chen C-Y, Quan Z-X. Coverage evaluation of universal bacterial primers using the metagenomic datasets. BMC Microbiol. 2012;12: 66. doi: 10.1186/1471-2180-12-66 22554309

53. Boyer SL, Flechtner VR, Johansen JR. Is the 16S–23S rRNA Internal Transcribed Spacer Region a Good Tool for Use in Molecular Systematics and Population Genetics? A Case Study in Cyanobacteria. Mol Biol Evol. 2001;18: 1057–1069. doi: 10.1093/oxfordjournals.molbev.a003877 11371594

54. Takeda M, Nakano F, Nagase T, Iohara K, Koizumi J. Isolation and Chemical Composition of the Sheath of Sphaerotilus natans. Biosci Biotechnol Biochem. 1998;62: 1138–1143. doi: 10.1271/bbb.62.1138 9692196

55. Takeda M, Nakamori T, Hatta M, Yamada H, Koizumi J. Structure of the polysaccharide isolated from the sheath of Sphaerotilus natans. Int J Biol Macromol. 2003;33: 245–250. doi: 10.1016/j.ijbiomac.2003.08.008 14607370

56. Kondo K, Umezu T, Shimura S, Narizuka R, Koizumi J, Mashima T, et al. Structure of perosamine-containing polysaccharide, a component of the sheath of Thiothrix fructosivorans. Int J Biol Macromol. 2013;59: 59–66. doi: 10.1016/j.ijbiomac.2013.04.013 23587998

57. Armbruster EH. Improved technique for isolation and identification of Sphaerotilus. Appl Microbiol. 1969;17: 320–321. 4180407

58. Nott MA, Driscoll HE, Takeda M, Vangala M, Corsi SR, Tighe SW. Biofilm microbiome and isolated Sphaerotilus montanus (strain KMKE) gene sequences from streams receiving organic airport deicer runoff. In: NCBI BioProject [Internet]. 2017. Available:

59. Odum HT. Primary Production in Flowing Waters. Limnol Oceanogr. 1956;1: 102–117.

60. Lampert W, Sommer U. Limnoecology: The Ecology of Lakes and Streams. 2nd ed. New York, United States: Oxford University Press; 2007.

61. Suzuki T, Kanagawa T, Kamagata Y. Identification of a Gene Essential for Sheathed Structure Formation in Sphaerotilus natans, a Filamentous Sheathed Bacterium. Appl Environ Microbiol. 2002;68: 365–371. doi: 10.1128/AEM.68.1.365-371.2002 11772646

62. Gridneva E, Chernousova E, Dubinina G, Akimov V, Kuever J, Detkova E, et al. Taxonomic investigation of representatives of the genus Sphaerotilus: descriptions of Sphaerotilus montanus sp. nov., Sphaerotilus hippei sp. nov., Sphaerotilus natans subsp. natans subsp. nov. and Sphaerotilus natans subsp. sulfidivorans subsp. nov., and an emended description of the genus Sphaerotilus. Int J Syst Evol Microbiol. 2011;61: 916–925. doi: 10.1099/ijs.0.023887-0 20495027

63. Koryak M, Stafford LJ, Reilly RJ, Hoskin RH, Haberman MH. The impact of airport deicing runoff on water quality and aquatic life in a Pennsylvania stream. J Freshw Ecol. 1998;13: 287–298.

64. ACRP. Understanding Microbial Biofilms in Receiving Waters Impacted by Airport Deicing Activities. Washington, D.C.: Transportation Research Board; 2014 p. 63. Report No.: 115. Available:

65. U.S. Geological Survey. USGS water data for the Nation. In: U.S. Geological Survey National Water Information System database [Internet]. 2019. Available:

66. Bernhardt ES, Likens GE. Dissolved Organic Carbon Enrichment Alters Nitrogen Dynamics in a Forest Stream. Ecology. 2002;86: 1689–1700.

67. Mohamed MN, Lawrence JR, Robarts RD. Phosphorus Limitation of Heterotrophic Biofilms from the Fraser River, British Columbia, and the Effect of Pulp Mill Effluent. Microb Ecol. 1998;36: 121–130. doi: 10.1007/s002489900099 9688774

68. Seder-Colomina M, Goubet A, Lacroix S, Morin G, Ona-Nguema G, Esposito G, et al. Moderate oxygen depletion as a factor favouring the filamentous growth of Sphaerotilus natans. Antonie Van Leeuwenhoek. 2015;107: 1135–1144. doi: 10.1007/s10482-015-0405-7 25666377

69. Lau AO, Strom PF, Jenkins D. Growth Kinetics of Sphaerotilus natans and a Floc Former in Pure and Dual Continuous Culture. J Water Pollut Control Fed. 1984;56: 41–51.

70. Barr Engineering Company. Determining the Aquatic Toxicity of Deicing Materials. Minneapolis, MN; 2013 Dec p. 64. Report No.: CR11-02.

71. Kaushal SS. Increased salinization decreases safe drinking water. Environ Sci Technol. 2016;50: 2765–2766. doi: 10.1021/acs.est.6b00679 26903048

72. Ramakrishna DM, Viraraghavan T. Environmental Impact of Chemical Deicers–A Review. Water Air Soil Pollut. 2005;166: 49–63. doi: 10.1007/s11270-005-8265-9

73. Schuler MS, Hintz WD, Jones DK, Lind LA, Mattes BM, Stoler AB, et al. How common road salts and organic additives alter freshwater food webs: in search of safer alternatives. J Appl Ecol. 2017;54: 1353–1361.

Článek vyšel v časopise


2020 Číslo 1
Nejčtenější tento týden