Wetlands

, Volume 34, Issue 1, pp 9–17 | Cite as

Methodologies for Extracellular Enzyme Assays from Wetland Soils

  • Christian Dunn
  • Timothy G. Jones
  • Astrid Girard
  • Chris Freeman
Article

Abstract

Measurement of extracellular enzymic activity in wetland soils can give an indication of the ecosystems biogeochemical processes, and rates of nutrient and carbon cycling. Analysis of these have allowed researchers to gain an understanding of the ecosystems’ microbial ecology and how it can be affected by environmental factors. Here we give a detailed description of the assays necessary to determine the activity of a suite of key hydrolase enzymes and phenol oxidases. These enzymes control the rates of decomposition and consequently the production of biogenic greenhouse gases. Knowing the processes responsible for the breakdown of organic matter is therefore essential if it becomes necessary to curb these emissions. Our protocols allow for cost effective analysis of a large number of samples and provide sufficient accuracy to determine differences between soil types. When coupled with contemporary microbial techniques these enzyme assays permit entire biochemical pathways to be determined, giving unparalleled knowledge on the processes involved in wetland ecosystems.

Keywords

Extracellular enzymes Peatlands Organic soils Phenol oxidases Hydrolases Enzyme assays 

Notes

Acknowledgments

This research was funded by the Knowledge and Economy Skills Scholarship, which is part funded by the European Social Fund through the European Union’s Convergence programme and administered by the Welsh Assembly Government. We would also like to thank George Meyrick of Energy and Environment Business Services for supporting this research.

References

  1. Bending GD, Read DJ (1997) Lignin and soluble phenolic degradation by ectomycorrhizal and ericoid mycorrhizal fungi. Mycol Res 101Google Scholar
  2. Blank RR, Derner JD (2004) Effects of CO2 enrichment on plant-soil relationships of Lepidium latifolium. Plant Soil 262:159–167CrossRefGoogle Scholar
  3. Burke RM, Cairney JWG (2002) Laccases and other polyphenol oxidases in ecto- and ericoid mycorrhizal fungi. Mycorrhiza 12Google Scholar
  4. Burns RG (1982) Enzyme-activity in soil: location and a possible role in microbial ecology. Soil Biol Biochem 14:423–427CrossRefGoogle Scholar
  5. Burns A, Ryder DS (2001) Response of bacterial extracellular enzymes to inundation of floodplain sediments. Freshw Biol 46:1299–1307CrossRefGoogle Scholar
  6. Corpe WA, Winters H (1972) Hydrolytic enzymes of some periphytic marine bacteria. Can J Microbiol 18:1483–1490PubMedCrossRefGoogle Scholar
  7. Corstanje R, Reddy KR (2004) Response of biogeochemical indicators to a drawdown and subsequent reflood. J Environ Qual 33:2357–2366PubMedCrossRefGoogle Scholar
  8. Crawford DL (1978) Lignocellulose decomposition by selected Streptomyces strains. Appl Environ Microbiol 35:1041–1045PubMedCentralPubMedGoogle Scholar
  9. Daatselaar MCC, Harder W (1974) Some aspects of regulation of production of extracellular proteolytic-enzymes be a marine bacterium. Arch Microbiol 101:21–34PubMedCrossRefGoogle Scholar
  10. De Cesare F, Garzillo AMV, Buonocore V, Badalucco L (2000) Use of sonication for measuring acid phosphatase activity in soil. Soil Biol Biochem 32:825–832CrossRefGoogle Scholar
  11. Deng S, Popova I (2011) Carbohydrate hydrolases. In: Dick RP (ed) Mehods of soil enzymology. Soil Science Society of America, Inc., Madison, pp 185–209Google Scholar
  12. Dick R, Burns RG (2011) A brief history of soil enzyme research. In: Dick RP (ed) Methods of soil enzymology. Soil Science Society of America, Wisconsin, pp 1–34Google Scholar
  13. Drenovsky RE, Feris KP, Batten KM, Hristova K (2008) New and current microbiological tools for ecosystem ecologists: towards a goal of linking structure and function. Am Midl Nat 160:140–159CrossRefGoogle Scholar
  14. Duarte B, Reboreda R, Cacador I (2008) Seasonal variation of extracellular enzymatic activity (EEA) and its influence on metal speciation in a polluted salt marsh. Chemosphere 73:1056–1063PubMedCrossRefGoogle Scholar
  15. Duckworth W, Coleman JE (1970) Physiochemical and kinetic properties of mushroom tyrosinase. J Biol Chem 245:1613PubMedGoogle Scholar
  16. Duran N, Rosa MA, D’Annibale A, Gianfreda L (2002) Applications of laccases and tyrosinases (phenoloxidases) immobilized on different supports: a review. Enzyme Microb Technol 31:907–931CrossRefGoogle Scholar
  17. Endo K, Hayashi Y, Hibi T, Hosono K, Beppu T, Ueda K (2003) Enzymological characterization of EpoA, a laccase-like phenol oxidase produced by Streptomyces griseus. J Biochem 133Google Scholar
  18. Evans CD, Jones TG, Burden A, Ostle N, Zielinski P, Cooper MDA, Peacock M, Clark JM, Oulehle F, Cooper D, Freeman C (2012) Acidity controls on dissolved organic carbon mobility in organic soils. Glob Chang Biol 18:3317–3331CrossRefGoogle Scholar
  19. Fenner N, Freeman C (2011) Drought-induced carbon loss in peatlands. Nat Geosci 4:895–900CrossRefGoogle Scholar
  20. Fenner N, Freeman C, Reynolds B (2005a) Hydrological effects on the diversity of phenolic degrading bacteria in a peatland: implications for carbon cycling. Soil Biol Biochem 37Google Scholar
  21. Fenner N, Freeman C, Reynolds B (2005b) Observations of a seasonally shifting thermal optimum in peatland carbon-cycling processes; implications for the global carbon cycle and soil enzyme methodologies. Soil Biol Biochem 37:1814–1821CrossRefGoogle Scholar
  22. Fenner N, Dowrick DJ, Lock MA, Rafarel CR, Freeman C (2006) A novel approach to studying the effects of temperature on soil biogeochemistry using a thermal gradient bar. Soil Use Manag 22:267–273CrossRefGoogle Scholar
  23. Freeman C, Nevison GB (1999) Simultaneous analysis of multiple enzymes in environmental samples using methylumbelliferyl substrates and HPLC. J Environ Qual 28:1378–1380CrossRefGoogle Scholar
  24. Freeman C, Liska G, Ostle NJ, Jones SE, Lock MA (1995) The use of fluorogenic substrates for measuring enzyme-activity in peatlands. Plant Soil 175:147–152CrossRefGoogle Scholar
  25. Freeman C, Liska G, Ostle NJ, Lock MA, Reynolds B, Hudson J (1996) Microbial activity and enzymic decomposition processes following peatland water table drawdown. Plant Soil 180:121–127CrossRefGoogle Scholar
  26. Freeman C, Liska G, Ostle NJ, Lock MA, Hughes S, Reynolds B, Hudson J (1997) Enzymes and biogeochemical cycling in wetlands during a simulated drought. Biogeochemistry 39:177–187CrossRefGoogle Scholar
  27. Freeman C, Nevison GB, Hughes S, Reynolds B, Hudson J (1998) Enzymic involvement in the biogeochemical responses of a Welsh peatland to a rainfall enhancement manipulation. Biol Fertil Soils 27:173–178CrossRefGoogle Scholar
  28. Freeman C, Evans CD, Monteith DT, Reynolds B, Fenner N (2001a) Export of organic carbon from peat soils. Nature 412:785–785PubMedCrossRefGoogle Scholar
  29. Freeman C, Ostle N, Kang H (2001b) An enzymic ‘latch’ on a global carbon store—a shortage of oxygen locks up carbon in peatlands by restraining a single enzyme. Nature 409:149–149PubMedCrossRefGoogle Scholar
  30. Freeman C, Kim SY, Lee SH, Kang H (2004a) Effects of elevated atmospheric CO2 concentrations on soil microorganisms. J Microbiol 42:267–277PubMedGoogle Scholar
  31. Freeman C, Ostle NJ, Fenner N, Kang H (2004b) A regulatory role for phenol oxidase during decomposition in peatlands. Soil Biol Biochem 36:1663–1667CrossRefGoogle Scholar
  32. Freeman C, Fenner N, Shirsat AH (2012) Peatland geoengineering: an alternative approach to terrestrial carbon sequestration. Philosophical transactions. Ser A Math Phys Eng Sci 370:4404–4421CrossRefGoogle Scholar
  33. Frogbrook ZL, Bell J, Bradley RI, Evans C, Lark RM, Reynolds B, Smith P, Towers W (2009) Quantifying terrestrial carbon stocks: examining the spatial variation in two upland areas in the UK and a comparison to mapped estimates of soil carbon. Soil Use Manag 25:320–332CrossRefGoogle Scholar
  34. Gao Y, Mao L, C-Y M, Zhou P, J-J C, Y-E Z, W-J S (2010) Spatial characteristics of soil enzyme activities and microbial community structure under different land uses in Chongming Island, China: geostatistical modelling and PCR-RAPD method. Sci Total Environ 408:3251–3260PubMedCrossRefGoogle Scholar
  35. Gorham E (1991) Northern Peatlands—role in the carbon-cycle and probable responses to climatic warming. Ecol Appl 1:182–195CrossRefGoogle Scholar
  36. Gramss G, Voigt KD, Kirsche B (1999) Oxidoreductase enzymes liberated by plant roots and their effects on soil humic material. Chemosphere 38Google Scholar
  37. Guilbault GG (1990) Practical fluorescence, 2nd edn. Marcel Dekker, New YorkGoogle Scholar
  38. Gutknecht JLM, Goodman RM, Balser TC (2006) Linking soil process and microbial ecology in freshwater wetland ecosystems. Plant Soil 289:17–34CrossRefGoogle Scholar
  39. Hullo MF, Moszer I, Danchin A, Martin-Verstraete I (2001) CotA of Bacillus subtilis is a copper-dependent laccase. J Bacteriol 183Google Scholar
  40. Jordan DB, Wagschal K (2010) Properties and applications of microbial beta-D-xylosidases featuring the catalytically efficient enzyme from Selenomonas ruminantium. Appl Microbiol Biotechnol 86Google Scholar
  41. Kang H, Freeman C, Lock MA (1998) Trace gas emissions from a north Wales fen—role of hydrochemistry and soil enzyme activity. Water Air Soil Pollut 105:107–116CrossRefGoogle Scholar
  42. Kang HJ, Freeman C, Ashendon TW (2001) Effects of elevated CO2 on fen peat biogeochemistry. Sci Total Environ 279:45–50PubMedCrossRefGoogle Scholar
  43. Kang HJ, Freeman C, Park SS, Chun J (2005) N-Acetylglucosaminidase activities in wetlands: a global survey. Hydrobiologia 532Google Scholar
  44. Kim S-Y, Kang H (2008) Effects of elevated CO2 on below-ground processes in temperate marsh microcosms. Hydrobiologia 605:123–130CrossRefGoogle Scholar
  45. Klose S, Bilen S, Tabatabai MA, Dick WA (2011) Sulfur cycle enzymes. In: Dick RP (ed) Methods of soil enzymology. Soil Science Society of America, WisconsinGoogle Scholar
  46. Kourtev PS, Ehrenfeld JG, Huang WZ (2002) Enzyme activities during litter decomposition of two exotic and two native plant species in hardwood forests of New Jersey. Soil Biol Biochem 34Google Scholar
  47. Lakowicz JR (2006) Principles of fluorescence spectroscopy, 3rd edn. Springer Science and Business Media, New YorkCrossRefGoogle Scholar
  48. Mackelprang R, Waldrop MP, DeAngelis KM, David MM, Chavarria KL, Blazewicz SJ, Rubin EM, Jansson JK (2011) Metagenomic analysis of a permafrost microbial community reveals a rapid response to thaw. Nature 480:368–371PubMedCrossRefGoogle Scholar
  49. Marx MC, Wood M, Jarvis SC (2001) A microplate fluorimetric assay for the study of enzyme diversity in soils. Soil Biol Biochem 33:1633–1640CrossRefGoogle Scholar
  50. Mason HS (1948) The chemistry of melanin; mechanism of the oxidation of dihydroxyphenylalanine by tyrosinase. J Biol Chem 172:83–99PubMedGoogle Scholar
  51. Mentzer JL, Goodman RM, Balser TC (2006) Microbial response over time to hydrologic and fertilization treatments in a simulated wet prairie. Plant Soil 284:85–100CrossRefGoogle Scholar
  52. Oshrain RL, Wiebe WJ (1979) Arylsulfatase activity in salt marsh soils. Appl Environ Microbiol 38:337–340PubMedCentralPubMedGoogle Scholar
  53. Pelaez F, Martinez MJ, Martinez AT (1995) Screening of 68 species of basidiomycetes for enzymes involved in lignin degradation. Mycol Res 99:37–42CrossRefGoogle Scholar
  54. Pind A, Freeman C, Lock MA (1994) Enzymatic degradation of phenolic materials in peatlands—measurement of Phenol Oxidase activity. Plant Soil 159:227–231CrossRefGoogle Scholar
  55. Pomerantz SH, Murthy VV (1974) Purification and properties of tyrosinases from vibrio-tyrosinaticus. Arch Biochem Biophys 160Google Scholar
  56. Press MC, Henderson J, Lee JA (1985) Arylsulfatase activity in peat in relation to acidic deposition. Soil Biol Biochem 17:99–103CrossRefGoogle Scholar
  57. Prosser JA, Speir TW, Stott DE (2011) Soil oxidoreductases and FDA hydrolysis. In: Dick RP (ed) Methods of soil enzymology. Soil Science Society of America Inc., Wisconsin, pp 103–124Google Scholar
  58. Siciliano SD, Lean DRS (2002) Methyltransferase: an enzyme assay for microbial methylmercury formation in acidic soils and sediments. Environ Toxicol Chem 21:1184–1190PubMedCrossRefGoogle Scholar
  59. Sinsabaugh RL (1994) Enzymatic analysis of microbial pattern and process. Biol Fertil Soils 17:69–74CrossRefGoogle Scholar
  60. Sinsabaugh RL, Findlay S (1995) Microbial-production, enzyme-activity, and carbon turnover in surface sediments of the Hudson River Estuary. Microbial Ecol 30:127–141CrossRefGoogle Scholar
  61. Sinsabaugh RL, Linkins AE (1990) Enzymatic and chemical-analysis of particulate organic-matter from a boreal river. Freshw Biol 23:301–309CrossRefGoogle Scholar
  62. Sinsabaugh RL, Antibus RK, Linkins AE, McClaugherty CA, Rayburn L, Repert D, Weiland T (1992) Wood decomposition over a 1st-order watershed—mass-loss as a function of lignocellulase activity. Soil Biol Biochem 24:743–749CrossRefGoogle Scholar
  63. Sinsabaugh RL, Osgood MP, Findlay S (1994) Enzymatic models for estimating decomposition rates and particulate detritus. J N Am Benthol Soc 13:160–169CrossRefGoogle Scholar
  64. Sinsabaugh RL, Klug MJ, Collins HP, Yeager PE, Peterson SO (1999) Characterizing soil microbial communities. In: Robertson GP, Coleman DC, Bledsoe CS, Sollins P (eds) Standard soil methods for long-term ecological research. Oxford University Press, New YorkGoogle Scholar
  65. Sinsabaugh, R. L., Lauber, C. L., Weintraub, M. N., Ahmed, B., Allison, S. D., Crenshaw, C., Contosta, A. R., Cusack, D., Frey, S., Gallo, M. E., Gartner, T. B., Hobbie, S. E., Holland, K., Keeler, B. L., Powers, J. S., Stursova, M., Takacs-Vesbach, C., Waldrop, M. P., Wallenstein, M. D., Zak, D. R. and Zeglin, L. H. (2008), Stoichiometry of soil enzyme activity at global scale. Ecology Letters, 11:1252–1264. doi:  10.1111/j.1461-0248.2008.01245.x
  66. Sinsabaugh RL, Hill BH, Shah JJF (2009) Ecoenzymatic stoichiometry of microbial organic nutrient acquisition in soil and sediment. Nature 462:795–798PubMedCrossRefGoogle Scholar
  67. Song K-Y, Zoh K-D, Kang H (2007) Release of phosphate in a wetland by changes in hydrological regime. Sci Total Environ 380:13–18PubMedCrossRefGoogle Scholar
  68. Tallis, J H, (1987) Fire and flood at Holme Moss: Erosion processes in an upland blanket mire. Journal of Ecology. 75:(4)1099–1129Google Scholar
  69. Thomas VK, Kuehn KA, Francoeur SN (2009) Effects of UV radiation on wetland periphyton: algae, bacteria, and extracellular polysaccharides. J Freshw Ecol 24:315–326CrossRefGoogle Scholar
  70. Turner BL, Baxter R, Whitton BA (2002) Seasonal phosphatase activity in three characteristic soils of the English uplands polluted by long-term atmospheric nitrogen deposition. Environ Pollut 120Google Scholar
  71. Whitehead PG, Crossman J (2012) Macronutrient cycles and climate change: key science areas and an international perspective. Sci Total Environ 434:13–17PubMedCrossRefGoogle Scholar
  72. Williams CJ, Shingara EA, Yavitt JB (2000) Phenol oxidase activity in peatlands in New York State: response to summer drought and peat type. Wetlands 20Google Scholar
  73. Williamson J, Mills G, Freeman C (2010) Species-specific effects of elevated ozone on wetland plants and decomposition processes. Environ Pollut 158:1197–1206PubMedCrossRefGoogle Scholar
  74. Wittmann C, Kahkonen MA, Ilvesniemi H, Kurola J, Salkinoja-Salonen MS (2004) Areal activities and stratification of hydrolytic enzymes involved in the biochemical cycles of carbon, nitrogen, sulphur and phosphorus in podsolized boreal forest soils. Soil Biol Biochem 36Google Scholar
  75. Woods AF (1899) The destruction of chlorophyll by oxidizing enzymesGoogle Scholar
  76. Ye R, Wright AL, Inglett K, Wang Y, Ogram AV, Reddy KR (2009) Land-use effects on soil nutrient cycling and microbial community dynamics in the Everglades Agricultural Area, Florida. Commun Soil Sci Plant Anal 40:2725–2742CrossRefGoogle Scholar

Copyright information

© Society of Wetland Scientists 2013

Authors and Affiliations

  • Christian Dunn
    • 1
  • Timothy G. Jones
    • 1
  • Astrid Girard
    • 1
    • 2
  • Chris Freeman
    • 1
  1. 1.The Wolfson Carbon Capture Laboratory, School of Biological SciencesBangor UniversityBangorUK
  2. 2.Sciences et MédecineUniversité de Pierre et Marie CurieParisFrance

Personalised recommendations