Advertisement

Biogeochemistry

, Volume 121, Issue 2, pp 287–304 | Cite as

Extracellular enzyme kinetics scale with resource availability

  • Robert L. Sinsabaugh
  • Jayne Belnap
  • Stuart G. Findlay
  • Jennifer J. Follstad Shah
  • Brian H. Hill
  • Kevin A. Kuehn
  • Cheryl R. Kuske
  • Marcy E. Litvak
  • Noelle G. Martinez
  • Daryl L. Moorhead
  • Daniel D. Warnock
Synthesis and Emerging Ideas

Abstract

Microbial community metabolism relies on external digestion, mediated by extracellular enzymes that break down complex organic matter into molecules small enough for cells to assimilate. We analyzed the kinetics of 40 extracellular enzymes that mediate the degradation and assimilation of carbon, nitrogen and phosphorus by diverse aquatic and terrestrial microbial communities (1160 cases). Regression analyses were conducted by habitat (aquatic and terrestrial), enzyme class (hydrolases and oxidoreductases) and assay methodology (low affinity and high affinity substrates) to relate potential reaction rates to substrate availability. Across enzyme classes and habitats, the scaling relationships between apparent Vmax and apparent Km followed similar power laws with exponents of 0.44 to 0.67. These exponents, called elasticities, were not statistically distinct from a central value of 0.50, which occurs when the Km of an enzyme equals substrate concentration, a condition optimal for maintenance of steady state. We also conducted an ecosystem scale analysis of ten extracellular hydrolase activities in relation to soil and sediment organic carbon (2,000–5,000 cases/enzyme) that yielded elasticities near 1.0 (0.9 ± 0.2, n = 36). At the metabolomic scale, the elasticity of extracellular enzymatic reactions is the proportionality constant that connects the C:N:P stoichiometries of organic matter and ecoenzymatic activities. At the ecosystem scale, the elasticity of extracellular enzymatic reactions shows that organic matter ultimately limits effective enzyme binding sites. Our findings suggest that one mechanism by which microbial communities maintain homeostasis is regulating extracellular enzyme expression to optimize the short-term responsiveness of substrate acquisition. The analyses also show that, like elemental stoichiometry, the fundamental attributes of enzymatic reactions can be extrapolated from biochemical to community and ecosystem scales.

Keywords

Ecological stoichiometry Extracellular enzymes Enzyme kinetics Microbial community Microbial metabolism 

Notes

Acknowledgments

RLS acknowledges support from the NSF Ecosystem Sciences program (DEB-0918718) and the Sevilleta LTER Program. KAK acknowledges support from NSF (DEB-0315686, DBI-0420965, DBI-0521018) and the Michigan Sea Grant College Program (NA76RG0133) under NOAA. CRK acknowledges support from a DOE BER Science Focus Area grant. MEL and RLS acknowledge support from DOE BER Grant number DE-SC0008088. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.

Supplementary material

10533_2014_30_MOESM1_ESM.docx (14 kb)
Supplementary material 1 (DOCX 14 kb)

References

  1. Abdelmagid HM, Tabatabai MA (1987) Nitrate reductase activity in soils. Soil Biol Biochem 19:421–428CrossRefGoogle Scholar
  2. Abichou T, Mahieu K, Chanton J, Romdhane M, Mansouri I (2011) Scaling methane oxidation: from laboratory incubation experiments to landfill cover field conditions. Waste Manag 31:978–986CrossRefGoogle Scholar
  3. Acosta-Martinez V, Tabatabai MA (2000) Arylamidase activity in soil. Soil Sci Soc Am J 64:215–221CrossRefGoogle Scholar
  4. Akatsuka T, Mitamura O (2011) Response of denitrification rate associated with wetting and drying cycles in a littoral wetland area of Lake Biwa, Japan. Limnology 12:127–135CrossRefGoogle Scholar
  5. Allison SD (2005) Cheaters, diffusion and nutrients constrain decomposition by microbial enzymes in spatially structured environments. Ecol Lett 8:626–635CrossRefGoogle Scholar
  6. Bar-Even A, Noor E, Savir Y, Liebermeister W, Davidi D, Tawfik DS, Milo R (2011) The moderately efficient enzyme: evolutionary and physicochemical trends shaping enzyme parameters. Biochemistry 50:4402–4410CrossRefGoogle Scholar
  7. Bartholemew GW, Alexander M (1981) Soil as a sink for atmospheric carbon monoxide. Science 212:1389–1391CrossRefGoogle Scholar
  8. Bender M, Conrad R (1993) Kinetics of methane oxidation in oxic soils. Chemosphere 26:687–696CrossRefGoogle Scholar
  9. Burns RG, DeForest JL, Marxsen JC, Sinsabaugh RL, Stromberger ME, Wallenstein MD, Weintraub MH, Zoppini A (2013) Soil enzyme research: current knowledge and future directions. Soil Biol Biochem 58:216–234CrossRefGoogle Scholar
  10. Cartes P, Jara AA, Demanet R, de la Luz Mora M (2009) Urease activity and nitrogen mineralization kinetics as affected by temperature and urea input rate in sourthern Chilean andisols. J Plant Sci Plant Nut 9:69–82Google Scholar
  11. Christian JR, Karl DM (1998) Ectoaminopeptidase specificity and regulation in Antarctic marine pelagic microbial communities. Aquatic Microb Ecol 15:303–310CrossRefGoogle Scholar
  12. Chróst RJ, Riemann B (1994) Storm-stimulated enzymatic decomposition of organic matter in benthic/pelagic coastal mesocosms. Mar Ecol Prog Series 108:185–192CrossRefGoogle Scholar
  13. Cleveland CC, Liptzin D (2007) C:N: P stoichiometry in soil: is there a “Redfield ratio” for the microbial biomass? Biogeochemistry 85:235–252CrossRefGoogle Scholar
  14. Cornish-Bowden A (2012) Fundamentals of Enzyme Kinetics, 4th edn. Wiley-Blackwell, WeinheimGoogle Scholar
  15. Costerton JW, Lewandowski Z, Caldwell DE, Korber DR, Lappinscott HM (1995) Microbial biofilms. Annu Rev Microb 49:711–745CrossRefGoogle Scholar
  16. Deng SP, Tabatabai MA (1994) Cellulase activity of soils. Soil Biol Biochem 26:1347–1354CrossRefGoogle Scholar
  17. Dote Y (2002) Kinetics of CH4 oxidation in mixed culture. Waste Manag Res 20:494–500CrossRefGoogle Scholar
  18. Dubey SK, Sinha ASK, Singh JS (2000) Spatial variation in the capacity of soil for CH4 uptake and population size of methane oxidizing bacteria in dry-land rice agriculture. Curr Sci 78:617–620Google Scholar
  19. Eivazi F, Tabatabai MA (1988) Glucosidases and galactosidases in soils. Soil Biol Biochem 20:601–606CrossRefGoogle Scholar
  20. Elias S, Banin E (2012) Multi-species biofilms: living with friendly neighbors. FEMS Microb Rev 36:990–1004Google Scholar
  21. Fanin N, Fromin N, Buatois B, Hattenschwiler S (2013) An experimental test of the hypothesis of non-homeostatic consumer stoichiometry in a plant litter–microbe system. Ecol Lett 16:764–772CrossRefGoogle Scholar
  22. Farrell RE, Gupta VVSR, Germida JJ (1994) Effects of cultivation on the activity and kinetics of arylsulfatase in Saskatchewan soils. Soil Biol Biochem 26:1033–1040CrossRefGoogle Scholar
  23. Faust K, Raes J (2012) Microbial interactions: from networks to models. Nat Rev Microb 10:538–550CrossRefGoogle Scholar
  24. Fontigny A, Billen G, Vives-Rigo J (1987) Some kinetic characteristics of exoproteolytic activity in coastal seawater. Estuarine Coastal and Shelf Sci 25:127–133CrossRefGoogle Scholar
  25. Foreman CM, Franchini P, Sinsabaugh RL (1998) The trophic dynamics of riverine bacterioplankton: relationships among substrate availability, ectoenzyme kinetics and growth. Limnol Oceanogr 43:1344–1352CrossRefGoogle Scholar
  26. Frankenberger WT, Tabatabai MA (1980) Amidase activity in soils: II kinetic parameters. Soil Sci Soc Am J 44:532–536CrossRefGoogle Scholar
  27. Frankenberger WT, Tabatabai MA (1991a) L-asparaginase activity of soils. Biol Fertility of Soils 11:6–12CrossRefGoogle Scholar
  28. Frankenberger WT, Tabatabai MA (1991b) L-glutaminase activity of soils. Soil Biol Biochem 23:869–874CrossRefGoogle Scholar
  29. German DP, Marcelo KRB, Stone MM, Allison SD (2012) The Michaelis-Menten kinetics of soil extracellular enzymes in response to temperature: a cross-latitudinal study. Global Change Biol 18:1468–1479CrossRefGoogle Scholar
  30. Grandy AS, Sinsabaugh RL, Neff JC, Stursova M, Zak DR (2008) Nitrogen deposition effects on soil organic matter chemistry are linked to variation in enzymes, ecosystems and size fractions. Biogeochemistry 91:37–49CrossRefGoogle Scholar
  31. Guo R, Conrad R (2008) Extraction and characterization of soil hydrogenases oxidizing atmospheric hydrogen. Soil Biol Biochem 40:1149–1154CrossRefGoogle Scholar
  32. Hashimoto LK, Kaplan WA, Wofsy SC, McElroy MB (1983) Transformations of fixed nitrogen and N2O in the Cariaco Trench. Deep Sea Res 30:575–590CrossRefGoogle Scholar
  33. Hill BH, Elonen CM, Seifert LR, May AA, Tarquinio A (2012) Microbial enzyme stoichiometry and nutrient limitation in US streams and rivers. Ecol Indic 18:540–551CrossRefGoogle Scholar
  34. Hill BH, Elonen CM, Anderson LE, Lehter JC (2014a) Microbial respiration and ecoenzyme activity in sediments from the Gulf of Mexico hypoxic zone. Aquatic Microb Ecol 72:105–116CrossRefGoogle Scholar
  35. Hill BH, Elonen CM, Jicha TM, Kolka RK, Lehto LLP, Sebestyen SD, Seifert-Monson LR (2014b) Ecoenzymatic stoichiometry and microbial processing of organic matter in northern bogs and fens reveals a common P-limitation between peatland types. Biogeochemistry. doi: 10.1007/s10533-014-9991-0 Google Scholar
  36. Hobbie JE, Hobbie EA (2012) Amino acid cycling in planktonic and soil microbes studied with radioisotopes: measured amino acids in soil do not reflect bioavailability. Biogeochemistry 107:339–360CrossRefGoogle Scholar
  37. Hobbie JE, Hobbie EA (2013) Microbes in nature are limited by carbon and energy: the starving survival lifestyle in soils and consequences for estimating microbial rates. Frontiers in Microbiol 4 article 324Google Scholar
  38. Holtan-Hartwig L, Dörsch P, Bakken LR (2000) Comparison of denitrifying communities in organic soils: kinetics of NO3 and N2O reduction. Soil Biol Biochem 32:833–843CrossRefGoogle Scholar
  39. Horz H-P, Raghubanshi AS, Heyer J, Kammann C, Conrad R, Dunfield PF (2002) Activity and community structure of methane-oxidising bacteria in a wet meadow soil. FEMS Microb Ecol 41:247–257CrossRefGoogle Scholar
  40. Huang YL, Zeng YH, Yu ZL, Zhang J, Feng H, Lin XC (2013) In silico and experimental methods revealed highly diverse bacteria with quorum sensing and aromatics biodegradation systems: a potential broad application on bioremediation. Biores Technol 148:311–316CrossRefGoogle Scholar
  41. Hwang S-J, Havens KE, Steinman AD (1998) Activity and community structure of methane-oxidising bacteria in a wet meadow soil. Freshw Biol 40:729–745CrossRefGoogle Scholar
  42. Juan YH, Chen LJ, Wu ZJ, Wang R (2009) Kinetics of soil urease affected by urease inhibitors at contrasting moisture regimes. J Plant Sci Plant Nut 9:125–133Google Scholar
  43. Kaiser C, Franklin O, Dieckmann U, Richter A (2014) Microbial community dynamics alleviate stoichiometric constraints during litter decay. Ecol Lett. doi: 10.1111/ele.12269 Google Scholar
  44. Kempes C, Dutkiewicz S, Follows M (2012) Growth, metabolic partitioning, and the size of microorganisms. Proc Natl Acad Sci 109:495–500CrossRefGoogle Scholar
  45. King GM (1999) Attributes of atmospheric carbon monoxide oxidation by Maine forest soils. Appl Environ Microbiol 65:5257–5264Google Scholar
  46. Kirkby CA, Kirkegaard JA, Richardson AE, Wade LJ, Blanchard C, Batten G (2011) Stable soil organic matter: a comparison of stable C:N:P: S ratios in Australian and other world soils. Geoderma 163:197–208CrossRefGoogle Scholar
  47. Klipp E, Heinrich R (1994) Evolutionary optimization of enzyme kinetic parameters; effect of constraints. J Theor Biol 171:309–323CrossRefGoogle Scholar
  48. Manzoni S, Trofymow JA, Jackson RB, Porporato A (2010) Stoichiometric controls on carbon, nitrogen, and phosphorus dynamics in decomposing litter. Ecol Monogr 80:89–106CrossRefGoogle Scholar
  49. Manzoni S, Schaeffer SM, Katul G, Porporato A, Schimel JP (2014) A theoretical analysis of microbial eco-physiological and diffusion limitations to carbon cycling in drying soils. Soil Biol Biochem 73:69–83CrossRefGoogle Scholar
  50. Martiny AC, Vrugt JA, Primeau FW, Lomas MW (2013) Regional variation in the particulate organic carbon to nitrogen ratio in the surface ocean. Global Biogeochem Cycles 27:723–731CrossRefGoogle Scholar
  51. Marx M-C, Wood M, Jarvis SC (2001) A microplate fluorimetric assay for the study of enzyme diversity in soils. Soil Biol Biochem 33:1633–1640CrossRefGoogle Scholar
  52. Marx M-C, Kandeler E, Wood M, Wermbter N, Jarvis SC (2005) Exploring the enzymatic landscape: distribution and kinetics of hydrolytic enzymes in soil particle-size fractions. Soil Biol Biochem 37:35–48CrossRefGoogle Scholar
  53. McCarthyl JJ, Garside C, Nevins JL (1992) Nitrate supply and phytoplankton uptake kinetics in the euphotic layer of a Gulf stream warm-core ring. Deep Sea Res 39:S393–S403CrossRefGoogle Scholar
  54. McLaren AD (1978) Kinetics and consecutive reactions of soil enzymes. In: Burns RG (ed) soil enzymes. Academic Press, New York, pp 97–116Google Scholar
  55. Michaelis L, Menten MI (1913) Die Kinetik der Invertinwirkung. Biochemische Zeitschrift 49:333–369Google Scholar
  56. Moorhead DL, Sinsabaugh RL (2006) A theoretical model of litter decay and microbial interaction. Ecol Monogr 76:151–174CrossRefGoogle Scholar
  57. Moorhead DL, Lashermes G, Sinsabaugh RL (2012) A theoretical model of C- and N-acquiring exoenzyme activities balancing microbial demands during decomposition. Soil Biol Biochem 53:131–141CrossRefGoogle Scholar
  58. Moscatelli MC, Lagomarsino A, Garzillo AMV, Pignatora A, Grego S (2012) β-Glucosidase kinetic parameters as indicators of soil quality under conventional and organic cropping systems applying two analytical approaches. Ecol Indic 13:332–337CrossRefGoogle Scholar
  59. Münster U (1994) Studies on phosphatase activities in humic lakes. Environ Int 20:49–59CrossRefGoogle Scholar
  60. Nannipieri P, Gelsomino A, Felici M (1991) Method to determine guaiacol oxidase activity in soil. Soil Sci Soc Am J 55:1347–1352CrossRefGoogle Scholar
  61. Nedoma J, Van Wambeke F, Strojsova A, Strojsova M, Duhamel S (2007) Affinity of extracellular phosphatases for ELF97 phosphate in aquatic environments. Mar Freshw Res 58:454–460CrossRefGoogle Scholar
  62. Nor YM (1982) Soil urease activity and kinetics. Soil Biol Biochem 14:63–65CrossRefGoogle Scholar
  63. Olson RJ (1981) 15 N tracer studies of the primary nitrite maximum. J Mar Res 39:203–226Google Scholar
  64. Quiquampoix H, Servagent-Noinville S, Baron M-H (2002) Enzyme adsorption on soil mineral surfaces and consequences for catalytic activity. In: Burns RG, Dick RP (eds) Enzymes in the environment. Marcel Dekker, New York, pp 285–306Google Scholar
  65. Rath J, Schiller C, Herndl GJ (1993) Ectoenzymatic activity and bacterial dynamics along a trophic gradient in the Caribbean Sea. Mar Ecol Prog Series 102:89–96CrossRefGoogle Scholar
  66. Rich JJ, King GM (1999) Carbon monoxide consumption and production by wetland peats. FEMS Microb Ecol 28:215–224CrossRefGoogle Scholar
  67. Saliot A, Cauwet G, Cahet G, Mazaudier D, Daumas R (1996) Microbial activities in the Lena River delta and Laptev Sea. Mar Chem 53:247–254CrossRefGoogle Scholar
  68. Schimel JP, Weintraub MN (2003) The implications of exoenzyme activity on microbial carbon and nitrogen limitation in soil: a theoretical model. Soil Biol Biochem 35:549–563CrossRefGoogle Scholar
  69. Seip KL, Reynolds CS (1995) Phytoplankton functional attributes along trophic gradient and season. Limnol Oceanogr 40:589–597CrossRefGoogle Scholar
  70. Senwo ZN, Tabatabai MA (1996) Aspartase activity of soils. Soil Sci Soc Am J 60:1416–1422CrossRefGoogle Scholar
  71. Sinsabaugh RL, Follstad Shah JJ (2012) Ecoenzymatic stoichiometry and ecological theory. Annu Rev Ecol Evol Syst 43:313–342CrossRefGoogle Scholar
  72. Sinsabaugh RL, Findlay S, Franchini P, Fischer D (1997) Enzymatic analysis of riverine bacterioplankton production. Limnol Oceanogr 42:29–38CrossRefGoogle Scholar
  73. Sinsabaugh RL, Lauber CL, Weintraub MN, Ahmed B, Allison SD, Crenshaw C, Contosta AR, Cusack D, Frey S, Gallo ME, Gartner TB, Hobbie SE, Holland K, Keeler BL, Powers JS, Stursova M, Takacs-Vesbach C, Waldrop M, Wallenstein M, Zak DR, Zeglin LH (2008) Stoichiometry of soil enzyme activity at global scale. Ecol Lett 11:1252–1264Google Scholar
  74. Sinsabaugh RL, Hill BH, Follstad Shah JJ (2009) Ecoenzymatic stoichiometry of microbial organic nutrient acquisition in soil and sediment. Nature 462:795–798CrossRefGoogle Scholar
  75. Sinsabaugh RL, Follstad Shah JJ, Hill BH, Elonen CM (2012) Ecoenzymatic stoichiometry of stream sediments with comparison to terrestrial soils. Biogeochemistry 111:455–467CrossRefGoogle Scholar
  76. Siuda W, Chróst RJ (2002) Decomposition and utilization of particulate organic matter by bacteria in lakes of different trophic status. Polish J Environ Studies 11:53–65Google Scholar
  77. Siuda W, Kiersztyn B, Chróst RJ (2007) The dynamics of protein decomposition in lakes of different trophic status—reflections on the assessment of the real proteolytic activity in situ. J Microb Biotechnol 17:897–904Google Scholar
  78. Somville M, Billen G (1983) A method for determining exoproteolytic activity in natural waters. Limnol Oceanogr 28:190–193CrossRefGoogle Scholar
  79. Sorrell BK, Downes MT, Stanger CL (2002) Methanotrophic bacteria and their activity on submerged aquatic macrophytes. Aquat Bot 72:107–119CrossRefGoogle Scholar
  80. Stark JM, Firestone MK (1996) Kinetic characteristics of ammonium-oxidizer communities in a California oak woodland-annual grassland. Soil Biol Biochem 28:1307–1317CrossRefGoogle Scholar
  81. Stone MM, Plante AF (2014) Changes in phosphatase kinetics with soil depth across a variable tropical landscape. Soil Biol Biochem 71:61–67CrossRefGoogle Scholar
  82. Stone MM, Weiss MS, Goodale CL, Adams MB, Fernandez IJ, German DP, Allison SD (2012) Temperature sensitivity of soil enzyme kinetics under N-fertilization in two temperate forests. Global Change Biol 18:1173–1184CrossRefGoogle Scholar
  83. Strickland MS, McCulley RL, Bradford MA (2013) The effect of a quorum-quenching enzyme on leaf litter decomposition. Soil Biol Biochem 64:65–67CrossRefGoogle Scholar
  84. Tabatabai MA, Garcia-Manzanedo AM, Acosta-Martinez V (2002) Substrate specificity of arylamidase in soils. Soil Biol Biochem 34:103–110CrossRefGoogle Scholar
  85. Thompson A, Sinsabaugh RL (2000) Matric and particulate phosphatase and leucine aminopeptidase activity in limnetic biofilms. Aquatic Microb Ecol 21:151–159CrossRefGoogle Scholar
  86. Toetz DW, Varga LP, Loughran ED (1973) Half-saturation constants for uptake of nitrate and ammonia by reservoir plankton. Ecology 54:903–908CrossRefGoogle Scholar
  87. Vega LM, Alvarez PJ, McLean RJC (2014) Bacterial signaling ecology and potential applications during aquatic biofilm construction. Microb Ecol 68:24–44CrossRefGoogle Scholar
  88. Wagai R, Kishimoto-Mo AW, Yonemura S, Shirato Y, Hiradate S, Yagasaki Y (2013) Linking temperature sensitivity of soil organic matter decomposition to its molecular structure, accessibility, and microbial physiology. Global Change Biol 19:1114–1125CrossRefGoogle Scholar
  89. Westerhoff HV, Hellingworth KJ, Van Dam K (1983) Thermodynamic efficiency of microbial growth is low but optimal for maximal growth rate. Proc Natl Acad Sci 80:305–309CrossRefGoogle Scholar
  90. Wetzel RG (1991) Extracellular enzymatic interactions: storage, redistribution and interspecific communication. In: Chrost RJ (ed) Microbial enzymes in aquatic environments. Springer-Verlag, New York, pp 6–28CrossRefGoogle Scholar
  91. Whalen SC, Reeburgh WS (2001) Carbon monoxide consumption in upland boreal forest soils. Soil Biol Biochem 33:1329–1338CrossRefGoogle Scholar
  92. Williams PJI (1973) The validity of the application of simple kinetic analysis to heterogeneous microbial populations. Limnol Oceanogr 18:159–165CrossRefGoogle Scholar
  93. Zhang YHP (2011) Substrate channeling and enzyme complexes for biotechnological applications. Biotechnol Adv 29:715–725CrossRefGoogle Scholar
  94. Zhang YL, Chen LJ, Sun CX, Li DP, Wu ZJ, Duan ZH (2010) Kinetic and thermodynamic properties or hydrolases in northeastern Chinese soils affected by temperature. Agrochimica 46:232–243Google Scholar
  95. Zhuang XL, Gao J, Ma AZ, Fu SL, Zhuang GQ (2013) Bioactive Molecules in Soil Ecosystems: masters of the Underground. Int J Mol Sci 5:8841–8868CrossRefGoogle Scholar

Copyright information

© Springer International Publishing Switzerland 2014

Authors and Affiliations

  • Robert L. Sinsabaugh
    • 1
  • Jayne Belnap
    • 2
  • Stuart G. Findlay
    • 3
  • Jennifer J. Follstad Shah
    • 4
  • Brian H. Hill
    • 5
  • Kevin A. Kuehn
    • 6
  • Cheryl R. Kuske
    • 7
  • Marcy E. Litvak
    • 1
  • Noelle G. Martinez
    • 1
  • Daryl L. Moorhead
    • 8
  • Daniel D. Warnock
    • 1
  1. 1.Biology DepartmentUniversity of New MexicoAlbuquerqueUSA
  2. 2.Southwest Biological Science CenterU.S. Geological SurveyMoabUSA
  3. 3.Cary Institute of Ecosystem StudiesMillbrookUSA
  4. 4.Watershed Sciences DepartmentUtah State UniversityLoganUSA
  5. 5.National Health and Environmental Effects Laboratory, Mid-Continent Ecology Division, Office of Research and DevelopmentU.S. Environmental Protection AgencyDuluthUSA
  6. 6.Department of Biological SciencesUniversity of Southern MississippiHattiesburgUSA
  7. 7.Bioscience DivisionLos Alamos National LaboratoryLos AlamosUSA
  8. 8.Department of Environmental ScienceUniversity of ToledoToledoUSA

Personalised recommendations