Effects of Heavy Metal Pollution of Apple Orchard Surface Soils Associated with Past Use of Metal-Based Pesticides on Soil Microbial Biomass and Microbial Communities


Apple orchard surface soils in Japan are polluted with copper (Cu), lead (Pb), and arsenic (As) due to long-term use of metal-based pesticides. We investigated the effects of heavy metals accumulated in the surface soils in apple orchards on the microbial biomass and the microbial communities. Soil samples were taken from a chestnut orchard (unpolluted control) and five apple orchards with different degrees of heavy metal pollution. Total concentrations of Cu, Pb, and As in soil ranged from 29 to 931 mg/kg, 35 to 771 mg/kg, and 11 to 198 mg/kg, respectively. The amount of microbial biomass carbon expressed on a soil organic carbon basis decreased with increasing concentrations of heavy metals. Thus, the heavy metals that accumulated in apple orchard surface soils had adverse effects on the soil microbial biomass. The analysis of phospholipid fatty acid (PLFA) composition indicated that the microbial community structure had changed because of the pesticide-derived heavy metals in soil. The relative abundance of gram-positive bacterial marker PLFAs increased and that of fungal marker PLFA decreased with increasing concentrations of heavy metals in soil. Denaturing gradient gel electrophoreses targeting the 16S ribosomal RNA gene of bacteria and the 18S ribosomal RNA gene of fungi also showed shifts in the composition of bacterial and fungal communities induced by soil pollution with heavy metals. However, the diversity of microbial communities was not significantly affected by the heavy metal pollution. This was attributable to the adaptation of the microbial communities in apple orchard surface soils to heavy metals derived from previously used pesticides.

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M. Aoyama and R. Tanaka, "Effects of Heavy Metal Pollution of Apple Orchard Surface Soils Associated with Past Use of Metal-Based Pesticides on Soil Microbial Biomass and Microbial Communities," Journal of Environmental Protection, Vol. 4 No. 4A, 2013, pp. 27-36. doi: 10.4236/jep.2013.44A005.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] M. Aoyama and T. Nagumo, “Factors Affecting Microbial Biomass and Dehydrogenase Activity in Apple Orchard Soils with Heavy Metal Accumulation,” Soil Science and Plant Nutrition, Vol. 42, No. 4, 1996, pp. 821-831. doi:10.1080/00380768.1996.10416629
[2] K. Chander and P. C. Brookes, “Effects of Heavy Metals from Past Applications of Sewage Sludge on Microbial Biomass and Organic Matter Accumulation in a Sandy Loam and Silty Loam UK Soil,” Soil Biology & Biochemistry, Vol. 23, No. 10, 1991, pp. 927-932. doi:10.1016/0038-0717(91)90172-G
[3] K. Chander and P. C. Brookes, “Residual Effects of Zinc, Copper and Nickel in Sewage Sludge on Microbial Biomass in a Sandy Loam,” Soil Biology & Biochemistry, Vol. 25, No. 9, 1993, pp. 1231-1239. doi:10.1016/0038-0717(93)90219-2
[4] A. Fliessbach, R. Martens and H. H. Reber, “Soil Microbial Biomass and Microbial Activity in Soils Treated with Heavy Metal Contaminated Sewage Sludge,” Soil Biology & Biochemistry, Vol. 26, No. 9, 1994, pp. 1201-1205. doi:10.1016/0038-0717(94)90144-9
[5] K. Chander, P. C. Brookes and S. A. Harding, “Microbial Biomass Dynamics Following Addition of Metal-Enriched Sewage Sludges to a Sandy Loam,” Soil Biology & Biochemistry, Vol. 27, No. 11, 1995, pp. 1409-1421. doi:10.1016/0038-0717(95)00074-O
[6] E. Bååth, “Effects of Heavy Metals in Soil on Microbial Processes and Populations: A Review,” Water, Air, & Soil Pollution, Vol. 47, No. 3-4, 1989, pp. 335-375. doi:10.1007/BF00279331
[7] H. Babich and G. Stotzky, “Heavy Metal Toxicity to Microbe-Mediated Ecologic Processes: A Review and Potential Application to Regulatory Policies,” Environmental Research, Vol. 36, No. 1, 1985, pp. 111-137. doi:10.1016/0013-9351(85)90011-8
[8] K. E. Giller, E. Witter and S. P. McGrath, “Toxicity of Heavy Metals to Microorganisms and Microbial Processes in Agricultural Soils: A Review,” Soil Biology & Biochemistry, Vol. 30, No. 10-11, 1998, pp. 1389-1414. doi:10.1016/S0038-0717(97)00270-8
[9] M. Aoyama and T. Nagumo, “Effects of Heavy Metal Accumulation in Apple Orchard Soils on Microbial Biomass and Microbial Activities,” Soil Science and Plant Nutrition, Vol. 43, No. 3, 1997, pp. 601-612. doi:10.1080/00380768.1997.10414786
[10] M. Aoyama and T. Nagumo, “Comparison of the Effects of Cu, Pb, and As on Plant Residue Decomposition, Microbial Biomass, and Soil Respiration,” Soil Science and Plant Nutrition, Vol. 43, No. 3, 1997, pp. 613-622. doi:10.1080/00380768.1997.10414787
[11] J. L. Kirk, L. A. Beaudette, M. Hart, P. Moutoglis, J. N. Klironomos, H. Lee and J. T. Trevors, “Methods of Studying Soil Microbial Diversity,” Journal of Microbiological Methods, Vol. 58, No. 2, 2004, pp. 169-188. doi:10.1016/j.mimet.2004.04.006
[12] S. Malik, M. Beer, M. Megharaj and R. Naidu, “The Use of Molecular Techniques to Characterize the Microbial Communities in Contaminated Soil and Water,” Environment International, Vol. 34, No. 2, 2008, pp. 265-276. doi:10.1016/j.envint.2007.09.001
[13] E. Bååth, A. Frostegård and H. Fritze, “Soil Bacterial Biomass, Activity, Phospholipid Fatty Acid Pattern, and pH Tolerance in an Area Polluted with Alkaline Dust Deposition,” Applied and Environmental Microbiology, Vol. 58, No. 12, 1992, pp. 4026-4031.
[14] E. Bååth, M. Diaz-Ravina, A. Frostegård and C. Campbell, “Effects of Metal-Rich Sludge Amendments on the Soil Microbial Community,” Applied and Environmental Microbiology, Vol. 64, No. 1, 1998, pp. 238-245.
[15] B. Frey, M. Stemmer, F. Widmer, J. Luster and C. Sperisen, “Microbial Activity and Community Structure of a Soil after Heavy Metal Contamination in a Model Forest Ecosystem,” Soil Biology & Biochemistry, Vol. 38, No. 7, 2006, pp. 1745-1756. doi:10.1016/j.soilbio.2005.11.032
[16] A. Frostegård, E. Bååth and A. Tunlid, “Shifts in the Structure of Soil Microbial Communities in Limed Forests as Revealed by Phospholipid Fatty Acid Analysis,” Soil Biology & Biochemistry, Vol. 25, No. 6, 1993, pp. 723-730. doi:10.1016/0038-0717(93)90113-P
[17] A. Frostegård, A. Tunlid and E. Bååth, “Phospholipid Fatty Acid Composition, Biomass, and Activity of Microbial Communities from Two Soil Types Experimentally Exposed to Different Heavy Metals,” Applied and Environmental Microbiology, Vol. 59, No. 11, 1993, pp. 3605-3617.
[18] M. B. Hinojosa, J. A. Carreira, R. García-Ruíz and R. P. Dick, “Microbial Response to Heavy Metal-Polluted Soils,” Journal of Environmental Quality, Vol. 34, No. 5, 2005, pp. 1789-1800. doi:10.2134/jeq2004.0470
[19] T. Pennanen, A. Frostegård, H. Fritze and E. Bååth, “Phospholipid Fatty Acid Composition and Heavy Metal Tolerance of Soil Microbial Communities along Two Heavy Metal-Polluted Gradinents in Coniferous Forest,” Applied and Environmental Microbiology, Vol. 62, No. 2, 1996, pp. 420-428.
[20] L. Zelles, “Fatty Acid Patterns of Phospholipids and Lipopolysaccharides in the Characterisation of Microbial Communities in Soil: A Review,” Biology and Fertility of Soils, Vol. 29, No. 2, 1999, pp. 111-129. doi:10.1007/s003740050533
[21] R. G. Joergensen and C. Emmerling, “Methods for Evaluating Human Impact on Soil Microorganisms Based on Their Activity, Biomass, and Diversity in Agricultural Soils,” Journal of Plant Nutrition and Soil Science, Vol. 169, No. 3, 2006, pp. 295-309. doi:10.1002/jpln.200521941
[22] I. C. Anderson, P. I. Parkin and C. D. Campbell, “DNA- and RNA-Derived Assessments of Fungal Community Composition in Soil Amended with Sewage Sludge Rich in Cadmium, Copper and Zinc,” Soil Biology & Biochemistry, Vol. 40, No. 9, 2008, pp. 2358-2365. doi:10.1016/j.soilbio.2008.05.015
[23] M. E. F. Boivin, G. D. Greve, S. A. E. Kools, A. W. G. van der Wurff, P. Leeflang, E. Smit, A. M. Breure, M. Rutgers and N. M. van Straalen, “Discriminating between Effects of Metals and Natural Variables in Terrestrial Bacterial Communities,” Applied Soil Ecology, Vol. 34, No. 2-3, 2006, pp. 103-113. doi:10.1016/j.apsoil.2006.03.009
[24] F. Gremion, A. Chatzinotas, K. Kaufmann, W. von Sigler and H. Harms, “Impacts of Heavy Metal Contamination and Phytoremediation on a Microbial Community during a Twelve-Month Microcosm Experiment,” FEMS Microbiology Ecology, Vol. 48, No. 2, 2006, pp. 273-283. doi:10.1016/j.femsec.2004.02.004
[25] Z. Li, J. Xu, C. Tang, J. Wu, A. Muhammad and H. Wang, “Application of 16S rDNA-PCR Amplification and DGGE Fingerprinting for Detection of Shift in Microbial Community Diversity in Cu-, Zn-, and Cd-Contaminated Paddy Soils,” Chemosphere, Vol. 62, No. 8, 2006, pp. 1374-1380. doi:10.1016/j.chemosphere.2005.07.050
[26] Y. P. Wang, J. Y. Shi, H. Wang, Q. Lin, X. C. Chen and Y. X. Chen, “The Influence of Soil Heavy Metals Pollution on Soil Microbial Biomass, Enzyme Activity, and Community Composition Near a Copper Smelter,” Ecotoxicology and Environmental Safety, Vol. 67, No. 1, 2007, pp. 75-81. doi:10.1016/j.ecoenv.2006.03.007
[27] X. Zhou, Z. He, Z. Liang, P. J. Stoffella, J. Fan, Y. Yang and C. A. Powell, “Long-Term Use of Copper-Containing Fungicide Affects Microbial Properties of Citrus Grove Soils,” Soil Science Society of America Journal, Vol. 75, No. 3, 2011, pp. 898-906. doi:10.2136/sssaj2010.0321
[28] C. A. Macdonald, C. D. Campbell, J. R. Bacon and B. K. Singh, “Multiple Profiling of Soil Microbial Communities Identifies Potential Genetic Markers of Metal-Enriched Sewage Sludge,” FEMS Microbiology Ecology, Vol. 65, No. 3, 2008, pp. 555-564. doi:10.1111/j.1574-6941.2008.00538.x
[29] C. A. Macdonald, I. M. Clark, F.-J. Zhao, P. R. Hirsch, B. K. Singh and S. P. McGrath, “Long-Term Impacts of Zinc and Copper Enriched Sewage Sludge Additions on Bacterial, Archaeal and Fungal Communities in Arable and Grassland Soils,” Soil Biology & Biochemistry, Vol. 43, No. 5, 2011, pp. 932-941. doi:10.1016/j.soilbio.2011.01.004
[30] A. Pérez-de-Mora, P. Burgos, E. Madejón, F. Cabrera, P. Jaeckel and M. Schloter, “Microbial Community Structure and Function in a Soil Contaminated by Heavy Metals: Effects of Plant Growth and Different Amendments,” Soil Biology & Biochemistry, Vol. 38, No. 2, 2006, pp. 327-341. doi:10.1016/j.soilbio.2005.05.010
[31] E. Smit, P. Leeflang and K. Wernars, “Detection of Shifts in Microbial Community Structure and Diversity in Soil Caused by Copper Contamination Using Amplified Ribosomal DNA Restriction Analysis,” FEMS Microbiology Ecology, Vol. 23, No. 3, 1997, pp. 249-261. doi:10.1111/j.1574-6941.1997.tb00407.x
[32] R. Turpeinen, T. Kairesalo and M. M. Haggblom, “Microbial Community Structure and Activity in Arsenic-, Chromium- and Copper-Contaminated Soils,” FEMS Microbiology Ecology, Vol. 47, No. 1, 2004, pp. 39-50. doi:10.1016/S0168-6496(03)00232-0
[33] M. Aoyama and S. Kuroyanagi, “Effects of Heavy Metal Accumulation Associated with Pesticide Application on the Decomposition of Orchard Grass in Soils,” Soil Science and Plant Nutrition, Vol. 42, No. 1, 1996, pp. 121-131. doi:10.1080/00380768.1996.10414695
[34] E. D. Vance, P. C. Brookes and D. S. Jenkinson, “An Extraction Method for Measuring Soil Microbial Biomass C,” Soil Biology & Biochemistry, Vol. 19, No. 6, 1987, pp. 703-707. doi:10.1016/0038-0717(87)90052-6
[35] J. Wu, R. G. Joergensen, B. Pommerening, R. Chaussod and P. C. Brookes, “Measurement of Soil Microbial Biomass C by Fumigation Extraction: An Automated Procedure,” Soil Biology & Biochemistry, Vol. 22, No. 8, 1990, pp. 1167-1169. doi:10.1016/0038-0717(90)90046-3
[36] E. G. Bligh and W. J. Dyer, “A Rapid Method of Total Lipid Extraction and Purification,” Canadian Journal of Biochemical Physiology, Vol. 37, No. 8, 1959, pp. 911-917. doi:10.1139/o59-099
[37] T. C. Balser and M. K. Firestone, “Linking Microbial Community Composition and Soil Processes in a California Annual Grassland and Mixed-Conifer Forest,” Biogeochemistry, Vol. 73, No. 2, 2005, pp. 395-415. doi:10.1007/s10533-004-0372-y
[38] G. Muyzer, E. C. D. Waal and A. G. Uitterlinden, “Profiling of Complex Microbial Populations by Denaturing Gradient Gel Electrophoresis Analysis of Polymerase Chain Reaction-Amplified Genes Coding for 16S rRNA,” Applied and Environmental Microbiology, Vol. 59, No. 3, 1993, pp. 695-700.
[39] E. J. Vainio and J. Hantula, “Direct Analysis of Wood-Inhabiting Fungi Using Denaturing Gradient Gel Electrophoresis of Amplified Ribosomal DNA,” Mycological Research, Vol. 104, No. 8, 2000, pp. 927-936. doi:10.1017/S0953756200002471
[40] M. S. Pedro, S. Haruta, M. Hazaka, R. Shimada, C. Yoshida, K. Hiura, M. Ishii and Y. Igarashi, “Denaturing Gradient Gel Electrophoresis Analyses of Microbial Community from Field-Scale Composter,” Journal of Bioscience and Bioengineering, Vol. 91, No. 2, 2001, pp. 159-165. doi:10.1263/jbb.91.159
[41] H. Yanai, “Excel Statistics: Multivariate Analysis Techniques,” OMS, Tokorozawa, Japan, 2005.
[42] J. C. Zak, M. R. Willing, D. L. Moorhead and H. G. Wildman, “Functional Diversity of Microbial Communities: A Quantitative Approach,” Soil Biology & Biochemistry, Vol. 26, No. 9, 1994, pp. 1101-1108. doi:10.1016/0038-0717(94)90131-7
[43] L. Zelles, “Phospholipid Fatty Acid Profiles in Selected Members of Soil Microbial Communities,” Chemosphere, Vol. 35, No. 1-2, 1997, pp. 275-294. doi:10.1016/S0045-6535(97)00155-0
[44] J. B. Brant, E. W. Sulzman and D. D. Myrold, “Microbial Community Utilization of Added Carbon Substrates in Response to Long-Term Carbon Input Manipulation,” Soil Biology & Biochemistry, Vol. 38, No. 8, 1997, pp. 2219-2232. doi:10.1016/j.soilbio.2006.01.022
[45] L. Zelles, Q. Y. Bai, R. X. Ma, R. Rackwitz, K. Winter and F. Beese, “Microbial Biomass, Metabolic Activity and Nutritional Status Determined from Fatty Acid Patterns and Poly-Hydroxybutyrate in Agriculturally-Managed Soils,” Soil Biology & Biochemistry, Vol. 26, No. 4, 1994, pp. 317-323. doi:10.1016/0038-0717(94)90175-9
[46] J. P. E. Anderson and K. H. Domsch, “A Physiological Method for the Quantitative Measurement of Microbial Biomass in Soil,” Soil Biology & Biochemistry, Vol. 10, No. 3, 1978, pp. 207-213. doi:10.1016/0038-0717(78)90098-6
[47] J. P. E. Anderson and K. H. Domsch, “Quantification of Bacterial and Fungal Contributions to Soil Respiration,” Archiv für Mikrobiologie, Vol. 93, No. 2, 1973, pp. 113-127. doi:10.1007/BF00424942
[48] R. M. C. P. Rajapaksha, M. A Tobor-Kaplon and E. Bååth, “Metal Toxicity Affects Fungal and Bacterial Activities in Soil Differently,” Applied and Environmental Microbiology, Vol. 70, No. 5, 2004, pp. 2966-2973. doi:10.1128/AEM.70.5.2966-2973.2004
[49] F. Altimira, C. Yánez, G. Bravo, M. González, L. A. Rojas and M. Seeger, “Characterization of Copper-Resistant Bacteria and Bacterial Communities from Copper-Polluted Agricultural Soils of Central Chile,” BMC Microbiology, Vol. 12, 2012, p. 193. doi:10.1186/1471-2180-12-193
[50] R. J. Ellis, P. Morgan, A. J. Weightman and J. C. Fry, “Cultivation-Dependent and -Independent Approaches for Determining Bacterial Diversity in Heavy-Metal-Contaminated Soil,” Applied and Environmental Microbiology, Vol. 69, No. 6, 2003, pp. 3223-3230. doi:10.1128/AEM.69.6.3223-3230.2003
[51] H. Deng, X. F. Li, W. D. Cheng and Y. G. Zhu, “Resistance and Resilience of Cu-Polluted Soil after Cu Perturbation, Tested by a Wide Range of Soil Microbial Parameters,” FEMS Microbiology Ecology, Vol. 70, No. 2, 2009, pp. 137-148. doi:10.1111/j.1574-6941.2009.00741.x
[52] L. Ranjard, A. Echairi, V. Nowak, D. Lejon, R. Nouaim and R. Chaussod, “Field and Microcosm Experiments to Evaluate the Effects of Agricultural Cu Treatment on the Density and Genetic Structure of Microbial Communities in Two Different Soils,” FEMS Microbiology Ecology, Vol. 58, No. 2, 2006, pp. 303-315. doi:10.1111/j.1574-6941.2006.00157.x

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