Microwave assisted biocidal extraction is an alternative method to measure microbial biomass of carbon from cultivated and non-cultivated soils

  • Janksyn Bertozzi
  • Diva S. AndradeEmail author
  • Cláudio C. Oliveira
  • Abdullahi Bala
  • João Henrique Caviglione
Environmental Microbiology - Research Paper


Developing simple and cost-effective methods for soil microbial biomass carbon (MBC) measurement eases routine laboratory analysis and enables large numbers of soil samples to be measured in a relatively short period of time. Thus, the objective of this study was to develop a microwave-assisted biocidal-extraction (MWE) method which does not employ CHCl3 as biocide and K2SO4 as C-extractor, to estimate MBC. First, the microorganisms of soil samples are killed using microwave (MW) irradiation at energy level of 800 J g−1 soil as biocide followed by microwave irradiation extraction (MWE) at 562 W (120 J g−1 soil for 1 min), using deionized water as solvent. Microbial biomass of carbon from two contrasting soils microwaved with 80, 100, and 140 J g−1 soil did not differ from those obtained by using the chloroform fumigation-extraction (CFE) method with 0.5 mol L−1 K2SO4 as extractant. To evaluate the robustness of the MWE method, twenty-six soil samples, from cultivated and non-cultivated areas, with clay contents from 70–690 g kg−1, organic carbon from 5.52 to 50.82 g C kg−1 and pH values from 3.9 to 6.8 were analyzed for MBC using MWE and CFE methods. There was a linear regression (MW = − 17.87 + 0.92*K2SO4; R2 = 0.705; p < 0.001) between MWE and CFE. The biocidal microwave-assisted extraction method using 120 J g−1 soil for 1 min is a cleaner method for evaluating MBC, because it does not require chloroform, potassium sulfate salt and takes a shorter time to extract a set of soil samples.


Chloroform fumigation-extraction Extractable carbon Microwave extraction Microwave energy Soil microorganisms 



The first author acknowledges a scholarship from National Council for Scientific and Technological Development (CNPq) at the Universidade Estadual de Maringá (UEM), PR, Brazil. This work was partially supported by the National Council for the Improvement of Higher Education (CAPES, 001). DSA is also research fellow of CNPq (312996/2017-9).

Author contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Janksyn Bertozzi, Diva S. Andrade, and João Henrique Caviglione. The first draft of the manuscript was written by Janksyn Bertozzi, Cláudio C. Oliveira, and Diva S. Andrade and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. 1.
    Alessi DS, Walsh DM, Fein JB (2011) Uncertainties in determining microbial biomass C using the chloroform fumigation–extraction method. Chem Geol 280:58–64. CrossRefGoogle Scholar
  2. 2.
    Araújo ASF, Santos VB, Monteiro RTR (2008) Responses of soil microbial biomass and activity for practices of organic and conventional farming systems in Piauí state. Europ J Soil Biol 44:225–230. CrossRefGoogle Scholar
  3. 3.
    Badalucco L, Gelsomino A, Dell’Orco S, Grego IS, Nannipieri P (1992) Biochemical characterization of soil organic compounds extracted by 0.5 M K2S04 before and after chloroform fumigation. Soil Biol Biochem 24:569–578. CrossRefGoogle Scholar
  4. 4.
    Brookes PC, Kragt JF, Powlson DS, Jenkinson DS (1985) Chloroform fumigation and the release of soil nitrogen: the effects of fumigation time and temperature. Soil Biol Biochem 17:831–835. CrossRefGoogle Scholar
  5. 5.
    de Araujo A (2010) Is the microwave irradiation a suitable method for measuring soil microbial biomass? Rev Environ Sci Biotechnol 9:317–321. CrossRefGoogle Scholar
  6. 6.
    De La Hoz A, Díaz-Ortiz A, Moreno A (2005) Microwave in organic synthesis. Termal and nonthermal microwave effects. Chem Soc Rev 34:164–178CrossRefGoogle Scholar
  7. 7.
    de Sousa RS, Nunes LAPL, JEL A, de Araujo ASF (2019) Maize rhizosphere soil stimulates greater soil microbial biomass and enzyme activity leading to subsequent enhancement of cowpea growth. Environ Sustainability 2:89–94. CrossRefGoogle Scholar
  8. 8.
    Diprose MF (2001) Some considerations when using a microwave oven as a laboratory research tool. Plant Soil 229:271–280CrossRefGoogle Scholar
  9. 9.
    Eberhardt U, Apel G, Joergensen RG (1996) Effects of direct chloroform fumigation on suspended cells of 14C and 32P labelled bacteria and fungi. Soil Biol Biochem 28:677–679. CrossRefGoogle Scholar
  10. 10.
    Ferreira AS, Camargo FAO, Vidor C (1999) Utilização de microondas na avaliação da biomassa microbiana do solo. Rev Bras Ciênc Solo 23:991–996. CrossRefGoogle Scholar
  11. 11.
    Ferriss RS (1984) Effects of microwave oven treatment on microorganisms in soil. Phytopathology 74:121–126. CrossRefGoogle Scholar
  12. 12.
    Franzluebbers AJ, Haney RL, Hons FM, Zuberer DA (1999) Assessing biological soil quality with chloroform fumigation-incubation: why subtract a control? Can J Soil Sci 79:521–528. CrossRefGoogle Scholar
  13. 13.
    Haney RL, Franzluebbers AJ, Hons FM, Hossner LR, Zuberer DA (2001) Molar concentration of K2SO4 and soil affect estimation of extractable C with chloroform fumigation-extraction Soil Biology. Biochemistry 33:1501–1507Google Scholar
  14. 14.
    Haney RL, Franzluebbers AJ, Hons FM, Zuberer DA (1999) Soil C extracted with water or K2SO4: pH effect on determination of microbial biomass. Can J Soil Sci 79:529–533. CrossRefGoogle Scholar
  15. 15.
    Hendricks CW, Pascoe N (1988) Soil microbial biomass estimates using 2450 MHz microwave irradiation. Plant Soil 110:39–47. CrossRefGoogle Scholar
  16. 16.
    Islam KR, Weil RR (1998) Microwave irradiation of soil for routine measurement of microbial biomass carbon. Biol Fertil Soils 27:408–416. CrossRefGoogle Scholar
  17. 17.
    Jenkinson DS, Powlson DS (1976) The effects of biocidal treatments on metabolism in soil—V: a method for measuring soil biomass. Soil Biol Biochem 8:209–213. CrossRefGoogle Scholar
  18. 18.
    Kappe CO (2008) Microwave dielectric heating in synthetic organic chemistry. Chem Soc Rev 37:1127–1139. CrossRefGoogle Scholar
  19. 19.
    Leite LFC, Oliveira FC, Araújo ASF, Galvão SRS, Lemos JO et al (2010) Soil organic carbon and biological indicators in an Acrisol under tillage systems and organic management in north-eastern. Brazil Soil Res 48:258–265. CrossRefGoogle Scholar
  20. 20.
    Maynaud G, Baudoin E, Bourillon J, Duponnois R, Cleyet-Marel J-C et al (2019) Short-term effect of 915-MHz microwave treatments on soil physicochemical and biological properties. Eur J Soil Sci 0. CrossRefGoogle Scholar
  21. 21.
    Mendes IC, Souza LM, Sousa DMG, Lopes AAC, Reis-Junior FB et al (2019) Critical limits for microbial indicators in tropical Oxisols at post-harvest: the FERTBIO soil sample concept. Appl Soil Ecol. CrossRefGoogle Scholar
  22. 22.
    Monz CA, Reuss DE, Elliotr ET (1991) Soil microbial biomass carbon and nitrogen estimates using 2450 MHz microwave irradiation or chloroform fumigation followed by direct extraction. Agric Ecosyst Environ 34:55–63. CrossRefGoogle Scholar
  23. 23.
    Moura RTA, Garrido MS, Sousa CS, Menezes RSC, Sampaio EVSB (2018) Comparison of methods to quantify soil microbial biomass carbon. Acta Sci, Agron 40. CrossRefGoogle Scholar
  24. 24.
    Nelson DW, Sommers LE (1982) Total carbon, organic carbon and organic matter. In: Page AL, Miller RH, Keeney D (eds) Methods of Soil Analysis, 2nd, vol 2. Am Soc Agron, Madison, pp 539–594Google Scholar
  25. 25.
    Plaza M, Turner C (2015) Pressurized hot water extraction of bioactives. TrAC Trends Anal Chem 71:39–54. CrossRefGoogle Scholar
  26. 26.
    Rennert T, Gockel KF, Mansfeldt T (2007) Extraction of water-soluble organic matter from mineral horizons of forest soils. J Plant Nutr Soil Sci 170:514–521. CrossRefGoogle Scholar
  27. 27.
    Reyes-Escogido L, Balam-Chi M, Rodrıíguez-Buenfil I, Valdes J, Kameyama L et al (2010) Purification of bacterial genomic DNA in less than 20 min using chelex-100 microwave: examples from strains of lactic acid bacteria isolated from soil samples. Antonie van Leeuwenhoek 98:465–474. CrossRefPubMedGoogle Scholar
  28. 28.
    Ross DJ (1989) Estimation of soil microbial C by a fumigation-extraction procedure: influence of soil moisture content. Soil Biol Biochem 21:767–772. CrossRefGoogle Scholar
  29. 29.
    Rotbart N, Borisover M, Bukhanovsky N, Nasonova A, Bar-Tal A et al (2017) Examination of residual chloroform interference in the measurement of microbial biomass C by fumigation-extraction. Soil Biol Biochem 111:60–65. CrossRefGoogle Scholar
  30. 30.
    Růžek L, Růžková M, Voříšek K, Kubát J, Friedlová M et al (2009) Chemical and microbiological characterization of Cambisols, Luvisols and Stagnosols. Plant Soil Environ 55:231–237. CrossRefGoogle Scholar
  31. 31.
    SAS II (1999) SAS/ STAT user’s guide Cary, N C, USA 12:Cary, N. C, USA. SAS Institute IncGoogle Scholar
  32. 32.
    Silva AS, Colozzi Filho A, Nakatani AS, Alves SJ, Andrade DS et al (2015) Microbial characteristics of soils under an integrated crop-livestock system. Rev Bras Ciênc Solo 39:40–48. CrossRefGoogle Scholar
  33. 33.
    Sparling G, Vojvodic-Vukovic M, Schipper LA (1998) Hot-water-soluble c as a simple measure of labile soil organic matter: the relationship with microbial biomass c. Soil Biol Biochem 30:1469–1472CrossRefGoogle Scholar
  34. 34.
    Sparling GP, West AW (1988) A direct extraction method to estimate soil microbial C: calibration in situ using microbial respiration and 14C labelled cells. Soil Biol Biochem 20:337–343. CrossRefGoogle Scholar
  35. 35.
    Vance ED, Brookes PC, Jenkinson DS (1987a) An extraction method for measuring soil microbial biomass. Soil Biol Biochem 19:703–707. CrossRefGoogle Scholar
  36. 36.
    Vance ED, Brookes PC, Jenkinson DS (1987b) Microbial biomass measurements in forest soils: determination of KC values and test of hypoteses to explain the failure of the chloroform fumigation-incubation method in acid soils. Soil Biol Biochem 19:689–696. CrossRefGoogle Scholar
  37. 37.
    Vega-Ávila A, Medina E, Paroldi H, Toro M, Baigori M et al (2018) Bioindicators of soil quality of open shrubland and vineyards. J Soil Sci Plant Nutr 18:1065–1079. CrossRefGoogle Scholar
  38. 38.
    Vela GR, Wu JF (1979) Mechanism of lethal action of 2,450-MHz radiation on microorganisms. Appl Environ Microbiol 37:550–553PubMedPubMedCentralGoogle Scholar
  39. 39.
    Wainwright M, Killham K, Diprose MF (1980) Effects of 2450 MHz microwave radiation on nitrification, respiration and S-oxidation in soil. Soil Biol Biochem 12:489–493. CrossRefGoogle Scholar
  40. 40.
    Wang W, Dalal RC, Moody PW (2001) Evaluation of the microwave irradiation method for measuring soil microbial biomass. Soil Sci Soc Am J 65:1696–1703CrossRefGoogle Scholar
  41. 41.
    Weil RR, Islam KR (2003) Comments on “evaluation of the microwave irradiation method for measuring soil microbial biomass”. Soil Sci Soc Am J 67:674–675CrossRefGoogle Scholar
  42. 42.
    Xiao D, Xiao S, Ye Y, Zhang W, He X et al (2019) Microbial biomass, metabolic functional diversity, and activity are affected differently by tillage disturbance and maize planting in a typical karst calcareous soil. J Soils Sediments 19:809–821. CrossRefGoogle Scholar
  43. 43.
    Zagal E (1989) Effects of microwave radiation on carbon and nitrogen mineralization in soil. Soil Biol Biochem 21:603–605. CrossRefGoogle Scholar
  44. 44.
    Zhou BW, Shin SG, Hwang K, Ahn J-H, Hwang S (2010) Effect of microwave irradiation on cellular disintegration of Gram positive and negative cells. Appl Microbiol Biotechnol 87:765–770. CrossRefPubMedGoogle Scholar

Copyright information

© Sociedade Brasileira de Microbiologia 2019

Authors and Affiliations

  1. 1.Universidade Tecnológica Federal do Paraná (UTFPR) campus LondrinaLondrinaBrazil
  2. 2.Department of Soil Science–PPG-Agricultura ConservacionistaInstituto Agronômico do Paraná (IAPAR)LondrinaBrazil
  3. 3.Universidade Estadual de Maringá (UEM)MaringáBrazil
  4. 4.Federal University of Technology, MinnaMinnaNigeria

Personalised recommendations