Sterilize Methods Comparison for Soils

Cost, Time, and Efficiency




Soil sterilization is generally used to eliminate or reduce microbial activity in studies involving microbial inoculations, soil enzymes, among others. Achieving an adequate sterility condition is not straightforward due to the variety of resistance structures that are generated in soil microbial ecosystems and the reservoirs that can form between soil aggregates. This is why finding an effective method to achieve good sterilization is important in methodological terms, so the present work aims to compare the effectiveness of three widely used methodologies to sterilize soil and to evaluate their cost/benefit in terms of time and inputs invested. Four treatments were tested: gamma irradiation, sterilization cycles at different times: three cycles of 1 h each and four cycles of 15 min each, and chloroform vapors. The evaluation and comparison of all samples sterilized by the different methodologies were based on the total aerobic heterotrophic bacterial count. The results of this study suggest that it is more efficient to use autoclaving methods because the process is more accessible in terms of equipment and methodologies, and the final results are the same. In the case of this work, sterilization with chloroform vapors had to be rejected. While the use of gamma radiation may be more efficient in terms of time, it can be a costly and inaccessible service for some laboratories that do not have the equipment. Therefore, the most viable options in terms of time, cost, and benefit are those using autoclaves. Among these, shorter treatment times mean a reduction in the cost of using the equipment, so the option of 15-minute cycles is desirable.


Gamma irradiation, Autoclave, Soil bacteria


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S. Mocali et al., “Short-Term Resilience of Soil Microbial Communities and Functions Following Severe Environmental Changes,” Agriculture (Switzerland), vol. 12, no. 2, Feb. 2022, doi: 10.3390/agriculture12020268.

D. Vullo, M. Wachsman, and L. Alché, MICROBIOLOGÍA EN PRÁCTICA. Manual de técnicas de laboratorio para la enseñanza de microbiología básica y aplicada. CIudad de Buenos Aires: Editorial Atlante S.R.Ll, 2000.

D. C. Wolf, T. H. Dao, H. D. Scott, and T. L. Lavy, “Influence of Sterilization Methods on Selected Soil Microbiological, Physical, and Chemical Properties,” J Environ Qual, vol. 18, no. 1, pp. 39–44, 1989, doi: 10.2134/jeq1989.00472425001800010007x.

T. Mahmood, S. Mehnaz, F. Fleischmann, R. Ali, Z. H. Hashmi, and Z. Iqbal, “Soil sterilization effects on root growth and formation of rhizosheaths in wheat seedlings,” Pedobiologia (Jena), vol. 57, no. 3, pp. 123–130, 2014, doi: 10.1016/j.pedobi.2013.12.005.

J. T. Trevors, “Sterilization and inhibition of microbial activity in soil,” J Microbiol Methods, vol. 26, no. 1–2, pp. 53–59, 1996, doi: 10.1016/0167-7012(96)00843-3.

A. Soni, I. Oey, P. Silcock, and P. Bremer, “Bacillus Spores in the Food Industry: A Review on Resistance and Response to Novel Inactivation Technologies,” Compr Rev Food Sci Food Saf, vol. 15, no. 6, pp. 1139–1148, Nov. 2016, doi: 10.1111/1541-4337.12231.

P. Setlow, “Spores of Bacillus subtilis: Their resistance to and killing by radiation, heat and chemicals,” in Journal of Applied Microbiology, Sep. 2006, pp. 514–525. doi: 10.1111/j.1365-2672.2005.02736.x.

P. Setlow, “Spore Resistance Properties,” 2014, doi: 10.1128/microbiolspec.

J. Hu et al., “Combined selective gamma irradiation and pulverized soil inoculation for ecologically relevant soil microfauna studies,” Applied Soil Ecology, vol. 169, Jan. 2022, doi: 10.1016/j.apsoil.2021.104223.

S. Mohy, T. M. Elameen, R. Khalaphallah, and N. M. S. Hassan, “Antifungal activity of cinnamon and mint extracts against white and basal rot of garlic,” SVU-International Journal of Agricultural Sciences, vol. 4, no. 4, pp. 136–144, Oct. 2022, doi: 10.21608/svuijas.2022.181411.1255.

P. Dietrich, S. Cesarz, N. Eisenhauer, and C. Roscher, “Effects of steam sterilization on soil abiotic and biotic properties,” Soil Org, vol. 92, no. 2, pp. 99–108, 2020, doi: 10.25674/so92iss2pp99.

J. Giampaoli, C. Brandan, R. Enrico, M. Coll Araoz, and V. Lencina, “Efecto de la esterilización con métodos físicos en suelo, sobre la flora micorrícica y en el cultivo del yacón (Smallanthus sonchifolius) en Horco Molle, Tucumán,” Rev. Agron. Noroeste Arg., vol. 34, pp. 36–39, 2014.

T. Mahmood, S. Mehnaz, F. Fleischmann, R. Ali, Z. H. Hashmi, and Z. Iqbal, “Soil sterilization effects on root growth and formation of rhizosheaths in wheat seedlings,” Pedobiologia (Jena), vol. 57, no. 3, pp. 123–130, May 2014, doi: 10.1016/J.PEDOBI.2013.12.005.

J. T. Trevors, “Sterilization and inhibition of microbial activity in soil,” J Microbiol Methods, vol. 26, no. 1–2, pp. 53–59, Jul. 1996, doi: 10.1016/0167-7012(96)00843-3.

J. N. Horton, M. Sussman, and W. W. Mushin, “THE ANTIBACTERIAL ACTION OF ANAESTHETIC VAPOURS,” Br J Anaesth, vol. 42, no. 6, pp. 483–487, Jun. 1970, doi: 10.1093/BJA/42.6.483.

P. J. R. Challen, D. E. Hickish, and J. Bedford, “CHRONIC CHLOROFORM INTOXICATION,” 1958.

R. J. Golden, S. E. Holm, D. E. Robinson, P. H. Julkunen, and E. A. Reese, “Chloroform Mode of Action: Implications for Cancer Risk Assessment,” 1997.

D. S. JENKINSON, “Studies on the Decomposition of Plant Material in Soil: Ii. Partial Sterilization of Soil and the Soil Biomass,” Journal of Soil Science, vol. 17, no. 2, pp. 280–302, 1966, doi: 10.1111/j.1365-2389.1966.tb01474.x.

G. S. Acosta Peñaloza, D. J. Palacios Rincón, and L. G. Carvajal Restrepo, “Evaluación de la esterilización húmeda y la esterilización por microondas de dos tipos de suelos,” Revista científica, vol. 1, no. 17, p. 87, 2013, doi: 10.14483/23448350.4567.

C. Fahey and S. L. Flory, “Soil microbes alter competition between native and invasive plants,” Journal of Ecology, vol. 110, no. 2, pp. 404–414, Feb. 2022, doi: 10.1111/1365-2745.13807.

A. Koyama, T. Dias, and P. M. Antunes, “Application of plant–soil feedbacks in the selection of crop rotation sequences,” Ecological Applications, vol. 32, no. 2, Mar. 2022, doi: 10.1002/eap.2501.

S. Lundell et al., “Plant responses to soil biota depend on precipitation history, plant diversity, and productivity,” Ecology, vol. 103, no. 10, Oct. 2022, doi: 10.1002/ecy.3784.

J. E. Hyun, S. Bin Lee, D. Y. Jung, S. R. Kim, S. Y. Choi, and I. Hwang, “Effects of sterilization methods on the survival of pathogenic bacteria in potting soil stored at various temperatures,” Food Sci Biotechnol, vol. 32, no. 1, pp. 111–120, Jan. 2023, doi: 10.1007/s10068-022-01173-1.

E. Ormeño-Orrillo and D. Zúñiga-Dávil, “Optimizacion del tiempo de esterilizacion de soportes basados en suelo y compost en la produccion de jnoculantes para leguminosas,” Rev Peru Biol, vol. 6, no. 2, pp. 181–184, 1999, doi: 10.15381/rpb.v6i2.8313.

M. C. Ogwu et al., “Changes in soil taxonomic and functional diversity resulting from gamma irradiation,” Sci Rep, vol. 9, no. 1, pp. 1–13, 2019, doi: 10.1038/s41598-019-44441-7.

A. F. Caballero Jaramillo, “Evaluación del efecto del control biológico, vaporización, fotólisis UV y fotocatálisis con TiO 2 sobre Fusarium oxysporum en matriz sólida (suelo) y líquida (agua),” Grado de Microbiólogo Industrial, Pontificia Universidad Javeriana, Bogotá, 2012.

P. Setlow, “Resistance of Bacterial Spores,” 2017. [Online]. Available:

F. M. Bento, F. A. O. Camargo, B. C. Okeke, and W. T. Frankenberger, “Comparative bioremediation of soils contaminated with diesel oil by natural attenuation, biostimulation and bioaugmentation,” Bioresour Technol, vol. 96, no. 9, pp. 1049–1055, Jun. 2005, doi: 10.1016/j.biortech.2004.09.008.

M. Viñas, J. Sabaté, M. J. Espuny, and A. M. Solanas, “Bacterial community dynamics and polycyclic aromatic hydrocarbon degradation during bioremediation of heavily creosote-contaminated soil,” Appl Environ Microbiol, vol. 71, no. 11, pp. 7008–7018, Nov. 2005, doi: 10.1128/AEM.71.11.7008-7018.2005.

L. J. Merini, V. Cuadrado, C. G. Flocco, and A. M. Giulietti, “Dissipation of 2,4-D in soils of the Humid Pampa region, Argentina: A microcosm study,” Chemosphere, vol. 68, no. 2, pp. 259–265, 2007, doi: 10.1016/j.chemosphere.2007.01.012.

A. Singh, O. P. Ward, and R. C. Kuhad, “Feasibility Studies for Microbial Remediation Hydrocarbon-Contaminated Soil,” in Soil Biology, 2005.

B. Velázquez-Martí and C. Gracia-López, “THERMAL EFFECTS OF MICROWAVE ENERGY IN AGRICULTURAL SOIL RADIATION,” Int J Infrared Millimeter Waves, vol. 25, no. 7, p. 1109, 2004.

R. Margesin, A. Zimmerbauer, and F. Schinner, “Monitoring of bioremediation by soil biological activities,” Chemosphere, vol. 40, pp. 339–346, 2000.

O. Schoefs, M. Perrier, and R. Samson, “Estimation of contaminant depletion in unsaturated soils using a reduced-order biodegradation model and carbon dioxide measurement,” Appl Microbiol Biotechnol, vol. 64, no. 1, pp. 53–61, Mar. 2004, doi: 10.1007/s00253-003-1423-3.

R. Margesin and F. Schinner, “Efficiency of Indigenous and Inoculated Cold-Adapted Soil Microorganisms for Biodegradation of Diesel Oil in Alpine Soils,” 1997.

C. H. Chaîneau, G. Rougeux, C. Yéprémian, and J. Oudot, “Effects of nutrient concentration on the biodegradation of crude oil and associated microbial populations in the soil,” Soil Biol Biochem, vol. 37, no. 8, pp. 1490–1497, Aug. 2005, doi: 10.1016/j.soilbio.2005.01.012.

D. Wolf and H. Skipper, “Soil Sterilization,” in Methods of Soil Analysis, Madison, USA: Soil Science Society of America, 2011, pp. 1–16.

J. Hu et al., “Combined selective gamma irradiation and pulverized soil inoculation for ecologically relevant soil microfauna studies,” Applied Soil Ecology, vol. 169, p. 104223, Jan. 2022, doi: 10.1016/J.APSOIL.2021.104223.

Y. Luo et al., “Effects of sterilization and maturity of compost on soil bacterial and fungal communities and wheat growth,” Geoderma, vol. 409, Mar. 2022, doi: 10.1016/j.geoderma.2021.115598.

A. Beloian, Methods of testing for sterility: Efficacy of sterilizers, sporicides, and sterilizing processes, 3rd ed. Philadelphia: Lea and Febiger, 1983.

A. E. Berns, H. Philipp, H. D. Narres, P. Burauel, H. Vereecken, and W. Tappe, “Effect of gamma-sterilization and autoclaving on soil organic matter structure as studied by solid state NMR, UV and fluorescence spectroscopy,” Eur J Soil Sci, vol. 59, no. 3, pp. 540–550, Jun. 2008, doi: 10.1111/J.1365-2389.2008.01016.X.

N. P. McNamara, H. I. J. Black, N. A. Beresford, and N. R. Parekh, “Effects of acute gamma irradiation on chemical, physical and biological properties of soils,” Applied Soil Ecology, vol. 24, no. 2, pp. 117–132, Oct. 2003, doi: 10.1016/S0929-1393(03)00073-8.




How to Cite

G. A. Querejeta, “Sterilize Methods Comparison for Soils: Cost, Time, and Efficiency”, Int. J. Methodol., vol. 2, no. 1, pp. 34–40, Apr. 2023.