Enhanced Biodegradation of Total Petroleum Hydrocarbons (TPHs) in Contaminated Soil using Biocatalyst

  • Owen, Jeffrey S. ;
  • Pyo, Sunyeon ;
  • Kang, Guyoung
  • Received : 2015.09.25
  • Accepted : 2015.10.28
  • Published : 2015.10.31


Biocatalytic degradation of total petroleum hydrocarbons (TPHs) in contaminated soil by hemoglobin and hydrogen peroxide is an effective soil remediation method. This study used a laboratory soil reactor experiment to evaluate the effectiveness of a nonspecific biocatalytic reaction with hemoglobin and H2O2 for treating TPH-contaminated soil. We also quantified changes in the soil microbial community using real-time PCR analysis during the experimental treatment. The results show that the measured rate constant for the reaction with added hemoglobin was 0.051/day, about 3.5 times higher than the constant for the reaction with only H2O2 (0.014/day). After four weeks of treatment, 76% of the initial soil TPH concentration was removed with hemoglobin and hydrogen peroxide treatment. The removal of initial soil TPH concentration was 26% when only hydrogen peroxide was used. The soil microbial community, based on 16S rRNA gene copy number, was higher (7.1 × 106 copy number/g of bacteria, and 7.4 × 105 copy number/g of Archaea, respectively) in the hemoglobin catalyzed treatment. Our results show that TPH treatment in contaminated soil using hemoglobin catalyzed oxidation led to the enhanced removal effectiveness and was non-toxic to the native soil microbial community in the initial soil.


Hemoglobin;Hydrogen peroxide;Soil remediation;Total petroleum hydrocarbon (TPH);Real time PCR


  1. Rice, R.H., Lee, Y.M., and Brown, W.D., 1983, Interactions of heme protein with hydrogen peroxide: Protein cross-linking and covalent binding of benzo[a]pyrene and 17B-estradiol, Arch. Biochem. Biophys., 221(2), 417-427.
  2. Stahl, D.A. and Amann, R.I., 1991, Development and application of nucleic acid probes. In: E. Stackebrandt and M. Goodfellow (eds.), Nucleic Acid Techniques in Bacterial Systematics, John Wiley & Sons, New York, p. 205-248.
  3. Takai, K. and Horikoshi, K., 2000, Rapid detection and quantification of members of the archaeal community by quantitative PCR using fluorogenic probes, Appl. Environ. Microbiol., 66, 5066-5072.
  4. Umezawa, T. and Higuchi, T., 1989, Aromatic ring cleavage by lignin peroxidase. In: N.G. Lewis and M.G. Paice (eds.), Plant Cell Wall Polymers Biogenesis and Biodegradation, American Chemical Society, Washington, DC, p. 503-518.
  5. Chung, N., Park, K., Stevens, S.K., and Kang, G., 2014, Verification of heme catalytic cycle with 5-aminosalicylic acid and its application to soil remediation of polycyclic aromatic hydrocarbons, Environ. Eng. Res., 19(2), 139-143.
  6. Danner, D.J., Brognac, P.J., Arceneaux, D., and Chandra Patel, V., 1973, The oxidation of phenol and its reaction product by horseradish peroxidase and hydrogen peroxide, Arch. Biochem. Biophys., 156, 759-763.
  7. Hong, J. and Cho, J., 2015, Environmental variables shaping the ecological niche of Thaumarchaeota in soil: Direct and indirect causal effects, PLoS One, 10(8):e0133763. doi:10.1371, 1-20.
  8. Kang, G., Park, K., Cho, J., Stevens, D.K., and Chung, N., 2015, Remediation of polycyclic aromatic hydrocarbons in soil using hemoglobin-catalytic mechanism, J. Environ. Eng., ASCE, ISSN 0733-9372/04015025-1-5. doi:10.1061/(ASCE)EE.1943-7870.0000955.
  9. Ministry of Environment (Korea), 2009, Official Test Method on Soil Pollution, ES 07552.1, 174-185.
  10. Nadkarni, M.A., Martin, F.E., Jacques, N.a., and Hunter, N., 2002, Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primers set, Microbiology, 148, 257-266.
  11. Oh, S.Y. and Shin, D.S., 2013, Treatment of diesel-contaminated soil by Fenton and persulfate oxidation with zero-valent iron, Soil Sed. Contam., 23(2), 180-193.
  12. Palmroth, M., Langwaldt, J.H., Aunola, T.A., Goi, A., Puhakka, J.A., and Tuhkanen, T.A., 2006, Treatment of PAH-contaminated soil by combination of Fenton’s reaction and biodegradation, J. Chem. Technol. Biotechnol., 81, 598-607.
  13. Casamayor, E., Massana, R., Benlloch, S., Øvreås, L., Díez, B., and Goddard, V.J., 2002, Changes in archaeal, bacterial and eukaryal assemblages along a salinity gradient by comparison of genetic fingerprinting methods in a multipond solar saltern. Environ. Microbiol., 4, 338-348.
  14. Chen, S., Stevens, D.K., and Kang, G., 1999, Pentachlorophenol and crystal violet degradation in water and soils using heme and hydrogen peroxide, Wat. Res., 33(17), 3657-3665.
  15. Chen, S., Stevens, D.K., Kang, G., and Hsieh, M., 2006, Treating soil PCP at optimal conditions using heme and peroxide, J. Environ. Eng., ASCE, 132(7), 704-708.
  16. Chen, S., Stevens, D.K., Kang, G., Hsu, J., and Wu, S., 2009, Kinetics of pentachlorophenol degradation in soil using heme and peroxide, J. Environ. Eng., ASCE, 135(4), 279-284.


Grant : 분말헤모글로빈을 이용한 다환방향족 탄화수소 (PAHs) 오염토양 정화기술