The Study of the Phytoremediation Efficiency of Crude Oil Contaminated Soil by Inoculating the Soil with Brachybacterium muris and Pseudomonas putida

Document Type : Research Paper


1 Persian Gulf and Oman Sea Ecological Research Institute, Iranian fisheries science Research Institute, Agricultural Education and Extension Research Organization, Bandar Abbas, Hormozgan, Iran

2 Soil Science Department, Faculty of Agriculture, Zanjan University of Zanjan, Zanjan, Iran.


Petroleum products are considered to be the most widely used and expensive chemicals in the modern world, but pollution from the extraction and transportation of crude oil has become a global environmental problem. In this experiment, the efficiency of phytoremediation, bioremediation and bio-enhanced phytoremediation in removing crude oil from soil was investigated. For this purpose, a factorial experiment was performed in a completely randomized design with three replications. Factors include three levels of soil oil pollution (zero, 4 and 8% by weight), four plant treatments (without plants, bermudagras (Cynodon dactylon), sorghum (Bicolor Sorghum) and barley (Hordeum vulgare)) and three bacterial treatments (without bacteria). Were native bacteria (Brachybacterium muris) and non-native bacteria (Pseudomonas putida). The results showed that the percentage of crude oil removal by plant cultivation, soil inoculation with bacteria and combined application of plant and bacteria increased significantly compared to the control. Plant culture was more effective than bacterial inoculation in reducing the concentration of petroleum products and the efficiency of bacteria increased significantly with plant culture. At each level of contamination, the highest percentage of oil removal was observed with the combined application of sorghum and Brachybacterium muris. In all plant treatments, the highest oil removal percentage was measured at 4% oil pollution and Brachybacterium muris inoculation. Oil pollution significantly reduced leaf dry weight and chlorophyll concentration, but the use of bacteria (especially native bacteria) significantly reduced the negative effects of oil pollution on plants compared to non-inoculation treatments. Oil pollution increased the proline concentration in the leaves of plants and decreased the proline concentration with the use of native bacteria. Establishment of plant with microorganisms can be considered as a key component of the strategy to remove hydrocarbons. Consequently, these bacterial and plant species can be used for the biodegradation of soils contaminated with crude oil.


Alexander, M. (1999). Biodegradation and Bioremediation. Academic Press INC., San Diego, USA.
Al-Hawas, G.H.S., Shukry, W.M., Azzoz, M.M. and Al-Moaik, R.M.S. (2012). The effect of sublethal concentrations of crude oil on the metabolism of Jojoba (Simmodsia chinensis) seedlings. International Research Journal of Plant Science, 3(4), pp.54-62.
Allison, L. E., and Moodie, C. D. (1965). Carbonate. In Methods of soil analysis (Black, C. A. ed.). Part 2, Am. Soc. Agron., Madison, WI. p. 1379-1396.
Andria, V., Reichenauer, T. G. and Sessitsch, A. (2009). Expression of alkane monooxygenase (alkB) genes by plant-associated bacteria in the rhizosphere and endosphere of Italian ryegrass (Lolium multiflorum L.) grown in diesel contaminated soil. Environmental Pollution, 157: 3347-3350.
Arnon, D.I. (1956). Photosynthesis byisolated chloroplast. IV. General concept and comparison of three photochemicalreactions. Biochimica Biophysica Acta, 20: 449-461.
Atlas, R. M. (2005). Handbook of media for environmental microbiology (Atlas, R. M. ed.). CRC Press. The United States of America. pp. 664.
Basumatary, B, Saikia, R., Bordoloi, S., Das, H. C. and Sarma, H. P. (2012). Assessment of potential plant species for phytoremediation of hydrocarbon contaminated areas of upper Assam, India. Journal of Chemical Technology and Biotechnology, 87:1329-1334.
Bates, L.S., Walden, R.P., and Teare, I.D. (1973). Rapid determination of free prolinefor water stress studies. Plant Soil, 39: 205-207.
Benyahia, F.,  Abdulkarim, M., Chaalal, Z. O. and Hasanain, H.. (2005). Bioremediation of Crude Oil Contaminated Soils: A Black Art or an Engineering Challenge? Process Safety and Environmental Protection, 83:364-370.
Bouyoucos, C. J. (1962). Hydrometer method improved for making particle size analysis of soils. Agronomy Journal, 54: 464-465.
Bremner, J. M. (1965). Total nitrogen. In Methods of soil analysis (Black, C. A. ed.). Part 2, American Society of Agronomy, Mandison, WI. p. 1148-1158.
Chapman, H. D. (1965). Cation exchange capacity. In Method of soil analysis (Black, C. A. ed.). Part 2, Am. Soc. Agron., Madison, WI. p. 891-901
Chen W, Li J, Sun X, Min J, Hu X. (2017). High efficiency degradation of alkanes and crude oil by a salt-tolerant bacterium Dietzia species CN-3. International Biodeterioration and Biodegradation, 118:110-8.
Cunningham, S. D., Anderson, T. A., Schwab, P. A. and Hsu, F. C. (1996). Phytoremediation of soils contaminated with organic pollutants. Advances in Agronomy, 56: 55-114.
Escalante, E. E., Gallegos-Martınez, M. E., Favela-Torres, E. and Gutierrez-Rojas, M. (2005). Improvement of the hydrocarbon phytoremediation rate by Cyperus laxus Lam. inoculated with a microbial consortium in a model system. Chemosphere, 59: 405–413.
Feng, N. X., Yu, J., Zhao, H. M., Cheng, Y. T., Mo, C. H., Cai, Q. Y. and Wong, M. H. (2017). Efficient phytoremediation of organic contaminants in soils using plant–endophyte partnerships. Science of the Total Environment, 1:352-368.
Gao, X. Z., Yu, S. C., Wu, K. C., Cheung, N. F. Y., Tam, P. Y. and Qian, M. H. (2006) Interactions of rice (Oryza sativa L.) and PAH-degrading bacteria (Acinetobacter sp.) on enhanced dissipation of spiked phenanthrene and pyrene in waterlogged soil. Science of the Total Environment, 372: 1-11.
Gusain, Y. S., Singh, U. S. and Sharma, A. K. (2015). Bacterial mediated amelioration of drought stress in drought tolerant and susceptible cultivars of rice (Oryza sativa L.). African Journal Biotechnology, 14: 764-773.
Hamdi, H., Benzarti, S., Manusadianasc, L., Aoyamaa, I. and Jedidi, N. (2007). Bioaugmentation and biostimulation effects on PAH dissipation and soil ecotoxicity under controlled conditions. Soil Biology and Biochemistry, 39:1926–1935.
Harmsen, J. (1991). Possibilities and limitations of landfarming for cleaning contaminated soils. In: Hinchee, R. E and Ollen-buttel, R. F. (Eds.). On site Bioreclamation. PP. 255–272.
Hemke, P. H. and Spark, D. L. (1996). Potassium. P 551-574. In D.L., Sparks et al., (Eds.). Method of soil analysis, Published by: Soil Science Societyof America, Inc. American Society of Agronomy, Inc. Madison, Wisconsin, USA.
Hewedy, A.M. (1999). Influence of singleand multi bacterial fertilizer on the growth and fruit yield of tomato. Egyptian Journal Applied Science, 14: 508-523.
Hirt, H., Shinozaki, K. (2004). Plant Responses to Abiotic Stress. Springer Science. 300p.
Huang, X.D., El-Alawi, Y., Penrose, D.M., Glick, B.R., and Greenberg, B.M. (2004). Responses of three grass species to creosote during phytoremediation. Environmental Pollution, 130: 453-463.
Idise, O., Ameh, J., Yakubu, S. and Okuofu, C. (2010). Modification of Bacillus cereus and Pseudomonas aeruginosa isolated from a petroleum refining effluent for increased petroleum product degradation. African Journal of Biotechnology, 9(22): 3303-3307.
Ilangovan, K., and Vivekanandan, M. (1992). Effect of oil pollution on soil respiration and growth of Vigna mungo (L.) Hepper, The Science of the Total Environment, 116: 187-194.
Ilyina, A., Castillo, S.M.I., Villarreal, S.J.A., Ramirez, E.G. and Candelas, R. (2003).Isolation of soil bacteria for bioremediation of hydrocarbon contamination. (Corporation Mrxicana de Investigacion en Materiales S.A. de C.V. (COMIMSA).
Kaul, S., Sharma, S.S. and Mehta, I.K. (2008). Free radical scavenging poten-tial of L-proline: evidence from in vitro assays. Amino Acids, 34: 315–320.
Kavi Kishor, P.B., Sangam, S., Amrutha, R.N., Sri Laxmi, P., Naidu, K.R., Rao, K.R.S.S., Rao, S., Reddy, K.J., Theriappan, P. and Sreenivasulu, N. (2005). Regulation of proline biosynthesis, degradation, uptake and transport in higher plants: Its implications in plant growth and abiotic stress tolerance. Current Science, 88: 424-438.
Kavousi Bafti, M., Asrar, Z., Hassanshahian, M., Keramat. B. (2014). A study of the effects of crude oil pollution and oil-degrading bacteria on some biochemical and growth factors of Zea mays. Iranian Journal of Plant Biology, 6: 71-84.
  Kirimura, K., Nakagawa, H., Tsuji, K., Kazuya, M., Kurane, R., Usami, S.H., (1999), Selective and continuous degradation of carbazole contained in petroleum oil by resting cells of Sphingomonas sp., CDH- Bioscience, Biotechnology and Biochemistry, 63(9): 1563-1568. 26.
Leahy, J.G., Colwell, R.R. (1990). Microbialdegradation of hydrocarbons in the environment. Microbial, 54: 305-315.
Lin, T. C., Pan, P. T., and Cheng, S. S. (2010). Ex situ bioremediation of oil-contaminated soil. Journal of hazardous materials, 176(1-3):27-34.
Lindsay, W. L. and Norvell, W.A. (1978). Development of a DTPA soil test for zinc, iron, manganese, and copper. Soil Science Society of America Journal, 42: 421-428.
Liste, H. H. and Felgentreu, D. (2006). Crop growth, culturable bacteria, and degradation of petrol hydrocarbons (PHCs) in a long-term contaminated field soil. Applied Soil Ecology, 31:43–52.
Liu, W, Luo, Y, Teng, Y and Li, Z. (2010). Phytoremediation of oilfield sludge after prepared bed bioremediation treatment. International Journal of Phytoremediation. 12: 268-278.
Liu, W., Sun, J., Ding, L., Luo, Y., Chen, M., and Tang, C. (2013). Rhizobacteria (Pseudomonas sp. SB) assist phytoremediation of oily-sludge-contaminated soil by tall fescue (Testuca arundinacea L.). Plant and soil, 371(1-2): 533-542.
Luepromchai, E., Lertthamrongsak, W., Pinphanichakarn, P., and Thaniyavarn, S. (2007). Biodegradation of PAHs in petroleum-contaminated soil using tamarind leaves as microbial inoculums. Journal of Science Technology, 29: 515-527.
Ma, H., Wang, A., Zhang, M., Li, H., Du, S., Bai, L. and Chen, S. (2018). Compared the physiological response of two petroleum-tolerant contrasting plants to petroleum stress. International Journal of Phytoremediation, 20(10): 1043-1048.
McFarland J. (1907). Nephelometer: an instrumentfor media used for estimating the number of bacteria in suspensions used for calculating the opsonic index and for vaccines. The Journal of the American Medical Association, 14:1176-8.
Media, V. F., Maestri, E., Marmiroli, M., Dietz, A. C. and Mc Cutcheon, S. C. (2003). Plant tolerances to contaminants. P. 189-233, In: S. C., Mc Catcheon, J. L., Schnoor (eds). Phytoremediation, transformation and control of contaminants, Wiley- Interscience. 1024pp.
Medina-Bellver, Antonio Delgado, P. M., Rodríguez-Sánchez, A., Reyes, E., Ramos, J. L. and Marqués. S. (2005). Evidence for in situ crude oil biodegradation after the Prestige oil spill. Environmental Microbiology, 7: 773–779.
Minai-Tehrani, D., Herfatmanesh, A., Azari-Dehkordi, F and Minooi, S. (2006). Effect of salinity on biodegradation of aliphatic fractions of crude oil in soil. Pakistan Journal Biology Science, 9:1531-1535.
Mishra, A. and Nautiyal, C. (2009) Functional diversity of the microbial community in the rhizosphere of chickpea grown in diesel fuelspiked soil amended with Trichoderma ressei using sole-carbon-source utilization profiles, World Journal of Microbiology and Biotechnology, 25: 1175–1180.
Moopam. P, (2010). Manaul of oceanographic observation and pollutant analyses methods. 3th ed., Kuwit, 321p.
Moubasher, H. A., Hegazy, A.K., Mohamed, N.H., Moustafa, Y.M., Kabiel, H.F. and Hamad, A.A. (2015). Phytoremediation of soils polluted with crude petroleum oil using Bassia scoparia and its associated rhizosphere microorganisms. International Biodeterioration and Biodegradation, 98: 113-120.
Mujahid, T. Y., Wahab, A., Padhiar, S. H., Subhan, S. A., Baloch, M. N., and  Pirzada, Z. A. (2015). Isolation and Characterization of Hydrocarbon Degrading Bacteria from Petrol Contaminated Soil. Journal of Basic and Applied Sciences, 11: 223-231.
Mukred, A.M., Hamid, A.A., Hamzah, A. and Yusoff, W.M.W. (2008). Development of three bacteria consortium for the bioremediation of crude petroleum-oil in contaminated water. Online Journal of Biological Sciences, 8(4): 73-79.
Njoku, K.L., Akinola, M.O. and Busari, T.O. (20120. Effect of time of application of spent oil on the growth and performance of maize (Zea mays). African Journal of Environmental Science and Technology, 6:67-71.
Olsen, S. R., Cole, C. V., Watanabe, F. S., and Dean, L. A. (1954). Estimation of available phosphorus in soil by extraction with sodium bicarbonate. USDA. Circ. 939. Gover, U. S. Prin. Office, Washington, DC, U. S. A.
Olukunle, O. F. and Oyegoke, T. S. (2016). Biodegradation of crude-oil by fungi Isolated from Cow Dung contaminated soils. Nigerian Journal Biotechnology, 31: 46 –58.
Palmroth, M. R. T., Pichtel, J. and Puhakka, J. A. (2002). Phytoremediation of subarctic soil contaminated with diesel fuel. Bioresource Technology, 84: 221-28.
Pena-Castro, J.M., Barrera-Figueroa, B.E., Fernandez, L.L., Ruiz, M.R., and Xoconostle, C.B. (2006) Isolation & identification of upregulated genes in bermudagrass roots (Cynodon dactylon L.) grown under petroleum hydrocarbon stress. Plant Science, 170: 724-731.
Peng, S., Zhou, Q., Cai, Z. and Zhang, Z. (2009). Phytoremediation of petroleum contaminated soils by Mirabilis Jalapa L. in a greenhouse plot experiment. Journal of Hazardous Materials, 168: 1490-1496.
Peretiemo-Clarke, B. O. and Achuba, F. I. (2007). Phytochemical effect of petroleum on peanut (Arachis hypogea) seedlings. Journal of Plant Pathology, 6: 179-182.
Radwan, S. S. (2009). Phytoremediatiom for oily desert soils. P. 289-298. In: Singh, A., Kuhad, R. C., Ward, O. P. (eds.). Advanced in Applied Bioremediation. Springer-Verlag Berlin Heidelberg. 361 pp.
Razmjoo, K., and Adavi, Z. (2012). Assessment of bermudagrass cultivars for phytoremediation of petroleum contaminated soils. International journal of phytoremediation, 14: 14-23.
Robinson, S. L., Novak, J. T., Widdowson, M. A., Crosswell, S. B. and Fetterolf, G. J. (2003). Field and laboratory evaluation of the impact of tall fescue on polyaromatic hydrocarbon degradation in an aged creosote-contaminated surface soil. Journal of Environmental Engineering, 129: 232-240.
Sarvi Moghanlo, V., Chorom, M., Falah, M., and Motamedy, H. (2012). Evaluation the effect of myccorhiza anddegrading bacteria in enhancing phytoremediation of oil compound in oil contaminated soil. Journal of Water and Soil, 26(4): 832-841. (In Persian with English abstract)
Tang, J., Wang, R., Niu, X. and Zhou, Q. (2010). Enhancement of soil petroleum emediation by using a combination of ryegrass (Lolium perenne) and different microorganisms. Soil and Tillage Research, 110: 87–93.
Tatari, M, Fotouhi Ghazvini, R, Moosavi, A and Babaei, G. (2018). Comparison of some physiological aspects of drought stress resistance in two ground cover genus. Journal of Plant Nutrition, 41: 1215-1226.
Walkley, A., and Black, T. A. (1934). An examination of the deligaref method for determination organic matter and a propose modification of the chromic acid titration method. Soil Science, 37: 29-38.
White, P. M., Wolf, D. C., Thoma, G. J. and Reynolds, C. M. (2006). Phytoremediation of alklated polycyclic aromatic hydrocarbons in a crude oilcontaminated soil. Water, Air and Soil Pollution, 169: 207-220.
Wilts, C. C., Rooney, W. L., Chen, Z., Schwab, A. P. and Banks, M. K. (1998). Greenhouse evaluation of agronomic and crude oil phytoremediation potential among alfalfa genotypes. Journal of Environmental Quality, 27(1): 169-73.
Wu, M., Dick, W. A., Li, W., Wang, X., Yang, Q., Wang, T., Xu, L.  Zhang, M. and Chen, L. (2016). Bioaugmentation and biostimulation of hydrocarbon degradation and the microbial community in a petroleum-contaminated soil. International Biodeterioration and Biodegradation, 107: 158-164.