Using 3,4-dichloro benzoic acid as growth substrate by Brevibacterium spp: Effect of some growth conditions

Main Article Content

Haya Allawi
Amjad Al Tarawneh
Haitham Qaralleh
Muhamad Al-limoun
Khalid Alsharafa


Brevibacterium spp.that was already isolated from wastewater treatment plant of Petra City, Jordan. While 1 and 2 mM of 3,4-dichloro benzoic acid (3,4-DCBA) were used, the biodegradation rate was 14.5 and 21.25 µM/h, respectively, and the remaining was 58% for both. The remaining ratio to the quantity of 3,4-DCBA used was taken in consideration, as a result, 1 mM concentration of 3,4-DCBA substrate was applied for the rest of experiments because of the remaining amount is 0.58 mM 3,4-DCBA while 1.15 mM remained from 2mM concentration used. Thereafter, Brevibacterium spp was grown in batch cultures using M9 minimal media plus 1 mM concentrations of 3,4-DCBA as carbon and energy source. 3,4-DCBA degradation was optimally achieved at a 37 oC incubation temperature, a pH of 7.0 and an agitation rate of 150 rpm. During carbon starvation, the increased biodegradation potential of Brevibacterium spp. was accompanied by a lessening in the acclimatization period besides enhancement of the biodegradation completion of 3,4-DCBA. The adaptation to 3,4-DCBA not only increased the degradation rate but also managed to reduce the time required for complete 3,4-DCBA degradation from 80 to nearly 40 hours. In conclusion, the acclimatized period, and carbon starvation and other optimal conditions were key factors for the enhancement of the degradation ability of 3,4-DCBA by Brevibacterium spp.


Download data is not yet available.


Metrics Loading ...

Article Details



Aiba, S., Shoda, M., & Nagatani, M. (1968). Kinetics of product inhibition in alcohol fermentation. Biotechnology and bioengineering, 10(6), 845-864.

Althunibat OY, Qaralleh H, Al-Dalin SY, Abboud M, Khleifat K, Majali IS, Aldal'in HK, Rayyan WA, Jaafraa A (2016). Effect of thymol and carvacrol, the major components of Thymus capitatus on the growth of Pseudomonas aeruginosa. J Pure Appl Microbiol. 1(10): 367-74.

Aljundi, IH., Khleifat, K.M., Khlaifat, AM., Ibrahim, AM, Tarawneh KA. (2010). Biodegradation of 2-chlorobenzoic acid by Klebsiella oxytoca: mathematical modeling and effect of some growth conditions. Indust. Eng. Chem. Res. 49 (16): 7159-7167

Ampe, F., Léonard, D., & Lindley, N. D. (1998). Repression of phenol catabolism by organic acids in Ralstonia eutropha. Appl. Environ. Microbiol., 64(1), 1-6.

Arensdorf JJ & Focht DD (1994) Formation of chlorocatechol meta cleavage products by a Pseudomonad during metabolism of monochlorbiphenyls. Appl. Environ. Microbiol. 60: 2884–2889

Banta, G., & Kahlon, R. S. (2007). Dehalogenation of 4—Chlorobenzoic Acid by Pseudomonas isolates. Indian journal of microbiology, 47(2), 139-143.

Dorn, E., and Knackmuss, HJ. (1978). Chemical structure and biodegradability of halogenated aromatic compounds: Two catechol 1, 2-dioxygenases from 3-chlorobenzoate-grown Pseudomonad[J], Biochem J 174, pp. 73–84.

Edwards, V. H. (1970). The influence of high substrate concentrations on microbial kinetics. Biotechnology and Bioengineering, 12(5), 679-712.

Eppert, I., Valdés-Stauber, N., Götz, H., Busse, M., & Scherer, S. (1997). Growth reduction of Listeria spp. caused by undefined industrial red smear cheese cultures and bacteriocin-producing Brevibacterium lines as evaluated in situ on soft cheese. Appl. Environ. Microbiol., 63(12), 4812-4817.

Funke, G., & Carlotti, A. (1994). Differentiation of Brevibacterium spp. encountered in clinical specimens. Journal of clinical microbiology, 32(7), 1729-1732.

Gruner, E., Pfyffer, G. E., & von Graevenitz, A. L. E. X. A. N. D. E. R. (1993). Characterization of Brevibacterium spp. from clinical specimens. Journal of clinical microbiology, 31(6), 1408-1412.

Häggblom, M. M. (1992). Microbial breakdown of halogenated aromatic pesticides and related compounds. FEMS Microbiology Reviews, 9(1), 29-71.

Hickey, W. J., & Focht, D. D. (1990). Degradation of mono-, di-, and trihalogenated benzoic acids by Pseudomonas aeruginosa JB2. Appl. Environ. Microbiol., 56(12), 3842-3850.

Hochster, R. (Ed.). (2012). Metabolic Inhibitors V1: A Comprehensive Treatise. Elsevier

Khleifat, K. M., Hanafy, A. M. M., & Al Omari, J. (2014). Prevalence and molecular diversity of Legionella pneumophila in domestic hot water systems of private apartments. British Microbiology Research Journal, 4(3), 306.

Khleifat, K. M., Sharaf, E. F., & Al-limoun, M. O. (2015). Biodegradation of 2-chlorobenzoic acid by enterobacter cloacae: Growth kinetics and effect of growth conditions. Bioremediation Journal, 19(3), 207-217.

Khleifat, K.M., (2006a). Biodegradation of linear alkylbenzene sulfonate by a two-member facultative anaerobic bacterial consortium. Enzyme and microbial technology, 39(5): 1030-1035.

Khleifat, K.M., (2006b). Biodegradation of phenol by Ewingella americana: Effect of carbon starvation and some growth conditions. Process Biochemistry, 41(9): 2010-2016.

Khleifat, K.M., (2006c). Biodegradation of sodium lauryl ether sulfate (SLES) by two different bacterial consortia. Current microbiology, 53(5): 444-448.

Khleifat, K. M. (2006d). Correlation Between Bacterial Hemoglobin and Carbon Sources: Their Effect on Copper Uptake by Transformed E. coli Strain ?DH5. Current microbiology, 52(1), 64-68.

Khleifat, K. M., Matar, S. A., Jaafreh, M., Qaralleh, H., Al-limoun, M. O., & Alsharafa, K. Y. (2019). Essential Oil of Centaurea damascena Aerial Parts, Antibacterial and Synergistic Effect. Journal of Essential Oil Bearing Plants, 22(2), 356-367.

Khleifat, K., & Abboud, M. M. (2003). Correlation between bacterial haemoglobin gene (vgb) and aeration: their effect on the growth and ??amylase activity in transformed Enterobacter aerogenes. Journal of applied microbiology, 94(6), 1052-1058.

Khleifat, K. M., Abboud, M. M., Al-Mustafa, A. H., & Al-Sharafa, K. Y. (2006a). Effects of carbon source and Vitreoscilla hemoglobin (VHb) on the production of ?-galactosidase in Enterobacter aerogenes. Current microbiology, 53(4), 277.

Khleifat, KM., Tarawneh, KA., Wedyan MA., Al-Tarawneh, AA., Al Sharafa K (2008). Growth Kinetics and Toxicity of Enterobacter cloacae Grown on Linear Alkylbenzene Sulfonate as Sole Carbon Source. Curr. Microbiol. 57:364–370

Khleifat, K. M., Abboud, M. M., & Al-Mustafa, A. H. (2006b). Effect of Vitreoscilla hemoglobin gene (vgb) and metabolic inhibitors on cadmium uptake by the heterologous host Enterobacter aerogenes. Process Biochemistry, 41(4), 930-934.

Khleifat, K. M., Nawayseh, K., Adjeroud, N. R., Khlaifat, A. M., Aljundi, I. H., & Tarawneh, K. A. (2009). Cadmium-resistance plasmid affected Cd+ 2 uptake more than Cd+ 2 adsorption in Klebsiella oxytoca. Bioremediation Journal, 13(4), 159-170.

Khleifat, K. M., Halasah, R. A., Tarawneh, K. A., Halasah, Z., Shawabkeh, R., & Wedyan, M. A. (2010). Biodegradation of linear alkylbenzene sulfonate by Burkholderia sp.: Effect of some growth conditions. Int J Agr Biol, 12, 17-25.

Khleifat, KM., (2007a). Effect of substrate adaptation, carbon starvation and cell density on the biodegradation of phenol by Actinobacillus sp. Fresenius Environ. Bull. 16 (7): 726-730.

Khleifat, K. M. (2007b). Biodegradation of phenol by Actinobacillus sp.: Mathematical interpretation and effect of some growth conditions. Bioremediation Journal, 11(3), 103-112.

Khleifat, K. M., Al-Majali, I., Shawabkeh, R., & Tarawneh, K. (2007). Effect of carbon and nitrogen sources on the biodegradation of phenol by Klebsiella oxytoca and growth kinetic characteristics. Fresenius Environmental Bulletin, 16(5), 1-7.

Lal, B., & Khanna, S. (1996). Degradation of crude oil by Acinetobacter calcoaceticus and Alcaligenes odorans. Journal of applied bacteriology, 81(4), 355-362.

Leven, L., & Schnürer, A. (2005). Effects of temperature on biological degradation of phenols, benzoates and phthalates under methanogenic conditions. International Biodeterioration & Biodegradation, 55(2), 153-160.

Leonard, D., & Lindley, N. D. (1998). Carbon and energy flux constraints in continuous cultures of Alcaligenes eutrophus grown on phenol. Microbiology, 144(1), 241-248.

Loh, K. C., & Wang, S. J. (1997). Enhancement of biodegradation of phenol and a nongrowth substrate 4-chlorophenol by medium augmentation with conventional carbon sources. Biodegradation, 8(5), 329-338.

Loh, K. C., & Tan, C. P. (2000). Effect of additional carbon sources on biodegradation of phenol. Bulletin of environmental contamination and toxicology, 64(6), 756-763.

Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Measurement of protein with the Folin phenol reagent. J Biol Chem, 193(1), 265-275.

Luedeking, R., & Piret, E. L. (1959). Transient and steady states in continuous fermentaion. Theory and experiment. Journal of biochemical and microbiological technology and engineering, 1(4), 431-459.

Onysko, K. A., Budman, H. M., & Robinson, C. W. (2000). Effect of temperature on the inhibition kinetics of phenol biodegradation by Pseudomonas putida Q5. Biotechnology and bioengineering, 70(3), 291-299

Margesin, R., & Schinner, F. (1997). Effect of temperature on oil degradation by a psychrotrophic yeast in liquid culture and in soil. FEMS Microbiology Ecology, 24(3), 243-249.

Matin, A., Baetens, M., Pandza, S., Park, C.H., Waggoner, S. (1999). Survival strategies in the stationary phase In: Microbial Ecology and Infectious Disease (Roserberg, E., Ed.), pp. 30-48. ASM Press. Washington, DC.

Majali, I. S., Oran, S. A., Khaled, M. K., Qaralleh, H., Rayyan, W. A., & Althunibat, O. Y. (2015). Assessment of the antibacterial effects of Moringa peregrina extracts. African Journal of microbiology research, 9(51), 2410-2414.

Marks, T. S., Smith, A. R., & Quirk, A. V. (1984). Degradation of 4-chlorobenzoic acid by Arthrobacter sp. Appl. Environ. Microbiol., 48(5), 1020-1025.

Neumann, G., Teras, R., Monson, L., Kivisaar, M., Schauer, F., & Heipieper, H. J. (2004). Simultaneous degradation of atrazine and phenol by Pseudomonas sp. strain ADP: effects of toxicity and adaptation. Appl. Environ. Microbiol., 70(4), 1907-1912.

Reardon, K. F., Mosteller, D. C., Rogers, J. B., DuTeau, N. M., & Kim, K. H. (2002). Biodegradation kinetics of aromatic hydrocarbon mixtures by pure and mixed bacterial cultures. Environmental Health Perspectives, 110(suppl 6), 1005-1011.

Romanov V & Hausinger RP (1994) Pseudomonas aeruginosa 142 uses a three-component ortho-halobenzoate 1,2-dioxygenase for metabolism of 2,4-dichloro- and 2-chlorobenzoate. J. Bacteriol. 176: 3368–3374

Qaralleh, H., Khleifat, K. M., Al-Limoun, M. O., Alzedaneen, F. Y., & Al-Tawarah, N. (2019). Antibacterial and synergistic effect of biosynthesized silver nanoparticles using the fungi Tritirachium oryzae W5H with essential oil of Centaurea damascena to enhance conventional antibiotics activity. Advances in Natural Sciences: Nanoscience and Nanotechnology, 10(2), 025016.

Shawabkeh, R., Khleifat, K. M., Al-Majali, I., & Tarawneh, K. (2007). Rate of biodegradation of phenol by Klebsiella oxytoca in minimal medium and nutrient broth conditions. Bioremediation Journal, 11(1), 13-19.

Schmidt, E., & Knackmuss, H. J. (1980). Chemical structure and biodegradability of halogenated aromatic compounds. Conversion of chlorinated muconic acids into maleoylacetic acid. Biochemical Journal, 192(1), 339-347.

Tarawneh, K. A., Halasah, Z. A., Khleifat, A. M., Batarseh, M. I., Khleifat, K. M., & Al-Mustafa, A. H. (2011). Evaluation of cefaclor oral suspensions stability using reversed phase high performance liquid chromatography and antimicrobial diffusion methods. Pakistan journal of pharmaceutical sciences, 24(3).

Urgun-Demirtas, M., Pagilla, K. R., Stark, B. C., & Webster, D. (2003). Biodegradation of 2-chlorobenzoate by recombinant Burkholderia cepacia expressing Vitreoscilla hemoglobin under variable levels of oxygen availability. Biodegradation, 14(5), 357-365.

Zeidan, R., Oran, S., Khleifat, K., & Matar, S. (2013). Antimicrobial activity of leaf and fruit extracts of Jordanian Rubus sanguineus Friv (Rosaceae). African Journal of Microbiology Research, 7(44), 5114-5118.

Zilli, M., Converti, A., Lodi, A., del Borghi, M. and Ferraiolo, G. (1993). Phenol removal from waste gases with a biological filter by Pseudomonas putida. Biotechnol. Bioeng, 41, 693–699.

Yap, L. F., Lee, Y. K., & Poh, C. L. (1999). Mechanism for phenol tolerance in phenol-degrading Comamonas testosteroni strain. Applied Microbiology and Biotechnology, 51(6), 833-840.

Yano, T., Nakahara, T., Kamiyama, S., & Yamada, K. (1966). Kinetic studies on microbial activities in concentrated solutions. Part. I. Effect of excess sugars on oxygen uptake rate of a cell free respiratory system. Agricultural and Biological Chemistry, 30(1), 42-48.

Yun, Q. I., Lin, Z. H. A. O., Ojekunle, Z. O., & Xin, T. A. N. (2007). Isolation and preliminary characterization of a 3-chlorobenzoate degrading bacteria. Journal of Environmental Sciences, 19(3), 332-337.

Most read articles by the same author(s)