Enhanced biodegradation of hexachlorocyclohexane (HCH) in contaminated soils via inoculation with Sphingobium indicum B90A

Abstract

Soil pollution with hexachlorocyclohexane (HCH) has caused serious environmental problems. Here we describe the targeted degradation of all HCH isomers by applying the aerobic bacterium Sphingobium indicum B90A. In particular, we examined possibilities for large-scale cultivation of strain B90A, tested immobilization, storage and inoculation procedures, and determined the survival and HCH-degradation activity of inoculated cells in soil. Optimal growth of strain B90A was achieved in glucose-containing mineral medium and up to 65% culturability could be maintained after 60 days storage at 30 degrees C by mixing cells with sterile dry corncob powder. B90A biomass produced in water supplemented with sugarcane molasses and immobilized on corncob powder retained 15-20% culturability after 30 days storage at 30 degrees C, whereas full culturability was maintained when cells were stored frozen at -20 degrees C. On the contrary, cells stored on corncob degraded gamma-HCH faster than those that had been stored frozen, with between 15 and 85% of gamma-HCH disappearance in microcosms within 20 h at 30 degrees C. Soil microcosm tests at 25 degrees C confirmed complete mineralization of [(14)C]-gamma-HCH by corncob-immobilized strain B90A. Experiments conducted in small pits and at an HCH-contaminated agricultural site resulted in between 85 and 95% HCH degradation by strain B90A applied via corncob, depending on the type of HCH isomer and even at residual HCH concentrations. Up to 20% of the inoculated B90A cells survived under field conditions after 8 days and could be traced among other soil microorganisms by a combination of natural antibiotic resistance properties, unique pigmentation and PCR amplification of the linA genes. Neither the addition of corncob nor of corncob immobilized B90A did measurably change the microbial community structure as determined by T-RFLP analysis. Overall, these results indicate that on-site aerobic bioremediation of HCH exploiting the biodegradation activity of S. indicum B90A cells stored on corncob powder is a promising technology.

Technicalities and Glitches of Terminal Restriction Fragment Length Polymorphism (T-RFLP)

“…Therefore, estimation and standardization of the amount of DNA is necessary for better resolution. To overcome the variability and improve the reproducibility of the data, apart from promoting the running of replicates Kaplan et al [12] and Dunbar et al [8] respectively proposed the methods for post-run data normalization based on the relative peak area and peak height of the generated TRFs.…”

“…It is an inexpensive, robust, reproducible, and rapid method for the study of microbial community structure. The potential of T-RFLP method for the study of community dynamics and structure is extensively studied for diverse habitats [5][6][7][8][9][10][11][12][13][14]. In addition to 16S rRNA gene, T-RFLP analysis also included genes representing a variety of functional groups [15][16][17].…”

The enzymatic basis for pesticide bioremediation

“…The simplest mechanism by which such a strategy could be realised is the use of HCH mineralising bacteria that express the enzymes encoded by the entire lin operon. In this regard, it is noteworthy that recent studies in which HCH contaminated soil was treated by the controlled release of a bacterium (Sphingobium indicum) containing the naturally occurring lin-operon led to signifi cant remediation of the pesticide residue (up to 95 %) [90].…”

Genomic tools in bioremediation

“…A large number of bacteria/biodegradation by sludge, have been reported for degradation of different industrial wastes, such as dyes [7], hydrocarbons, especially related to petrochemical wastes [8][9][10], tanneries [11], chlorinated aromatics [12], distillery [13], pesticides [14][15][16][17][18], heavy metals [19]. Similarly, there are phenomenon such as chemotaxis and its relevance in bioremediation has been reported using pure culture system in a model study [20].…”