Use of natural materials for the inhibition of iron oxidizing bacteria involved in the generation of acid mine drainage

Obligate autotrophic and acidophilic characteristics of iron oxidizing bacteria were exploited in order to prevent or attenuate the generation of acid mine drainage. Inhibition of biomass growth was performed by variation of 9K medium by increasing ferrous iron concentration (substrate inhibition), by addition of limestone (inhibition by pH increase) and olive pomace (inhibition by organic compounds). Inhibition tests of batch growth were performed according to a full factorial design with three factors (ferrous iron, limestone and olive pomace) taken at two levels. Blank tests were also performed in the same operating conditions but without inoculum. All tests were monitored by measuring cell and ferrous iron concentrations, pH and redox potential. Biomass characterization was performed by determining the sequence of 16S ribosomal RNA gene that denoted a 100% homology with the sequence of Acidithiobacillus ferrooxidans, one of the main bacteria responsible for acid mine drainage. Experimental data of cell growth denoted a strong inhibition in all the operating conditions, except the case when larger ferrous iron concentrations were used. Blank tests allowed isolation of the effect of chemical speciation from the effect due to metabolic activities. In particular, inhibition by limestone seemed to be due not only to pH increase, but also to a reduced availability of ferrous iron substrate, while inhibition by pomace may be related to the antioxidant properties of polyphenols. Interactions among operating conditions, biomass growth and chemical parameters were specifically assessed by analysis of variance. Estimates of significant effects (95%) for cell concentration denoted that only main effects related to limestone and pomace addition determined a significant diminution of cell concentration with a dynamic increasing trend.