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I~ <br />- 2 3 - <br />' 3.2.1 Innoculation. The effect of innoculating two samples <br />with an active culture of Thiobacillus ferrooxidans on pH and SOq produc- <br />tion is shown in Figure 6. Sample Equity-A1 had a large negative net <br />neutralization potential according to the acid/base accounting tests <br />(-433 tonnes CaC03/1000 tonnes), while the Endako sample had a large posi- <br />tive potential (+218 tonnes CaC03/1000 tonnes). In both cases the pH level <br />and SOq production were not significantly different for innoculated <br />compared to non-innoculated samples. Moreover, cumulative acidity produced <br />in the 4 weeks after innoculation were similar for the Equity samples <br />(138 mg as CaC03 for the non-innoculated and 134 mg as CaC03 for the <br />innoculated samples). Leachate tested on the tenth week proved negative <br />for the presence of viable iron oxidizing bacteria suggesting that the <br />bacteria may have been washed from the cell or unable to survive on the <br />samples tested. <br />This finding would contradict Bruynsteyn (30), who found solid <br />residues are more effective than supernatant as innoculi, and in e~zcess of <br />99% of the microorganisms are attached to solid surfaces, not subject to <br />washout. <br />The supernatant innocula in our experiment were examined for the <br />presence of viable bacteria prior to addition to the humidity cells. Nhile <br />exact numbers are not known, a viable population was added to the test <br />cells. <br />Several investigators have noted a lag time for establishment of <br />acid generating bacteria. Kleinmann and Crerar (2), in previously cited <br />work, found from 1-3 1/2 weeks necessary to achieve acidification. Sterile <br />material took up to 3 months to reach pH below 4. Harrison (31) in a study <br />of the microbial succession of bacteria in a coal refuse pile found iron <br />oxidizing bacteria near the top of the pile in a low pH environment. <br />(pH 2.6) after eight weeks. <br />These references suggest that some evidence of increased pyrite <br />oxidation (increased sulfate production, greater cumulative acidity, or <br />lower pH) should have been found in the 4 weeks of leachate samples. <br />collected from the innoculated experiments. Very rapid pyrite ozieation <br /> <br />