In order to develop an optimised coal production waste bioleaching unit, a sampling campaign was carried out on the existing waste dump. Eleven samples from various surface locations of the heap were used for enrichment cultures at three temperatures (30°C, 42°C and 48°C). Interestingly, none of the 11 cultures at 42°C grew well (low cell numbers and poor pyrite dissolution). Bioreactors inoculated with pooled enrichments showed the same results, indicating that the waste heap harboured organisms capable of growth and mineral oxidation at 30°C and 48°C but not 42°C. Analysis of the 48°C culture used for subsequent reactor tests using a variety of molecular microbiology methods (CE-SSCP, T-RFLP and high-throughput sequencing) revealed the culture was dominated by Sulfobacillus thermosulfidooxidans, Acidithiobacillus caldus and an unknown member of the Acidimicrobiaceae. Further tests at 42°C using a well-characterised bi-oleaching consortium, BRGM-KCC, performed well in initial reactors. However, during each batch the relative abundance of Leptospirillum ferriphilum decreased, and its numbers declined gradually with each subsequent subculture indicating some form of accrued inhibition. Coupled with the absence of a 42°C culture from the dump it was theorised that the waste was inhibitory to Leptospirillum spp. In order to further explore the possible reasons for the absence of Leptospi-rillum in the enrichments and bioreactors, a detailed analysis of the waste heap was undertaken using a range of geochemical and molecular biology tools. Results suggest the role of an as-yet unidentified organic carbon compound from the coal itself.