Recent understanding of microbial retention within heap bioleaching systems has highlighted the importance of quantifying microbial growth and activity in both the bulk flowing solution and in the ore-associated phases. Typically, industrial heap bioleaching operations report variations in process conditions such as inoculum preparation and concentration and elevated copper concentrations in the recycled irrigation solution. In this paper, a mini-column reactor system containing pre-constructed and agglomerated, low-grade ore samples representing grab samples from a larger heap, were used to investigate the effect of a selection of physico-chemical and operating conditions on microbial growth, colonisation and substrate utilisation kinetics, considering both the planktonic and sessile populations of Acidithiobacillus ferrooxidans. The factors studied included inoculum size, inoculum cultivation conditions, availability of ferrous iron in the bulk flowing solution and copper concentration in the bulk flowing solution. The microbial population in the interstitial phase, i.e. associated with, but not bound to, the ore, remained the most abundant within the heap under all physico-chemical conditions considered. A comparison of the tests with different inoculum sizes found that a smaller inoculum size resulted in an increased delay in microbial growth and ferrous iron oxidation, but similar apparent maximum specific growth rates and iron oxidation rates. In contrast to the microbial culture grown on pyrite, a delay in microbial activity was observed for the culture grown on ferrous iron. However, greater microbial cell densities were reached, in the interstitial and attached phases compared with the pyrite-grown culture. The introduction of 6 g L-1 cupric ions into the feed solution containing 0.2 g L-1 ferric iron resulted in decreased microbial growth rate in the interstitial phase but not in the attached phase. Where the pyrite culture was pre-exposed to cupric ion, the microbial growth rate in the interstitial and attached phases was significantly enhanced. Nevertheless, the presence of cupric ion in the irrigation solution resulted in a decrease in microbial ferrous iron oxidation rate, irrespective of pre-culture conditioning. This study emphasises the important role played by the stagnant interstitial phase during the colonisation of a low-grade heap, particularly under adverse conditions for microbial growth and activity. It also highlights the role of inoculum culture conditions on the potential trade-off between increased heap colonisation and increased lag periods in microbial activity during heap start-up. (C) 2015 Published by Elsevier Ltd.