## 4 Discussion
### Possible Role of Siderophores  
Actinobacteria have long been considered suitable candidates for biodegradation studies due to their high metabolic capability [43; 44; 45; 46]. Two properties in particular are of relevance in this case, (a) the number and diversity of degradative pathways for hydrocarbons, and (b) the production of secondary metabolites such as biosurfactants [47; 48; 49] and siderophores [26; 50; 51]. These properties allow actinobacteria to survive under a wide range of environmental conditions and to adapt to, or even change, local conditions by secreting metabolites. As such, the presence of actinobacteria and its secretion of metabolites will generally affect the microbial community. This is difficult to investigate in soil samples, however, as the distribution of bacteria and metabolites will depend on the soil's properties and mobilisation potential. In this study, siderophores clearly appeared to act as a promoting factor on the hydrocarbon degradation potential. Recently, it has also been demonstrated that citrate and DFOB, two typical microbial metabolites relevant for metal mobilisation and transport, were able to mobilise a number of metals and metalloids, depending on the conditions [52]. As such, the availability of trace elements of importance to microbial life may be affected by such processes. Both actinobacterial strains investigated in this study are proven producers of siderophores, which bind Fe\({}^{3+}\) and other trivalent metals, thereby affecting their bioavailability [26; 53; 54]. As such, we included iron treatment and DFOB siderophore biostimulation within our test variants of experiment B. Theoretically, the presence of Fe\({}^{3+}\) should suppress siderophore production, allowing microorganisms to have a non-iron limiting metabolism [55]; however, the effects of Fe\({}^{2+}\), DFOB and siderophores may differ. While Fe\({}^{2+}\) can support reductive processes, thus making other metals or metalloids bioavailable, siderophores, including DFOB, predominantly mobilise trivalent metals or metalloids, although the results obtained have been somewhat contradictory [26; 53; 55]. In our own study, a significant decrease in hydrocarbon concentration was observed following biostimulation by DFOB, even without bioaugmentation (variant BD), thereby suggesting a clear positive effect by siderophores on biostimulation of autochthonous microflora and their biodegradation activity. Similar observations have also been reported for siderophores of a _Rhodococcus_ strain, which can stimulate the growth of other bacterial organisms [50]. Test variants where Fe\({}^{3+}\) was added (BG3 and BR3), on the other hand, produced no significant hydrocarbon decrease when compared with those variants where only Fe\({}^{2+}\) was added (BG1 and BR1) or where no iron was added (variants BG2 and BR2). This suggests a positive effect on hydrocarbon degradation following production of siderophores (likely to have been repressed in variants BG3 and BR3 by high levels of Fe\({}^{3+}\)). One test of this would be to assess the concentration of water-extractable (presumably bioavailable) iron obtained as fraction 1 of the sequential BCR extraction. Despite a slight increase in water-extractable iron in all variants, concentration variability was relatively high and only variants BG3 and BR2 exhibited significant results (_p_ < 0.1). Grouping the variants together to promote the differences revealed a significant increase in water-extractable iron after treatment with _G. rubripertincta_ CWB2 (joint variants BG1 and BG2; _t_-test, \(p\) = 0.064) but no significant change following addition of _R. erythropolis_ S43 (joint variants BR1 and BR2; _t_-test, \(p\) = 0.118). Consideration of each strain as a factor (Table 3) indicated significance for _G. rubripertincta_ CWB2 only. A second possible mechanism includes either mobilisation or detoxification of other metals [26; 53] (see Table 1 for a list of water-extractable metals).