3.3 Influence of Fe amounts and compounds on biofilm development
Further gravimetrical determinations as well as ion chromatography (ICP-OES) verified that Fe2+ is somehow stored into the biofilm matrix and not rinsed out with the nutrient medium flowing through (data not shown). Here, most of the iron was incorporated in biofilms of experiments E1 and E3 (\(c\) = 2.5 mg/L Fe2+). Several hypotheses were postulated that describe the uptake and influence of iron into the biofilm. For instance, (Kang & Kirienko, 2018) confirm an uptake of iron via siderophores (iron carriers) as well as the storage of iron inPseudomonas aeruginosa biofilms. (Rizzi et al., 2019) report thatBacillus subtilis utilizes the formation of biofilms and the production of siderophores to take up iron (Fe) from the medium, likewise to ensure normal growth. Thereby the authors define iron (Fe) as the most important metal in biology (Rizzi et al., 2019). Additionally, (Oh, Andrews, & Jeon, 2018) found out that iron promotes biofilm formation through oxidative stress and that it stimulates EPS production in Campylobacter jejuni . Hence, in their study an addition of iron significantly supported the formation of microcolonies in the early stage as well as the differentiation into mature biofilm structures, which is reflected here both by the OCT as well as the analysis of the structural biofilm parameters.
Additionally, measurements of the individual biofilms via attenuated total reflectance infrared spectroscopy (ATR-IR) were performed (data presented in SI Figure 2). It was verified that experiments E1 and E3 (\(c\) = 2.5 mg/L Fe2+) as well as E2 and E4 (\(c\) = 0.25 mg/L Fe2+) are correlating, respectively, which coincidences with the results of the growth experiment (Figure 2). According to literature, Fe is mainly incorporated into the biofilms via polymorphs of iron oxide-hydroxides (x-FeOOH) (Chan, Stasio, Welch, Fakra, & Banfield, 2004; Florea et al., 2011; Neu et al., 2010; Omoike, Chorover, Kwon, & Kubicki, 2004). Thereby, in experiments E1 and E3 an incorporation of \(\alpha\)-FeOOH took place, whereas in E2 and E4 \(\beta\)‑FeOOH was stored into the biofilms. This was found in (Wagner, 2011) and (Ivleva, Wagner, Horn, Niessner, & Haisch, 2010), too, whereby the authors documented an incorporation of \(\gamma\)-FeOOH into waste water biofilms. As maintained by (Wagner, 2011) and (Möhle et al., 2007), cross-linking of iron with the biofilm matrix ensured an increased stability of the biofilms. \(\alpha\)‑FeOOH is a highly reactive compound and (Omoike et al., 2004) proved that an interaction of Bacillus subtilisbiofilms with \(\alpha\)-FeOOH ensures an energetically stable connection for further EPS- and cell adhesion. This made it possible for the biofilms in E1 and E3 (\(c\) = 2.5 mg/L Fe2+) to grow increasingly and to remain stable even at a high flow velocity (\(u\) = 3.75 cm/s) without sloughing events of microcolonies. With its loose structure, \(\beta\)-FeOOH exhibited the ability to store high amounts of water (Mei, Liao, Wang, & Xu, 2015). Thus, the low addition of iron(II) in E2 and E4 (\(c\) = 0.25 mg/L Fe2+) as well as the storage of \(\beta\)-FeOOH into the matrix could be reasons for the reduced accumulation of biofilm mass. Potentially, the type of x-FeOOH incorporation is concentration-dependent and dependent of the bacterial organism or its (cell) surface because absorptions were clearly distant from each other, as pointed in (Mei u. a., 2015), too.