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.