1. Introduction
Biofilms are aggregates composed of microorganisms, extracellular
polymeric substances (EPS) as well as extracellular DNA. Mainly, they
appear at interfaces in watery environments (Flemming, Wingender, &
Szewzyk, 2007). Within the last decade, more attention was paid to
biofilms due to their unique properties and potential applications as
productive systems (Edel, Horn, & Gescher, 2019; Rosche, Li, Hauer,
Schmid, & Buehler, 2009). On one hand, these communities have several
beneficial features, e.g. (i) in cleaning waste water (Van Loosdrecht &
Heijnen, 1993); (ii) by producing valuable (platform) chemicals (Cuny et
al., 2019), (iii) methane (as fuel) (Yeung et al., 2017) or (iv)
bioplastics (Hackbarth et al., 2020), too. On the contrary, biofilms can
have adverse effects, i.e. on human health by growing on implants or by
blocking industrial settings such as water pipes (Azeredo et al., 2016).
In order to optimize biofilm-technological processes it is necessary to
understand biofilm proliferation and behavior under certain conditions.
Direct analysis of bioflm behavior or rather structure can be performed
using different imaging techniques such as confocal laser scanning
microscopy (CLSM), scanning electron microscopy (SEM) or atomic force
microscopy (AFM) (Allen, Habimana, & Casey, 2018; Azeredo et al., 2016;
Bridier, Meylheuc, & Briandet, 2013; Dutta Sinha, Das, Tarafdar, &
Dutta, 2017). While CLSM can be used for investigating the biofilm
matrix composition (e.g., DNA and EPS) in a range of several
micrometers, SEM together with energy-dispersive X-ray spectroscopy
(EDX) examines biofilms up to 1 nm resolution and their elemental
composition. AFM determines for instance adhesion forces between biofilm
and the substratum as well as cohesive strength (Allen et al., 2018;
Azeredo et al., 2016). Optical coherence tomography (OCT) is an
application becoming increasingly relevant for the analysis of biofilms’
mesoscopic structure as seen from the raising number of publications
(Blauert, Horn, & Wagner, 2015; Dreszer et al., 2015; Haisch &
Niessner, 2007; Wagner & Horn, 2017; Weiss, Obied, Kalkman, Lammertink,
& van Leeuwen, 2016). Advantages among other imaging techniques are the
high optical resolution together with a fast acquisition of 3D datasets
of translucent tissues and materials in situ despite large
representative volumes. Mesoscopic biofilms are valuable for e.g.,
modeling of permeate fluxes in membrane systems (Derlon,
Peter-Varbanets, Scheidegger, Pronk, & Morgenroth, 2012) or substrate
turnover in biofilm reactors (Li, Wagner, Lackner, & Horn, 2016; Wagner
& Horn, 2017). Since a statistical survey of biofilm replicates is
inevitable for e.g., optimizing parameters in a biofilm production
reactor, OCT is the imaging modality of choice for the identification
and quantification of structural biofilm parameters.
Nutrients and hydrodynamics are some of the main effectors in biofilm
lifecycle. Several studies have been performed which focus on the
influence of different ions and flow velocities on biofilm behavior
(Guvensen, Demir, & Ozdemir, 2013; Park, Jeong, Lee, Kim, & Lee, 2011;
Paul, Ochoa, Pechaud, Liu, & Liné, 2012; Sehar, Naz, Das, & Ahmed,
2016; Song & Leff, 2006; P. Stoodley, Dodds, Boyle, & Lappin-Scott,
1999; P Stoodley, Cargo, Rupp, Wilson, & Klapper, 2002). Bivalent
cations such as Ca2+ and Mg2+ are
known to promote growth and stability of biofilms (Guvensen et al.,
2013; Sehar et al., 2016; P. Stoodley et al., 1999). Additionally, iron
(Fe2+) may be of concern regarding biofilm
development. Iron is an essential trace element and component of
iron-sulfur-complexes in various enzymes. Moreover, a couple of bacteria
utilize Fe3+ as an electron acceptor within the
respiratory chain (Riemer, Hoepken, Czerwinska, Robinson, & Dringen,
2004). Furthermore, iron is essential for almost all living organisms
and forms a cofactor in many cellular proteins, which are involved in
electron transport, detoxification of reactive oxygen species (ROS) or
DNA synthesis (Neilands, 1974). For that reason, at least a minimum of
iron availability should be necessary for the maturation process of
biofilms. The role of iron has been studied by several research
institutes, too, mainly applying pathogenic Pseudomonas
aeruginosa or static biofilms (e.g., agar plate- and microtiterplate
biofilms) with diverging results (Banin, Vasil, & Greenberg, 2005;
Berlutti et al., 2005; Kang & Kirienko, 2018; Musk, Banko, &
Hergenrother, 2005; Ranmadugala, Ebrahiminezhad, Manley-harris, &
Ghasemi, 2017; L. Yang et al., 2007). Additionally, most studies merely
focus on the performance of those biofilms instead of physical structure
- although structure and function are closely linked to each other. For
instance, Möhle et al. (2007) described a positive effect on the
biofilms’ stability grown in a rotating disc reactor when higher amounts
of iron sulfate (10 mg/L) were available (Möhle et al., 2007). Further
studies showed that a limitation of iron (Singh, Parsek, Greenberg, &
Welsh, 2002; Weinberg, 2004) as well as an excess of iron (Lin, Shu,
Huang, & Cheng, 2012; Musk et al., 2005; L. Yang et al., 2007) in the
environment inhibited the formation and development of biofilms in
contrast to suspended cells.
In this study, the effect of Fe2+ in the cultivation
medium on the development and maturation of Bacillus subtilisflow cell biofilms is investigated in order to evaluate biofilm behavior
in terms of structure in 3D and non-invasively. Hence, two different
Fe2+ concentrations were used to study the effect on
the biofilm’s morphology and maturation. This work strengthens the
fundamental knowledge about biofilm physical structure and their
interaction with interfaces. Furthermore, it highlights iron
(Fe2+) as an (trace) element, which can be used to
control biofilm development and maturation.