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.