# Streamer formation dynamics with mixed bacterial species: effects of cultivation conditions, hydrodynamics, and species

Zenamarkos Sendekie, Patrice Bacchin, Rob Lammertink, João Crespo

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Zenamarkos Sendekie, Patrice Bacchin, Rob Lammertink, João Crespo. Streamer formation dynamics with mixed bacterial species: effects of cultivation conditions, hydrodynamics, and species. AIP Advances, 2024, 14 (7), 10.1063/5.0211034. [https://doi.org/10.1063/5.0211034](https://doi.org/10.1063/5.0211034). [hal-04681596](https://hal.science/hal-04681596v1)

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# Streamer formation dynamics with mixed bacterial species: Effects of cultivation conditions, hydrodynamics, and species

Zenamarkos B. Sendekie 💻 ; Patrice Bacchin 💻 ; Rob G. H. Lammertink 💻 ; João G. Crespo

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# Streamer formation dynamics with mixed bacterial species: Effects of cultivation conditions, hydrodynamics, and species

Cite as: AIP Advances 14, 075213 (2024); doi: 10.1063/5.0211034

Submitted: 7 May 2024 • Accepted: 25 June 2024 •

Published Online: 11 July 2024

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Zenamarkos B. Sendekie,<fnref n="1" />,<fnref n="2" />,<fnref n="3" />,<fnref n="4" />,ᵃ⁾ ID Patrice Bacchin,<fnref n="2" /> ID Rob C. H. Lammertink,<fnref n="3" /> ID and João G. Crespo<fnref n="4" />

## AFFILIATIONS

## ABSTRACT

Most bacterial species synthesize extracellular polymeric substances (EPS) with diverse compositional, structural, and functional characteristics. When under sustained hydrodynamic flow, bacteria form streamers, which are filamentous matrix structures porous in nature. So far, investigations on streamers have been limited to pure culture bacterial species, overlooking the aggregate nature of bacterial flocs and biofilms. The aim of this work is to analyze the effects of the cultivation conditions (controlling the EPS synthesis), the hydrodynamics, and the bacterial species type on streamer formation by pure and mixed culture using microfluidic separators. *Enterobacter A47* (EPS-producing bacterium) and *Cupriavidus necator* (non-EPS producing bacterium) are used for the experimental work. It has been found that the EPS secreted by the bacteria and flow conditions play a very significant role in streamer formation dynamics. Strong flow conditions (i.e., high flow rates and small constrictions with tortuous architecture) favor the fast development of streamers, whereas intermediate flow rates result in sustained growth for longer filtration times, leading to dense streamers. Our analysis confirms that the presence of EPS in the bacterial suspension critically controls streamer formation by forming bacterial aggregates, or flocs, and bridging between different aggregates. We also found that streamer formation is significantly enhanced with mixed bacterial culture, which may be attributed to the symbiotic relationships influencing the concentration and characteristics of EPS and the material behavior in general.

© 2024 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1063/5.0211034

## I. INTRODUCTION

Bacterial biofilms are assemblies of micro-organisms in which cells are encased in a self-excreted matrix of extracellular polymeric substances (EPSs) sticking to each other on living or non-living surfaces. The formation of bacterial biofilm begins with the adhesion of individual bacteria to a surface, and the biofilm colonizes the surface and grows through cell division and enclosure by polysaccharide matrices.<fnref n="1" /> Recently, the study of biofilms has attracted significant interdisciplinary attention, mainly motivated by the fact that this form of bacterial collection can play both positive and negative roles in natural and engineered systems. For instance, biofilms can help

in bioremediation (in the removal of organics) in waste-water treatment, in CO₂ sequestration,<fnref n="2" />,<fnref n="3" /> and in enhancing biogas and methane yield in an anaerobic digester.<fnref n="4" /> On the other hand, biofilms can play negative roles such as in shipping industries causing biofouling,⁵ in membrane water purification systems for drinking water production forming active biofilm on the porous surface,⁶ and in clinical environments and medical devices (such as in heart stents and catheters) posing clogging and persistent infection risks aggravated by their significant resistance to antibiotics and antimicrobial agents when in biofilm form.⁷⁻¹⁰

The mechanisms by which bacteria form biofilm on surfaces and the conditions promoting this process are not yet fully

<footnote n="1">¹Faculty of Chemical and Food Engineering, Bahir Dar Institute of Technology, P.O. Box 26, Bahir Dar, Ethiopia</footnote>

<footnote n="2">²Laboratoire de Génie Chimique, Université de Toulouse, CNRS, INPT, UPS, Toulouse, France</footnote>

<footnote n="3">³Soft Matter, Fluidics and Interfaces, MESA+ Institute for Nanotechnology, Faculty of Science and Technology, University of Twente, Enschede, The Netherlands</footnote>

<footnote n="4">⁴LAQV-REQUIMTE, DQ, FCT, Universidade NOVA de Lisboa, 2829-516 Caparica, Portugal</footnote>

<footnote>ᵃ⁾ Author to whom correspondence should be addressed: zenamarkosbs@gmail.com</footnote>
understood. However, some studies have demonstrated that the biofilm formation process on a porous surface is affected by a number of factors such as the bacterial cell type, the surface properties of the bacteria, their physiology, the pH and ionic strength of the bacterial suspension, and the flow conditions.<sup>11-21</sup> The complex interplay between these biological and physico-chemical factors controls the biofilm formation process. Depending on these conditions, bacteria form biofilms with different architectures and morphologies on filters. Stoodley and co-workers<sup>22</sup> have reported that, when under flow through filters, bacteria form filamentous structures called streamers, porous-matrix structures formed mainly by externally imposed shear flow moving through a background flow. They are dominantly positioned in the bulk flow, not at the walls or stagnation points.<sup>1,23</sup> Recently, experimental studies using microfluidic devices have demonstrated that bacterial streamers can form in laminar flow conditions in zig-zag microchannels with different curvature angles<sup>24</sup> and in arrays of microchannels with different geometries and channel sizes.<sup>23</sup> Rusconi and co-workers<sup>25</sup> showed that hydrodynamics, especially the presence of secondary flow in the system, plays an important role in the development of streamers. On the other hand, Hassanpourfard et al.<sup>26</sup> investigated streamer distribution in a microfluidic device with cylindrical micro-pillars along the z axis using confocal laser scanning microscopy and reported that no localization of streamers was found; they found that streamers were distributed at different heights. Battin et al.<sup>27</sup> confirmed the presence of secondary flow around the grains of the fluidic devices using numerical simulations (COMSOL Multiphysics). Such contradictory findings suggest that the mechanism of streamer formation in relation to channel geometry or media topography remains poorly understood. Furthermore, the surface chemistry of the media significantly affects streamer inception and evolution;<sup>28-30</sup> Ghosh et al.<sup>31</sup> reported that smooth surfaces are more attractive to biofilms and to the resulting streamer formation by shearing due to flow-induced deformation affecting the initial stages of biofouling and resulting in streamer detachment on rough surfaces.

Bacteria of all taxa are known to synthesize diverse intracellular and extracellular biopolymers. Few intracellular biopolymers are studied with limited use, while there has been a vast range of studies on extracellular biopolymers with valuable structural and functional properties having different applications and promising prospects. Extracellular biopolymers are collectively referred to as extracellular polymeric substances, or exopolysaccharides (EPS). EPS play crucial roles in the development and stability of biofilms at solid-liquid interfaces such as adhesion, bacterial cell aggregation, water retention, biofilm cohesion, nutrient source and energy sink, protective barrier to various antibiotics, binding of enzymes, and sorption of organic compounds and inorganic ions.<sup>32-34</sup> The extent of EPS synthesis varies with bacterial species and the physico-chemical incubation conditions. The physico-chemical factors playing a central role in the yield of EPS include pH, temperature, incubation time, and medium composition (carbon, nitrogen, and cation sources).<sup>32</sup> Carbon availability, with the limitation of other nutrients such as nitrogen, oxygen, or phosphorus, controls EPS synthesis for most bacterial species.<sup>35,36</sup> A high carbon-to-nitrogen ratio is usually required to achieve a high yield of EPS synthesis.<sup>37,38</sup>

On the other hand, the initiation phenomena for streamer formation and quantitative evaluation of the formation and evolution dynamics remain puzzles until now. Some researchers argued that

streamer formation occurs only in turbulent flows,<sup>22,39-42</sup> while others confirmed later, with the advent of microfluidic devices, that it also occurs in creeping flow regimes.<sup>1,5,26,33,34,43-47</sup> These observations confirm that bacterial streamers can form under a variety of flow conditions, ranging from creeping to turbulent flow regimes.<sup>31</sup> Furthermore, the investigations on streamers until now have been limited to pure culture bacterial species, overlooking the aggregative nature of the economically relevant biofouling systems, which may most often involve mixed bacterial species,<sup>27,47-51</sup> which brings significant biological complexity to the analysis. The studies so far mostly did not account for inter-species interactions, disregarding cell-to-cell signaling,<sup>52</sup> which in turn may influence the material composition of the EPS. The nature or material composition of the EPS significantly determines the structure and hydrodynamics of the streamers.<sup>31</sup> Hence, investigations on streamer formation dynamics considering mixed bacterial species are still lacking and are challenging, demanding interdisciplinary developments of theoretical and experimental techniques for their qualitative analyses and quantitative evaluations at different spatio-temporal scales of scrutiny.

In this study, we present the effects of bacteria cultivation conditions, hydrodynamics, and bacterial species type (pure vs mixed species) on the bacterial streamer formation during filtration in microfluidic filters to get time- and space-resolved dynamic insights. The microfluidic systems mimic important engineering systems such as biomedical devices<sup>53-55</sup> and filtration units.<sup>1,56</sup> The cultivation conditions influence EPS synthesis, which in turn has a dominant role in streamer development. *Enterobacter A47* species is used for the cultivation conditions analysis. The bacterium *Enterobacter A47* has been studied for its synthesis of fucose-rich EPS (FucoPol) using glycerol as a carbon source.<sup>57-60</sup> The main control parameter in this study is the carbon-to-nitrogen ratio in the nutrient. Glycerol and ammonium phosphate are used as carbon and nitrogen sources, respectively. We also analyzed the extent of streamer formation by using mixed bacterial culture (a binary mixture) and compared it with the formation by pure culture. In the binary mixture, *Enterobacter A47* is mixed with *Cupriavidus necator*, a non-EPS producing species with a larger size compared to *Enterobacter A47*. *Cupriavidus necator* produces intracellular polyhydroxyalkanoates (PHA) when excess sugar is available. The experiments are performed at relatively high flow velocities (0.14 to 0.83 m/s) using different geometries of microchannels in order to investigate the effect of the hydrodynamics on the streamer formation.

## II. MATERIALS AND METHODS

### A. Microfluidic device

The microfluidic devices used for this work were designed using CleWin 4 software and fabricated in the MESA Plus (MESA+) nanolab at the University of Twente, The Netherlands. The dry reactive-ion etching process is used to fabricate the chips, and the devices are fabricated from silicon and glass wafers. Dry Reactive-Ion Etching (DRIE) of Silicon is used to etch the channels where the analysis takes place, the upstream and downstream of the channels, and the inlets and outlets. Anodic bonding is used for the bonding of the silicon wafer on which the microchannels are engraved with the Borofloat glass wafer after manually aligning and prebonding. The bonding takes place at 400 °C in a vacuum at a high voltage (1000 V).
<figure><img src="image_11.png" /><figcaption>**FIG. 1.** Microfluidic device: (a) assembly of the chip inside the chip holder; (b) different designs of the microchannels (shaded regions are flow areas). D denotes dead-end flow, S straight channels, A and P alternatively aligned square and polygon pillars, and (c) the design parameters for a 5 µm straight channel chip.</figcaption></figure>

This temperature is low enough to prevent plastic deformation of the glass wafer and, thereby, the possible closing of the channels. After the anodic bonding process, dicing is performed using a Disco-DAD dicing saw to get individual chips from the wafers. For use in filtration experiments, the chips are assembled in a chip holder that has inlets and outlets to which nano-ports and tubes are connected (Fig. 1).

The microchannels used for this experimental work have widths ranging from 2 to 5 µm with different geometries: straight microchannels (DS), microchannels with staggered square pillars (DA), and microchannels with staggered polygon pillars (DP). The microfluidic device assembly, the different designs of the microchannels, and the design parameters for a 5 µm straight channel chip are shown in Fig. 1. All the main channels and the microchannels have a depth of 20 µm (i.e., a single layer system).

## B. Bacterial suspension

*Enterobacter A47* (DSM 23139), a Gram-negative motile bacterium containing flagella, is grown on glycerol by-products from the biodiesel industry to study the effects of cultivation conditions (which influence the production of EPS) on streamer formation during filtration with microfluidic devices. The bacterium *Enterobacter A47* (DSM 23139) is rod-shaped with dimensions of 0.7–0.8 µm × 1.2–2.5 µm.⁶¹

*Enterobacter A47* produces a fucose-containing EPS (FucoPol) by cultivation in mineral medium supplemented with glycerol.⁶⁰ EPS synthesis by *Enterobacter A47* has been shown to be influenced by both the initial glycerol and nitrogen concentrations and by the nutrients' feeding rate.⁶⁰ Therefore, two types of *Enterobacter A47* cultures are cultivated by using different substrate ratios to control the EPS production.

The first culture (EPS-producing culture) is grown on a medium supplemented with glycerol by-product (40 gl⁻¹), while nitrogen concentration is kept low (<0.1 gl⁻¹). The second culture (a non-EPS producing culture) is cultivated with the same medium

<figure><img src="image_12.png" /><figcaption>**FIG. 2.** Experimental setup for investigating the streamer formation phenomenon. The bacterial suspension in the syringe is pumped through the microfluidic filter, and images are captured by the camera.</figcaption></figure>

and cultivation conditions but with an increased nitrogen concentration (1.27 g/l) to suppress the EPS production.

Mixed bacterial culture (*Enterobacter A47* and *Cupriavidus necator* in a 1:1 ratio) is also cultivated aerobically in the same medium as the first case to study streamer formation. The cultivation is performed at a controlled temperature of 30 °C and a pH of 7. Both bacterial species have a cell concentration of 6 g per dry weight after the growth phase. *Enterobacter A47* produces EPS, while *Cupriavidus necator* produces intracellular polyhydroxyalkanoates (PHA) when there is excess sugar. *Cupriavidus necator* is a non-EPS producing bacterium and is larger in size compared to *Enterobacter A47*.

## C. Filtration conditions

Filtration experiments are performed in dead-end mode at constant flow velocity by using a syringe pump (Harvard Apparatus PHD 2000). The filtration process is recorded using a light sensitive camera (Olympus). The dynamics of streamer formation during filtration is recorded by taking images every 30 s. The experimental setup for the filtration to study streamer formation phenomena is shown in Fig. 2. During filtration, flow conditions are kept in a laminar regime, and the maximum Reynolds number is 6.64, calculated at the maximum flow velocity (0.83 m/s) in the feed channel. The flow velocity is varied from 0.14 to 0.83 m/s (corresponding to flow rates from 0.05 to 0.30 ml/min).

## III. RESULTS

Physical effects (i.e., flow conditions and geometry of the micro-separator) and biological factors (i.e., bacterial type and cultivation conditions) influence streamer formation dynamics during the filtration of bacterial suspensions through micron-sized separators. In this experimental work, we used microfluidic devices to investigate how flow, channel geometry, and bacterial cultivation conditions, in particular the substrate composition, affect streamer development in both pure and mixed bacterial cultures. The experiments are performed at a relatively high flow regime (Re ≤ 6.64). Constant flow velocity filtration experiments are performed using three different geometries of the separation section with both pure and mixed bacterial cultures. These geometries are used to study the
effect of the connectivity of the microchannels and the sharp edges of the pillars on the extent of streamer formation (Sec. III A 1). These geometry effects are mainly related to the secondary flow that occurs at the edges of the pillars. The microchannel size is also varied (2 and 5 µm) to investigate the effect of the constriction on the streamer formation dynamics. The effect of the cultivation conditions is presented in Sec. III A 2. The flow velocity is varied from relatively low to high values (i.e., 0.14, 0.28, and 0.55 m/s) in experiments

with pure culture suspensions (Sec. III A 3) and abruptly increased to 0.83 m/s after forming a dense streamer at 0.55 m/s to analyze the effect of a sudden change in hydrodynamic conditions. Filtration experiments with mixed bacterial suspensions are performed at intermediate flow velocity (i.e., 0.28 m/s), at which high streamer formation is observed with pure bacterial suspensions (Sec. III B). Two microchannel geometries are used for filtration experiments with mixed culture: microchannels with staggered square pillars and

<figure><img src="image_13.png" /><figcaption>FIG. 3. (a) Temporal variation of the streamer formation for two different channel sizes and geometries. The images are captured at 0, 60, and 90 min. (b) Temporal variation of the streamer formation for staggered square channel geometry with a 2 µm size.</figcaption></figure>
polygon pillars. The following sections present the effects of all these parameters on the streamer formation dynamics.

# A. Pure culture

## 1. Geometry effect

Experiments are performed with microchannels having different tortuosity and channel sizes at a constant flow velocity of 0.55 m/s. A very low accumulation of bacteria and streamer formation is observed for non-EPS producing *Enterobacter A47* pure cultures cultivated at low C:N ratios. Figure 3(a) shows a 2D micrograph of bacterial accumulation for two channel sizes (2 and 5 µm) and two channel configurations (straight channels and staggered square pillars). The accumulation of materials on the upstream side of the filtering zone is observed frequently, and the possible reasons for this could be the glycerol remaining in the suspension, protein agglomeration, the presence of inorganic residues, contamination from the chips and connections, or a combination of these factors. Relatively dense streamers were formed when filtering the same bacterial species with tortuous channels (channels with alternatively aligned pillars). Figure 3(b) demonstrates the temporal increment of the density of streamers with time in staggered square pillar channels.

Figure 4 shows that the average streamer length increased more rapidly with the tortuous microchannels. The average streamer length is calculated by determining the area covered by the streamers and dividing the value by the width of the channel. The area covered by the streamers is calculated by ImageJ software. The tortuous nature of the microchannels has a more significant effect on streamer formation than the decrease in channel size, which is mainly due to the presence of secondary flow.²⁵ The inset image shows the attachment and gliding phenomena of bacteria during the inception of the streamers. The average streamer length for the tortuous channels is about 34 µm, while for the 2 µm channels, it is 6 µm after 100 min of filtration time. Recent studies have demonstrated that the presence

<figure><img src="image_14.png" /><figcaption>FIG. 4. Effect of geometry on bacterial streamer formation for filtration experiments performed at 0.55 m/s.</figcaption></figure>

of complex surface geometries enhances the formation of flexible streamers, which causes clogging in flow systems.⁵³,⁶²,⁶³ This could enhance streamer growth through the filtration of bacteria and other components in the system by interception from the bulk fluid.²⁷

In these systems, the hydrodynamics control the formation and the three-dimensional structure of the bacterial streamers, while bacterial motility is insignificant since the experiments are performed at relatively high flow rates. Therefore, the flow field around the pillars of the different channel geometries plays a major role in shaping the streamers in different structures. The tortuous nature of the flow (Fig. 3) actively enables the development of dense streamers close to the outlets of the channels. In contrast, long and highly porous filaments are formed by the flow in straight channels. This shows that flow determines the three-dimensional structure of bacterial streamers. The temporal oscillation in average streamer length for tortuous channels in Fig. 4 is due to the detachment of streamers from the channel outlets by the flow caused by the difference in pressure gradient in the background flow and in the streamer jets.⁶⁴ The streamer breakup is thus purely a flow instability phenomenon in the viscous filamentous jets.

## 2. Cultivation conditions effect

The presence of EPS in pure cultures has been confirmed and observed using various electron microscopy techniques. The EPS are characterized by their adsorption ability, biodegradability, and hydrophilicity/hydrophobicity. The main components in EPS, though it is a dynamically evolving entity, are carbohydrates, proteins, humic substances, and nucleic acids.⁶⁵,⁶⁶ The combination of the above special characteristics and the contents of the main components of EPS crucially affect the physicochemical properties of microbial aggregates, including the structure, surface characteristics, mass transfer, adsorption ability, and stability. Substrate type and nutrient levels have substantial effects on the microbial communities and their metabolism, influencing the production and composition of EPS.⁶⁷⁻⁷¹ Furthermore, EPS composition varies with species origin, growth temperature, and hydrodynamic shear.⁶⁵,⁶⁶,⁷²⁻⁷⁴ In this study, we analyze the effect of the presence of EPS on streamer formation. The C:N ratio in the substrate (which is kept at 50:1 for the EPS-producing culture and at 4:1 for the non-EPS producing culture) is the main controlling parameter for the EPS synthesis by the bacteria. The synthesis of EPS by *Enterobacter A47* bacterium in nitrogen limited cultivation conditions may be related to the absence of nitrogen, restricting other metabolic pathways and leaving place for EPS synthesis.⁶⁰ Fig. 5 shows very rapid streamer formation in the downstream zone of the separation section during filtration with the EPS-producing culture with straight microchannels, while there is no streamer formation by the non-EPS producing culture. The time scale for the streamer formation is in the order of a few minutes in the former case; the average streamer length reaches about 50 µm in just 5 min of filtration time. The streamers are very long but highly porous in structure. They are also very dynamic undulating in the background flow.

The inset images in Fig. 5 show the extent of the difference in streamer formation between the EPS-producing culture and the non-EPS producing culture after an hour of filtration time. Therefore, it can certainly be noted that EPS secretion by bacteria is the main cause, if not the only one, of streamer formation during filtration through constrictions.
<figure><img src="image_15.png" /><figcaption>FIG. 5. Effect of cultivation conditions on streamer formation for experiments performed with two cultures cultivated in different C:N nutrients. The filtration experiments were performed with straight channel geometry.</figcaption></figure>

<figure><img src="image_16.png" /><figcaption>FIG. 6. Effect of flow velocity for experiments performed with EPS producing culture using DS5 channel geometry.</figcaption></figure>

### 3. Flow velocity effect

Filtration experiments using EPS-producing pure culture, *Enterobacter A47*, are performed at three different flow rates of 0.05, 0.10, and 0.20 ml/min (corresponding velocities of 0.14, 0.28, and 0.55 m/s) to analyze the hydrodynamics effect on the bacterial streamer formation. The experiments are performed with 5 µm wide straight channels in dead-end mode. Figure 6 shows the average streamer length with time, and it can be noted that streamer formation is favored at intermediate flow velocity (0.28 m/s), with the average streamer length reaching up to 125 µm. The average streamer length at the highest flow velocity (i.e., 0.55 m/s) becomes almost constant (about 65 µm) after 20 min of filtration time, except for small oscillations. These oscillations are due to the balance between the formation and the subsequent detachment of streamers by the flow. The average streamer length increases throughout the filtration time at the lowest flow velocity (0.14 m/s), where no detachment of streamers by the flow is observed.

In order to analyze further the effect of the flow on the streamer stability and long-term fate, the flow velocity was suddenly increased from 0.55 to 0.83 m/s (corresponding flow rate of 0.30 ml/min in Fig. 7), after the formation of dense streamers, and the average streamer length was evaluated with time (Fig. 7). It can be noted from Fig. 7 that the average streamer length drops substantially and then starts to increase, revealing that the background fluid flow plays a significant role in the stability of bacterial streamers.44,63,75 Valiei et al.63 hypothesized instability of the streamer jets for failure or detachment, while Biswas et al. proposed instability due to necking as a mechanism for localized failure75 or due to void growth at the attachment surfaces.76 Streamer breakage or failure could also occur due to clogging of the channels after the formation of dense streamers, thereby resulting in localized failure by the stick-slip mechanism.44 The instabilities may arise from localized pressure cavities, tearing, and bacteria losses. The interaction between the

background flow and the structure of the streamers may change with time as the streamers get denser, which may create localized flow channels for the fluid, causing instability and failure. The biological life phenomena of the system in the streamers further complicate the analysis. Hence, a detailed investigation of the nucleation and growth of such instabilities and final failure mechanisms at the nanoscale is still a scientific challenge. The inset images in Fig. 7 demonstrate that the streamers are dense with less detachment at 0.55 m/s, while there is significant streamer detachment at 0.83 m/s and the structure is more porous.

<figure><img src="image_17.png" /><figcaption>FIG. 7. Effect of a sudden increase in flow velocity on the average streamer length after streamer formation. The experiments were performed with DS5 channel geometry (i.e., straight channels with a 5 µm channel width).</figcaption></figure>
## B. Mixed bacterial culture filtration

Almost all of the studies reported so far on the production of EPS and streamer formation are mainly conducted by employing pure bacterial cultures.<sup>1,26,63,77</sup> However, aggregative forms of bacteria, such as flocs or biofilms, are often supposed to comprise mixed species. The important interactions between microbes, i.e., inter-species and intra-species interactions, have been overlooked. Few researchers have reported that using mixed cultures over single cultures promotes EPS synthesis.<sup>78–81</sup> The cooperative growth of bacterial cells that occurs in mixed cultures of EPS-producing and non-EPS producing bacterial strains can influence the concentration and characteristics of the EPS.<sup>82</sup> The presence of certain non-EPS producing bacterial species along with EPS producing species in a mixed culture can stimulate the production of EPS. Figure 8 shows the comparison between the streamer formation dynamics during filtration of EPS producing pure culture (*Enterobacter A47*) and mixed bacterial cultures of *Enterobacter A47* and *Cupriavidus necator* at intermediate velocity (0.28 m/s) using microchannels with staggered square and polygonal pillars.

Very long streamers (about 330 µm) are formed by the mixed culture at a filtration time of 80 min with the staggered square pillar geometry. The corresponding average streamer length is about 240 µm for the pure culture, which shows about a 37% increment in average streamer length with the mixed culture. The initial stages of streamer formation show similar trends for both cultures. One possible reason for this could be that the initial stages of the bacterial adhesion process may be dominated by the *Enterobacter A47* species during filtration with the mixed culture. On the other hand, the initial stages of streamer formation may be dominated by the geometry of the micro-separator rather than the bacterial species during the filtration of both pure and mixed cultures. After a certain filtration time (about 20 min), the streamer formation is more

pronounced for the mixed culture. At later stages, higher concentrations of biological particles (bacteria) in mixed culture may enhance adhesion; cell-to-cell signaling or “co-evolutionary” relationships may also impact streamer formation due to an increase in carrying capacity through niche construction.<sup>27</sup> Scheidweiler *et al.*<sup>26</sup> cultured *Pseudomonas flourescens* bacterium in LB and M9 media and reported that significantly smaller flocs were formed in the latter case and streamer formation was not observed; this shows that growth conditions principally control streamer formation.

It has been reported that when two or more microbial strains form a biofilm, the presence of EPS may not only assist in establishing the biofilm but also promote greater biofilm growth than the comparable single species biofilm.<sup>83</sup> The EPS promotes not only the adhesion and growth of the cells that synthesize it but also those of other microbial species. Symbiotic relationships between the microbial strains may also be beneficial for EPS production in the mixed culture. These relationships between EPS and non-EPS producing bacterial strains could be useful for producing EPS with higher floculation efficiency, which increases the adhesion probability of the bacterial cells or aggregates. Mixed culture of EPS-producing and non-EPS producing bacterial strains may also reveal higher viscosity and higher molecular weight EPS production than that of pure culture,<sup>83</sup> which in turn enhances the streamer formation process.

# IV. DISCUSSION

## A. Role of EPS on bacterial streamer formation

In many systems, for instance, membrane bioreactors, the presence of EPS is a serious problem due to fouling, which occurs by pore clogging, floc adhesion, and cake layer and/or streamer formation.<sup>84</sup> The important factors determining the extent and severity of the fouling in membrane systems handling materials containing EPS are the concentration and the chemical characteristics of the EPS.<sup>85,86</sup> Different strategies have been proposed to mitigate this membrane fouling problem such as control of EPS production, modification of EPS characteristics, and removal of EPS from the system.<sup>86</sup> The implementation of these strategies requires a detailed analysis of the different components and the chemical and functional properties of the EPS to understand its role in fouling. EPS production control is challenging because EPS secretion is a general attribute of microorganisms in both natural systems (e.g., wastewater sludge) and in laboratories with controlled conditions occurring in both eukaryotes and prokaryotes.

Because of its special characteristics, the presence of EPS in microbial suspensions highly influences the properties of the suspension during flow. Provided that EPS is available with the desired properties, it can play fundamental roles in the matrix, including adherence to surfaces, aggregation of bacterial cells, formation of flocs and biofilms, cell-cell recognition, resistance against external forces such as shear by acting as structural elements, protective barrier for cells, water retention to avoid desiccation of cells, sorption of exogenous organic compounds and inorganic ions, etc. The biopolymers in EPS play an important role in the flocculation of particles by bridging mechanisms that encourage aggregation. They can also reduce the particle surface charge by using oppositely charged cations and/or bioflocculants, reducing the distance between the particles and making attractive forces more effective than repulsive

<figure><img src="image_18.png" /><figcaption>FIG. 8. Streamer formation dynamics by pure culture (*Enterobacter A47*) and by mixed culture (*Enterobacter A47* and *Cupriavidus necator*) using 5 µm wide channels with rectangular pillars and polygonal pillars at 0.28 m/s flow rate.</figcaption></figure>
forces between the particles. Therefore, EPS takes part in the aggre-
gation of particles through the charge neutralization mechanism.
The higher the molecular weight of the biopolymers (which is the
case for *Enterobacter A47*), the more effective the bridging mecha-
nism is. Flocculation with high molecular weight EPS involves more
adsorption sites.

The production of EPS by the *Enterobacter A47* bacterium
and its composition are largely influenced by a number of factors
that govern bacterial metabolism, including the cell growth phase,
the carbon and nitrogen sources and their ratios, the pH of the
system, the temperature of the system, and aeration (aerobic or
anaerobic conditions).⁶⁰ Increasing the initial nitrogen concentra-
tion led to higher *Enterobacter A47* growth, but it was detrimental
to EPS synthesis. Higher nitrogen concentrations not only signifi-
cantly reduced EPS production but also resulted in the synthesis of
polymers with a very different composition, namely, lower sucrose
content and higher glucose content;⁶⁰ this may be linked with the
production of an enzyme that degrades biopolymers.

A profound emphasis is given to the carbon-to-nitrogen ratio
in relation to EPS production because it has a significant effect on
microbial metabolism and, hence, on biopolymer production. The
carbon-to-nitrogen ratio has been varied over a wide range in order
to analyze its effect on EPS synthesis and streamer formation dur-
ing flow through microchannels. Increasing the carbon-to-nitrogen
ratio from 4:1 to 50:1 largely increased EPS production. Filtration
of bacterial suspensions of the two types (non-EPS producing and
EPS producing) through microchannels shows that the presence
of EPS in the system significantly controls streamer formation. Ye
et al. (2011b)⁸⁷ reported that decreasing the carbon-to-nitrogen ratio
from 100 to 20 (favorable for EPS production) resulted in decreased
carbohydrate content and increased protein content in the EPS. Dur-
maz and Sanin (2001)⁶⁷ also reported that a carbon-to-nitrogen ratio
of 5 in activated sludge resulted in EPS rich in proteins but low in
carbohydrates; increasing the ratio to 40 led to a sharp decrease in
the amount of protein.

Our analysis of the effect of EPS presence in bacterial suspen-
sion filtration shows that EPS plays a crucial role in streamer for-
mation, as demonstrated in Fig. 5. All the characteristics of the EPS
described earlier may have important roles in this dynamics. The
EPS can have a significant role in the formation of bacterial aggre-
gates, or flocs, which increases the adhesion probability and hence
the streamer formation. The biopolymers can also play a bridging
role between different bacterial aggregates, and the entanglements
among themselves and with other molecules lead to streamer for-
mation. The formation of tinny filamentous structures connecting
to form nets growing into streamer jets initiates the process, and
our experiments confirm this hypothesis, leading to the conclusion
that the EPS produced by bacteria a the main cause of streamer
formation. The production of EPS is in turn controlled by the
carbon-to-nitrogen ratio in the substrate. Therefore, the cultivation
decision of bacteria control the streamer formation phenomenon
during flow through constrictions.

Our study also shows that the presence of non-EPS producing
bacterial species (*Cupriavidus necator*) along with EPS-producing
species (*Enterobacter A47*) in a mixed culture enhances streamer
formation. This could be related to the cooperative growth that
occurs in the mixed culture of the bacterial species, which influences
the concentration and characteristics of the EPS.⁸² The different

characteristics of EPS may increase the adhesive properties of
surfaces. Symbiotic relationships between the two bacterial strains
might also enhance EPS production by the EPS-producing species.
Furthermore, EPS synthesis may increase due to an increase in
substrate utilization efficiency, which results from the secretion of
certain enzymes by the microbes helping them fulfill their nutrient
requirements. The flow properties of the mixed cultures (such as the
viscosity) could be largely influenced by these phenomena, and this
could enhance the extent of adhesion and streamer formation.

B. Hydrodynamic effects of streamer formation

Analysis of the rheological characteristics of the cultivation
broth of *Enterobacter A47* bacterium showed that there is a consid-
erable viscosity buildup, and the cultivation broth developed a non-
Newtonian behavior essentially due to EPS concentration, molecular
weight, chemical composition, the formation of new interactions
between individual EPS molecules, and the other components of the
broth. These new interactions and the resulting viscosity buildup can
have a prominent impact on the formation and morphology of the
bacterial streamers.

Concomitant with EPS synthesis and its paramount effect on
bacterial streamer formation, hydrodynamic conditions play impor-
tant roles in streamer inception dynamics. In this regard, we have
analyzed the parametric effects of the channel size, the architecture
of the micro-separator, and the flow velocity. The dynamics of the
streamer formation and the resulting morphology is highly influ-
enced by the size and geometry of the filter. The temporal growth
of the bacterial streamers is quantified by evaluating their average
length. The dynamics of the streamers in the flow system is analyzed
in terms of the area differences between two consecutive images
captured during the filtration experiment. The morphological dif-
ferences between streamers formed by different filter geometries are
analyzed by evaluating the “particles” size distribution.

In general, small channel sizes and increased tortuosity of chan-
nels resulted in rapid streamer formation and growth (Fig. 4). This
could be related to an increase in the capture efficiency of the bac-
teria as the channel size decreased and approached the size of the
bacteria and the role played by the secondary flow in the entangle-
ment between the polymer chains as the channels became tortuous
with sharp edge corners. The streamers formed with straight chan-
nels by the EPS-producing bacterial suspension of *Enterobacter*
*A47* are long and highly porous. In contrast, the streamers formed
during filtration using tortuous microchannels are relatively short
and dense in structure. This shows that the filter geometry largely
influences the morphology of the filamentous structures formed by
the bacterial aggregates. This morphological difference between the
streamers formed by the two geometries is analyzed by calculating
the “particle” size distribution by binarizing the images captured
after an hour of filtration. The “particle” size distribution evalua-
tion gives an estimation of how segregated or densely populated
the streamer jets are. Figures 9(a) and 9(b) present the results of
the “particle” size distribution for streamers formed with straight
microchannels (non-tortuous) and tortuous geometry, respectively.

The results show that the “particles” are largely distributed
for the streamers formed with both geometries and form a non-
Gaussian distribution. The “particle” sizes for streamers formed
using tortuous microchannels range from 0.5 to 350 µm, while the
<figure><img src="image_19.png" /><figcaption>FIG. 9. Demonstration of the particle size distribution for streamers formed by mixed cultures with (a) straight microchannels and (b) tortuous microchannels.</figcaption></figure>

corresponding values for streamers formed with straight microchannels range from 0.5 to 163 µm in diameter. A large number of small particles and a small number of relatively large “particles” are obtained from streamers formed with straight microchannels. Compared to this, the structure formed by the tortuous geometry results in a small number of small “particles” and a relatively large number of large “particles.” The mean diameter of “particles” for streamers formed with straight microchannels is 1.217 µm, while the corresponding value for streamers formed with tortuous microchannels is 3.701 µm. The sphericities of “particles” formed with straight and tortuous microchannels are 0.64 and 0.65, respectively. These data indicate that very long and highly porous

streamers are formed during filtration with straight microchannels [Fig. 9(a)]. On the contrary, the tortuosity of microchannels results in relatively large “particle” sizes, implying that the streamers are dense in structure. This could be related to the role of the sharp corners of the pillars (or the secondary flow) in the aggregation of the bacterial cells wrapped by the large molecular weight biopolymers.

High flow velocity enhanced the rapid formation and growth of streamers during the initial periods of the filtration experiments (Fig. 6). However, there is no further growth of the streamers, and it seems to reach a limit with small fluctuations in the average length due to the detachment of some streamer jets by the background
flow. Relatively low velocity filtration of the bacterial suspension resulted in a slow but steady increase in the average streamer length with no fluctuations. The longest streamers (about 130 µm using straight microchannels) are formed while filtering at intermediate flow velocity (0.28 m/s), with some fluctuations at long filtration times. Therefore, flow velocity plays an important role in the kinetics of streamer growth and the resulting average streamer length. A sudden increase in velocity (from 0.55 to 0.83 m/s) after the formation of dense streamers resulted in the depletion of the streamers due to detachment by the background fluid flow. This could be related to the instability created in the streamer jets by the flow due to the pressure gradient difference between the fluid flow and the streamer jets.

As the streamers thicken, they appear to become more “rigid.” It has been observed during the filtration experiments that they cease to undulate in the flow. Frequent detachment of streamer jets is also observed as the filtration experiment proceeds. Figure 10 shows the area differences between the consecutive images, indicating the streamer formation dynamics. There are two possible sources for the area difference: one is due to the formation of new streamers, and the other possibility is the mobility of the streamers by the background flow.

Higher fluctuations in area coverage by streamers in the initial stages of the filtration experiment can be noted in Fig. 10 for both straight and tortuous channels. This is due to the high mobility or undulation of the streamers by the fluid flow and the rapid growth of streamers during this period, both of which were clearly observed during the filtration experiments. This fluctuation is more pronounced and lasts longer (more than an hour) for straight channels, while for tortuous channels, the area difference shows a steady increase after about 20 min. This shows that denser and lower mobility streamers are formed by the tortuous microchannels. At the end of the filtration (after 70 min), a limit is reached, and the area differences are leveled-off for both cases. This is because the streamer

formation dynamics reach equilibrium due to the balance between the formation and the detachment of streamers in the fluid flow.

## V. CONCLUSION

In this work, we present the effects of bacterial cultivation conditions and hydrodynamics on streamer formation during the flow of both pure (EPS-producing) and mixed (EPS producing and non-EPS producing) bacterial suspensions through constrictions. The carbon-to-nitrogen ratio is used as a control parameter to analyze the effects of cultivation conditions on the bacterial species, which influence EPS synthesis and streamer formation. Complex microchannel geometries are used, and the flow velocities are varied in order to analyze the effect of the hydrodynamics on the streamer formation.

The results show that the substrate type and nutrient levels supplied to bacterial species influence the synthesis and composition of EPS, which in turn controls streamer formation. Nitrogen limited cultivation conditions favor EPS synthesis, which is the main factor in streamer formation. Rapid and pronounced streamers are developed during filtration with EPS producing culture. The tortuosity of microchannels plays a significant role in streamer formation and the morphology of the streamer due to the occurrence of secondary flow, which enhances the clogging in flow systems. Tortuous microchannels augment the rapid formation of dense streamers. Furthermore, moderate hydrodynamic conditions (i.e., intermediate flow velocities) favor streamer growth and stability, which are mainly related to the background fluid flow. Studies on streamer formation have so far overlooked the important interactions between microbes. The streamer formation dynamics comparison between pure and mixed bacterial cultures shows that streamer formation is more pronounced in the mixed culture. In summary, the results from our experimental study demonstrated that bacterial streamer formation is influenced by both biological (such as the type of culture and the cultivation conditions) and hydrodynamic (such as the flow conditions and the micro-separator geometry) conditions.

## ACKNOWLEDGMENTS

The authors acknowledge the financial support of the Education, Audio-visual and Culture Executive Agency (EACEA) division of the European Union under the program of the EUDIME (European Doctorate in Membrane Engineering, http://www.eudime.unical.it) grant in the Erasmus Mundus framework.

## AUTHOR DECLARATIONS

### Conflict of Interest

The authors have no conflicts to disclose.

### Author Contributions

**Zenamarkos B. Sendekie:** Conceptualization (equal); Data curation (equal); Formal analysis (equal); Investigation (equal); Methodology (equal); Validation (equal); Visualization (equal); Writing – original draft (equal); Writing – review & editing (equal). **Patrice Bacchin:** Conceptualization (equal); Formal analysis (equal); Funding acqui-

<figure><img src="image_20.png" /><figcaption>FIG. 10. The areal dynamics of the streamers showing their involvement or undulation in the background fluid flow for both pure and mixed cultures.</figcaption></figure>
sition (equal); Methodology (equal); Project administration (equal); Resources (equal); Software (equal); Supervision (equal); Validation (equal); Writing - review & editing (equal). **Rob G. H. Lammertink:** Conceptualization (equal); Formal analysis (equal); Funding acquisition (equal); Project administration (equal); Resources (equal); Supervision (equal); Writing - review & editing (equal). **João G. Crespo:** Conceptualization (equal); Formal analysis (equal); Funding acquisition (equal); Methodology (equal); Project administration (equal); Resources (equal); Supervision (equal); Writing - review & editing (equal).

## DATA AVAILABILITY

The data that support the findings of this study are available within the article.

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