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Keywords:

  • attachment;
  • biofilm;
  • Gram-negative bacteria;
  • biofouling;
  • exopolymeric substances;
  • microgravity

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions and outlook
  8. Acknowledgements
  9. References

Microorganisms tend to form biofilms on surfaces, thereby causing deterioration of the underlaying material. In addition, biofilm is a potential health risk to humans. Therefore, microorganism growth is not only an issue on Earth but also in manned space habitats like the International Space Station (ISS). The aim of the study was to identify physiological processes relevant for Micrococcus luteus attachment under microgravity conditions. The results demonstrate that simulated microgravity influences physiological processes which trigger bacterial attachment and biofilm formation. The ISS strains produced larger amounts of exopolymeric substances (EPS) compared with a reference strain from Earth. In contrast, M. luteus strains were growing faster, and Earth as well as ISS isolates produced a higher yield of biomass under microgravity conditions than under normal gravity. Furthermore, microgravity caused a reduction of the colloidal EPS production of ISS isolates in comparison with normal gravity, which probably influences biofilm thickness and stability as well.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions and outlook
  8. Acknowledgements
  9. References

Microorganisms tend to aggregate on surfaces and form biofilms which can have detrimental effects on materials (e.g. biocorrosion; Gu, 2007) or even endanger human health (e.g. medical implants; Padera, 2006). Material scientists have developed numerous strategies to prevent biofilm formation such as using antiadhesive materials and/or materials pretreated with organic or inorganic antimicrobial agents (Zhao et al., 2009). However, these antifouling materials, if they are not detrimental for nontarget cells, simply delayed the formation of biofilm. However, an efficient strategy to limit biofouling is to regularly remove the formed biofilm using high mechanical stress and/or strong biocides. Unfortunately, this is not always possible. For example, mechanical cleaning and sterilization procedures cannot be carried out on all surfaces including those of implants as well as surfaces inside the International Space Station (ISS). These two particular environments, medical implants and ISS surfaces, also share another characteristic: reduced gravity conditions.

Microgravity has been shown to have various effects on eukaryotic cell functions, but the effect of this environment on microorganisms is not as well characterized (Lynch & Matin, 2005; Crawford-Young, 2006). Low shear force affects microbial gene expression, cell morphology, physiological processes and pathogenesis (Nickerson et al., 2004). Experiments conducted with microbial liquid cultures under microgravity conditions revealed a shorter lag phase in comparison with standard conditions, as well as prolonged exponential phase (Kacena et al., 1999), formation of cellular aggregates (Purevdorj-Gage et al., 2006), a modification of the production of secondary metabolites (Fang et al., 1997), a decreased adhesion to target cells (Thomas et al., 2002), and a decreased sensitivity to antibiotics (Lynch et al., 2006). The Mir space station was heavily colonized by biofilm, which damaged quartz windows, corroded various metals and caused polymer deterioration, contributing to the shortened lifetime of that station (Matin & Lynch, 2005; Gu, 2007). A space flight experiment confirmed the increased chance of formation of stable biofilm under microgravity conditions (McLean et al., 2001). A ground-based experiment under low-shear modelled microgravity showed that Escherichia coli biofilms were thicker than their normal-gravity counterparts and exhibited increased resistance to stresses such as salt, ethanol or antibiotics (Lynch et al., 2006). However, the impact of microgravity conditions on initial cell attachment has not as yet been investigated. Under normal gravity conditions, microbial cells interact with the surface via different mechanisms depending on the distance between the cell and the surface. Apart from sedimentation and Brownian motion, Van der Waals forces mainly lead to attachment within a distance of >50 nm. At shorter distances (10–20 nm), electrostatic interactions start to play a role, and specific interactions become relevant for distances below 15 nm.

In this study, we characterized growth behaviour, cell wall properties and secretion pattern of exopolymeric substances (EPS) of Micrococcus luteus grown under normal as well as simulated microgravity conditions. The aim was to correlate these parameters with the ability of the bacteria to attach and form biofilm.

Micrococcus luteus is a Gram-positive, nonmotile, spherical, saprotrophic bacterium that belongs to the family of the Micrococcaceae. The obligate aerobe M. luteus is found in soil, dust, water and air, and as part of the normal flora of the mammalian skin. The bacteria also colonize the human mouth, mucosae, oropharynx and upper respiratory tract. Although M. luteus is nonpathogenic (risk class 1 organism), it should be considered a nosocomial pathogen in immunocompromised patients. Micrococcus luteus is resistant to reduced water potential and can tolerate desiccation and high salt concentrations. It has been shown to survive in oligotrophic environments for extended periods of time (Greenblatt et al., 2004). Not surprisingly, M. luteus was found on board the Mir station and the ISS during numerous microbial surveys (see review by Gu, 2007). The organisms were detected in the air system, growing on surfaces, and were involved in the biodegradation of polymeric materials.

This study presents a comparison of planktonic growth, cell wall characteristics and production of EPS of three M. luteus strains: the type reference strain (DSMZ20030) and two isolates from the ISS (LT100 and LT110).

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions and outlook
  8. Acknowledgements
  9. References

Random positioning machine (RPM)

The RPM is a laboratory instrument for the simulation of microgravity. Originally, the machine was developed by Hoson (Hoson et al., 1992) and manufactured by the Dutch Space, Leiden, Netherlands. Samples mounted on a platform randomly change the position in the three-dimensional (3D) space on the machine controlled by dedicated software running on a personal computer. The movement of the experimental platform suspended in the centre of two perpendicular cardanic frames is realized by two independent running engines. These engines are controlled by feedback signals from encoders, mounted on the motor axes, and by ‘null position’ sensors on the frames. Rotation rate (ω) and geometrical distance from the centre of rotation (R) yield ‘g-contours’ through the equation g2R/g0 (g0=9.81 m s−2), which provides guidelines for the design and layout of experimental packages and for the interpretation of the results. The RPM, also called the 3D clinostat, has been a standard apparatus to simulate microgravity for many years and several reference experiments have been carried out demonstrating that results from the RPM are comparable with the results obtained in space (More & Cogoli, 1996). The RPM was operated in a random walk (basic mode) with a rotation speed of 60° s−1 in a temperature-controlled room (25 ± 1 °C).

Microbial strains and culture conditions

Three M. luteus strains were studied. Dr P. Landini (University of Milan) provided the two ISS isolates, LT100 and LT110. The type reference strain was provided by the German Resource Centre for Biological Material (M. luteusT DSMZ20030). Crystal violet test on microtiter plates was used to assess the ability of the three strains to form biofilm under normal gravity conditions. Results showed that, under normal gravity conditions, the three strains formed a similar amount of biofilm (OD605 nm: 0.024 ± 0.006 for M. luteusT, 0.006 ± 0.012 for LT100 and 0.013 ± 0.012 for LT110; n=7). Bacteria were cultivated in tryptic soy broth (TSB) and frozen stocks were reconstituted. TSB 5 mL was inoculated with frozen stock and bacteria were allowed to grow overnight. The bacterial suspension was dispersed on tryptic soy agar plates and incubated at 25 °C. Three colonies were picked from one plate and grown separately overnight in liquid medium. The medium was made of (per liter) 10 g TSB, 3.5 g NaNH4HPO4, 3.7 g KH2PO4, 7.5 g K2HPO4, 0.25 g MgSO4, 2.8 mg FeSO4, 1.47 mg CaCl2, 1.98 mg MnCl2, 2.38 mg CoCl2, 0.17 mg CuCl2 and 0.29 mg ZnCl2. First, a solution of phosphate and sodium salts was prepared, the pH was adjusted to 7.1, and the medium was autoclaved. Filter-sterilized stock solutions of glucose, MgSO4 and the remaining salts were added to the cold medium. Fresh medium was inoculated with the overnight culture (dilution 1 : 100) and transferred into silicon tubes (diameter 6 mm, length 25 cm, thickness 1 mm). Control experiments showed similar growth rates within the silicon tubes compared with the baffled flasks, indicating that oxygen diffusion through the fine silicone tube was not growth-limiting. The silicon tubes were sealed with clamps. Particular attention was paid to avoid the presence of gas bubbles, which would create shear forces under the RPM. Tubes in which gas bubbles were detected at the end of the experiment were discarded. Tubes loaded with medium without bacteria were used as negative controls. Silicon tubes were placed at 25 °C either under normal gravity conditions on a shaker (70 r.p.m.) or on the RPM. Bacteria were allowed to grow until they reached the beginning of the stationary phase (c. 10 h). At this point the tubes were opened, the culture was transferred into fresh medium (dilution 1 : 100; OD600 nm=0.02) and grown under either normal or microgravity conditions until the middle of the exponential phase was reached (about 7 h). A summary of the experimental design is presented in Fig. 1. The three M. luteus strains were exposed to four types of culturing procedures: (1) preculture under normal gravity and culture under normal gravity (GG), (2) preculture under normal gravity and culture under microgravity (Gμg), (3) preculture under microgravity and culture under normal gravity (μgG) and (4) preculture under microgravity and culture under microgravity (μgμg).

image

Figure 1.  Experimental design.

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Growth rates and yield of biomass

Growth rates were determined by measuring OD600 nm of samples taken at three or more time points during the exponential growth phase, each point being from independent triplicate experiments. The total yield of biomass was estimated by measuring the OD600 nm at the beginning of the stationary phase.

Composition of EPS

The cell suspension was pelleted (8500 g, 15 min, Heraeus) and the medium discarded. Colloidal and capsular EPS were extracted according to the protocol of Hirst et al. (2003). Briefly, the cell pellet was resuspended in distilled water to extract the colloidal fraction. After 4 h the suspension was centrifuged, and the supernatant containing the colloidal EPS was frozen for later analysis. The cell pellet was resuspended in 0.1 M EDTA to extract the capsular fraction. After 12 h, the cells were centrifuged and the supernatant containing the capsular fraction was frozen for later analysis. EPS is composed mainly of polysaccharides and proteins. Proteins were quantified using the microBCA kit (Thermo Scientific) where bovine albumin served as standard. Polysaccharides were quantified according to the method of Dubois et al. (1956) using glucose as standard. EPS abundance was standardized with OD600 nm, which measures the amount of cells present in the culture.

Cell wall characteristics

Because bacterial surface properties could not be investigated directly, the cell wall of M. luteus was characterized using the microbial adhesion to solvent (MATS) method. According to the extended DLVO theory (named after Derjaguin, Landau, Verwey and Overbeek), the free energy of hydrophobic interaction (ΔGbsb) between two similar cells (b) immersed in solvent (s) can be calculated as

  • image(1)

where inline image is the apolar adhesive (Lifshitz–van der Waals, LW) interaction energy between the bacteria and the solvent, inline image is the polar cohesive interaction energy between the electron acceptors and the electron donors of the bacteria, inline image is the polar cohesive interaction energy between the electron acceptors and the electron donors of the solvent, inline image is the polar adhesive interaction energy between the electron acceptors of the bacteria and the electron donors of the solvent and inline image is the polar adhesive interaction energy between the electron donors of the bacteria and the electron acceptors of the solvent (van Oss, 1995). By comparing bacterial cell affinity to monopolar and apolar solvents with similar γLW it is possible to quantify the electron donor/electron acceptor interactions and the surface hydrophobicity of the bacterial cell surface. Bellon-Fontaine et al. (1996) developed and named this method ‘microbial adhesion to solvents’ (MATS). The monopolar solvent can be acidic (electron-accepting) or basic (electron-donating), but each pair of solvents has a similar component of Lifshitz–van der Waals surface tension (Table 1). For example, by comparing adhesion of bacterial cells to chloroform and hexadecane it is possible to estimate γs, i.e. the electron-donor property of the bacteria. Similarly, comparison of the adhesion of bacterial cells to diethyl ether and hexane gives a qualitative measurement of γs+, i.e. the electron-acceptor property of the bacteria.

Table 1.   Surface tension properties of MATS solvents (Bellon-Fontaine et al., 1996)
SolventFormulaγLW (mJ m−2)γ+ (mJ m−2)γ (mJ m−2)Type of solvent
ChloroformCHCl327.23.80Electron acceptor
HexadecaneC16H3427.700Apolar
Diethyl etherC4H10O216.7016.4Electron donor
HexaneC6H1418.400Apolar

Practically, bacterial cells were harvested by centrifugation (8500 g, 15 min, Heraeus), washed twice in 0.1 M potassium phosphate buffer, pH 7, and resuspended in the buffer to obtain a solution with an OD400 nm of approximately 0.7 (A0). Bacterial suspension (1.2 mL) was vortexed for 90 s with 0.2 mL of appropriate solvents (chloroform, hexadecane, diethyl ether, hexane). The emulsion was allowed to stand for 15 min and the OD of the aqueous phase (A) was monitored. The percentage of adherence was calculated as: % adherence=(1−A/A0) × 100.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions and outlook
  8. Acknowledgements
  9. References

Microgravity influences growth rate

Compared with normal gravity, simulated microgravity conditions increased the maximal growth rate of both M. luteusT and the ISS isolates (Table 2). Simulated microgravity conditions also influenced the yield of biomass. Under normal gravity conditions, total biomass at the beginning of the stationary phase was 1.7 and 1.8 OD for M. luteusT and LT100, respectively. Under microgravity conditions, total biomass reached 3.9 and 3.3 OD for M. luteusT and LT100, respectively. By contrast, microgravity conditions did not affect the yield of the ISS isolate LT110 (0.8 and 0.9 for microgravity and normal gravity conditions, respectively).

Table 2.   Maximal growth rates of Micrococcus luteus type strain (T) and space isolates (LT100 and LT110) placed under normal gravity (G) and simulated microgravity (μg) conditions. Average ± SD (n=3)
 GGμgGGμgμgμg
PrecultureGμgGμg
CultureGGμgμg
Micrococcus luteusT0.26 ± 0.020.21 ± 0.040.31 ± 0.040.31 ± 0.02
Micrococcus luteus LT1000.19 ± 0.060.30 ± 0.030.30 ± 0.020.28 ± 0.05
Micrococcus luteus LT1100.19 ± 0.030.27 ± 0.060.22 ± 0.060.27 ± 0.04

Microgravity influences abundance and composition of EPS

The ISS isolates LT110 formed significantly more colloidal carbohydrates than the Earth reference M. luteusT strain regardless of whether they were grown under simulated microgravity or normal gravity conditions (Fig. 2). Under normal gravity conditions, the ISS isolate LT100 also formed more colloidal proteins than the M. luteusT strain (Fig. 3). Both LT110 and LT100 produced more colloidal carbohydrates and proteins under normal gravity conditions compared with microgravity conditions (Figs 2 and 3). The response of M. luteus to microgravity conditions was a decrease in production of colloidal EPS (Figs 2 and 3). The EPS capsular fractions did not contain any detectable proteins. The abundance of carbohydrates in the capsular fraction was higher for M. luteusT under persistent microgravity conditions (Fig. 2). Under simulated microgravity conditions, the ratio of colloidal vs. capsular carbohydrates decreased significantly for M. luteusT and LT100. This result showed that the carbohydrates were more tightly bound to cells growing under microgravity conditions than normal gravity.

image

Figure 2.  Concentration of carbohydrates in the colloidal and capsular fractions of EPS of Micrococcus luteus type strain and space isolates (LT100 and LT110). The abundance of carbohydrates was standardized with OD600 nm, indicating the amount of cells present in the culture. Bars represents average values ± 95% confidence level (n=3).

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image

Figure 3.  Concentration of proteins in the colloidal fraction of EPS of Micrococcus luteus type strain and space isolates (LT100 and LT110). The abundance of proteins was standardized with OD600 nm, indicating the amount of cells present in the culture. Bars represents average values ± 95% confidence level (n=3).

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Microgravity influences cell wall characteristics

Due to large measurement uncertainties inherent in the use of the MATS method with hydrophilic cells, we could not detect significant differences between the M. luteus cell walls grown under different gravity conditions (Fig. 4). The cell walls of M. luteusT grown under simulated microgravity seemed to harbour fewer electron acceptors and more electron donors compared with cells grown under normal gravity conditions, possibly indicating that their cell walls became less hydrophilic when grown under microgravity. A similar trend was observed for LT110 (Fig. 4).

image

Figure 4.  Cell wall characterization of Micrococcus luteus type strain and space isolates (LT100 and LT110) according to the MATS method. Upper graph: microbial adherence to chloroform (colour bars) and to hexadecane (white bars). Lower graph: microbial adherence to diethyl ether (colour bars) and to hexane (white bars). Bars represents average values ± 95% confidence level (n=3).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions and outlook
  8. Acknowledgements
  9. References

Biofilm formation can be divided into two main stages: cell adhesion and biofilm proliferation. Cell adhesion depends on the probability of a cell coming into close vicinity of a surface. On the one hand, microgravity conditions decrease the probability of cell attachment by suppressing sedimentation. On the other, the probability of attachment is directly proportional to the amount of planktonic cells. In our study, this was estimated by the growth rate and the yield of biomass. Micrococcus luteus strains grew faster under microgravity conditions. Furthermore, M. luteusT and LT100 had higher yield of biomass under microgravity than under normal gravity. In general, increased growth kinetics and higher overall end point biomass under microgravity conditions represent a greater health risk for astronauts and an amplified deterioration of the materials present in the ISS. Furthermore, the higher density of cells that can be reached under microgravity compared with normal gravity, increases the probability of the organisms attaching to surfaces and biofilm formation.

The mechanism by which microgravity affects bacteria growth remains largely unknown. The most accepted explanation for the sensitivity of bacteria to microgravity is that they respond indirectly to the quiescent fluid environment which surrounds the cell cultured in suspension (Klaus et al., 1997). Such an undisturbed fluidic microenvironment is reached by two gravity-dependent phenomena: (1) sedimentation of the cells and (2) convection of less dense fluid in the vicinity of the cells. Microgravity conditions reduced these two phenomena by six orders of magnitude, creating evenly distributed cell cultures and mixed-density fluids that remain separated. In the absence of convection, the Brownian motion (diffusion) becomes the dominant transport mechanism. Motile organisms have the possibility to counteract the action of microgravity by actively moving within the medium. Reviews in the literature (Benoit & Klaus, 2007) show a strong correlation between motility and the effect of microgravity. In general, microgravity causes an increase in the amount of nonmotile cell in liquid compared with normal gravity. Exceptions to this finding were mainly found with motile bacteria (Salmonella, E. coli, Bacillus, Pseudomonas) or when experiments were conducted on solid agar. Our finding of increased growth rate and yield of biomass of the nonmotile M. luteus in liquid medium is in agreement with the data from the literature.

Initial cell attachment depends on the surface properties of both substrates and cell. As for most Gram-positive bacteria, the cell wall of M. luteus contains anionic polymers connected by covalent bonds through a linking oligomer with a peptidoglycan, the main structural polymer of cell walls (Naumova & Shashkov, 1997). Anionic polymers account for 10–60% of the weight of the cell wall and can be divided into three groups: teichoic acids, teichuronic acids and other carbohydrate-containing polymers. In this study, it was not possible to draw a clear conclusion about the influence of gravity on the cell wall of M. luteus. The cell walls grown under microgravity seemed to harbour fewer electron acceptors and more electron donors than the cell walls of bacteria grown under normal gravity. This observation could be due to a significant enrichment of anionic polymers in the cell walls of M. luteusT grown under microgravity conditions. A major feature of all anionic polymers is that they give a negative charge to the bacterial surface, which is important for physiological functions. The negative charge allows the cells to spread actively across the environment, take up cations and store them to maintain their cationic homeostasis. Anionic polymers also regulate the activity of autolytic enzymes, which are important for growth and bacterial cell division. Secondary functions of anionic polymers include their involvement in phage reception and immunogenicity (Naumova & Shashkov, 1997).

Under normal gravity conditions, the ISS isolate LT110 produced more colloidal carbohydrates compared with the M. luteusT strain. Strains isolated from the ISS produced larger amounts of EPS compared with the Earth reference strain. EPS is known to act as a protective barrier for cells against stress factors such as dehydration, UV and gamma radiation (Elasri & Miller, 1999; Niemira & Solomon, 2005; Chang et al., 2007). In this respect, the space environment is harsher than Earth's environment, which might explain why growth onboard the ISS selected strains forming high amounts of EPS. Various studies have shown an increased resistance of bacteria grown under microgravity to stresses such as ethanol, hyperosmosis, low pH or antibiotics compared with normal gravity conditions (see review by Nickerson et al., 2004). For E. coli, the microgravity effect is regulated by the σs transcription factor, which makes bacteria resistant to multiple stresses (Lynch et al., 2004). Escherichia coli formed more copious biofilms under microgravity compared with normal gravity conditions, and these biofilms were more resistant to stresses such as NaCl, ethanol, penicillin and chloramphenicol (Lynch et al., 2006). The authors did not directly quantify the EPS, but the observations suggest an increased EPS secretion under microgravity conditions. This was not the case in our experiment with M. luteus. Bacteria secrete colloidal EPS to shape their microenvironment and degrade macromolecules. A mathematical model has been developed to quantify the influence of gravity-dependent physical factors on extracellular transport processes (Klaus, 1998). This model showed a quasi-stable accumulation of byproducts around the cells in free suspension. Under microgravity, the bacteria would be able to create a favourable microenvironment more rapidly, as the excreted cofactors and/or enzymes may reach the necessary concentration sooner than under normal gravity. Therefore, it is not surprising that the bacteria produced less colloidal EPS under microgravity because they also need less to shape their microenvironment.

Numerous studies have demonstrated that the production of EPS is largely influenced by the shear stress working on the cells. Biofilm grown on high shear force tends to produce less viable cells and larger amounts of EPS, which is denser and less porous than biofilm produced at low shear force (Liu & Tay, 2002; Ramasamy & Zhang, 2005). Shear force also increases erosion and sloughing events. In contrast, absence of shear force may significantly affect the 3D structure of the biofilm. In our study, the formation of biofilm was not monitored directly, but our findings are in agreement with what is known about the relation between shear stress and EPS secretion. LT110 produced significantly fewer colloidal carbohydrates and LT100 produced significantly fewer colloidal proteins under simulated microgravity conditions, which represent a low shear stress environment. This indicates a possible decrease of biofilm abundance and stability under microgravity conditions. However, this hypothesis prediction should be viewed with caution because M. luteusT did not decrease its colloidal EPS production under microgravity. In stark contrast, Lynch et al. (2006) observed increased biofilm formation by E. coli under microgravity conditions.

Conclusions and outlook

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions and outlook
  8. Acknowledgements
  9. References

In the study, we investigated the fouling potential of M. luteus under normal and microgravity conditions. Micrococcus luteus strains isolated from the ISS were able to produce large amounts of EPS compared with the Earth reference strain. We also observed an increase in the growth rate and total biomass yield under microgravity conditions. These physiological changes may increase the chance of M. luteus attaching to surfaces. However, under microgravity, the LT110 produced fewer colloidal carbohydrates and LT100 produced fewer colloidal proteins compared with normal gravity conditions, which probably influences biofilm thickness and stability. Further experiments will focus on direct microscopic observations of biofilm formation of M. luteus under microgravity conditions.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions and outlook
  8. Acknowledgements
  9. References

Special thanks go to Aline Hunziker and Kathrin Grieder (Empa) for technical assistance, Stéphane Richard as well as Kriss Westphal (ETHZ) for their advice on the RPM and Linda Thoeny (Empa) for the manuscript revision.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions and outlook
  8. Acknowledgements
  9. References