Characterizing the Cell Surface Properties of Hydrocarbon-Degrading Bacterial Strains, a Case Study

Abstract

This chapter describes some of the most common methods used to characterize the cell surface properties of the bacterial cells. As a case study, the focus of this chapter is on Sphingomonas spp., Sph2, which is a Gram negative and hydrophilic bacterial strain. The species used in this research was isolated from groundwater at a phenol-contaminated site. This hydrocarbon-degrading strain that can participate in bioremediation of polluted environments belongs to Sphingomonadaceae family. This group of bacteria is unique among Gram-negative cells because of having glycosphingolipids (GSL) instead of the lipopolysaccharide (LPS) layer in their cell wall. To characterize this strain, its surface properties were examined using potentiometric titration, modelling surface protonation sites using ProtoFit, zeta potential measurements, and attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy. There is no published detailed study about cell wall characteristics of Sph2 yet, and this research reports such information for the first time. In addition, to investigate effects of the solution ionic strength on Sph2 adhesion behavior on metal oxides, its biofilm formation on hematite, as the model mineral, was evaluated in three different ionic strengths; ≈200 mM, 100 mM, and 20 mM. The ATR-FTIR analysis showed that despite the unique cell wall chemistry of Sph2 among the Gram-negative strains, its surface functional groups are similar to other bacterial species. Hydroxyl, carboxyl, phosphoryl, and amide groups were detected in Sph2 infrared spectra. The potentiometric titration results showed that Sph2 PZC is approximately 4.3. Optimizing the titration data based on ProtoFit non-electrostatic model (NEM) provided compatible results to the infrared spectroscopy analysis and four pKa values were identified; 3.9 ± 0.3, 5.9 ± 0.2, 8.9 ± 0.0, and 10.2 ± 0.1, which could be assigned to carboxyl, phosphate, amine, and hydroxyl groups, respectively. Zeta potential measurements demonstrated that changing the ionic strength from ≈200 mM to ≈20 mM shifts the zeta potential by ≈−20 mV. Direct observation showed that this alteration in the ionic strength coincides with a tenfold increase in the number of Sph2 attached cells to the hematite surface. This could be attributed to both electrostatic interactions between the cell and surface, and conformational changes of Sph2 surface biopolymers. In addition to reporting Sph2 cell wall characterization results for the first time, this study highlights importance of ionic strength in the cell adhesion to the mineral surfaces, which directly influence biofilm formation, bioremediation, and bacterial transport in aqueous systems.

Appendices

Appendix

Preparing the Hematite Surface for Cell Adhesion Studies (Synthesis, Coating, and Characterization)

Hematite was prepared by heating an acidic solution of FeCl_3. A STOE STADI P X-ray powder diffractometer and a Perkin Elmer Spectrum Spotlight FTIR imaging system for Fourier-transform infrared spectroscopy (FTIR) were used to analyze the synthetized materials. For XRD analysis, copper K alfa was the radiation source; a range of 10–70 degrees and a step size of 0.02 degrees were the test parameters. In FTIR experiments, the spectrum resolution was 4 cm⁻¹, covering the range of 4000–400 cm⁻¹ wave numbers, and 150 scans were collected for each sample.

To determine the point of zero charge (PZC) of the synthetic metal oxide, potentiometric titration was done. An automated potentiometric titrator (Metrohm, 718 STAT, Titrino) was used. During titrations, acid (HCl, 0.1 M) and base (NaOH, 0.1 M) were added by a computer-controlled microburette with a dispensing volume of 0.01 ml. The titrator was adjusted to add successive acid or base when the absolute value of the potential drift was equal to or less than 5 mV/min. The sample suspensions were purged with N₂ gas to remove carbon dioxide from the system for approximately 2 h before titration, which was performed in an N₂ atmosphere. In these tests, a magnetic stirrer provided continuous stirring and the suspension temperature was kept at 25 °C during the titration period. Surface hydrophobic/hydrophilic properties of the synthetic minerals were obtained by measuring the water-drop contact angle in air. Contact angles were obtained using the sessile drop method and a KRÜSS DSA 100 drop-shape analysis system. An aliquote of 3 μl of UHQ water was added to the mineral surfaces at room temperature. The contact angle between the surface and a tangent drawn on the drop surface, passing through the triple point of atmosphere-liquid-solid, was measured. Iron oxides’ hydrophilic nature stems from their surface hydroxyl groups. In general, surfaces with a water-drop contact angle of less than 90 degrees are hydrophilic; nevertheless, for the surfaces studied, the expected water-drop contact angles were considerably less.

The coating process involved the direct deposition of mineral particles from an aqueous suspension by evaporation, which has been explained in detail in previous publications (Pouran et al. 2014, 2017). After this step, the coated polystyrene surfaces were assessed using optical microscopy (Zeiss, Axiovision), direct imaging, and contact-angle measurements to determine their hydrophobicity (as described above). The ATR-FTIR , attenuated total reflection-Fourier transform infrared, technique using a Specac Silver Gate Essential Single Reflection ATR System and XPS, and X-ray photoelectron spectroscopy (KRATOS-Axis 165) were also used to compare the chemical properties of altered surfaces with those of reference polystyrene and mineral surfaces (Pouran et al. 2014).

Bacterial Strains, Growth Conditions, and Sample Preparation

Six bacterial strains were isolated for bacterial-adhesion and attached-growth studies. Rhodococcus spp., RC92 and RC291, both Gram-positive, were isolated from soil samples from a polluted gasworks site in northeast England. The bacteria Pseudomonas spp. (Pse1 and Pse2) and Sphingomonas spp. (Sph1 and Sph2) were isolated from groundwater at a phenol-contaminated site in the West Midlands (England). The strains Pse1, Pse2, Sph1, and Sph2 are Gram-negative. They have been classified using comparative 16S rRNA sequencing (Geoghegan et al. 2008). All strains were maintained on a solid R2A medium (Oxoid).

The bacterial strains were grown in an AB10 medium, which is a defined medium with known exact chemical composition. The carbon source was 2 mM of glucose, and the incubation time was 96 h at 20 °C on a shaker at 150 rpm. After incubation, cells were harvested by centrifugation in an early stationary phase and washed in 10 ml of sterile 0.9% NaCl solution. Samples of washed and resuspended strains (in 0.9% NaCl), with an optical density (OD) of 0.01 at λ = 600 nm, were resuspended in the AB10 medium. The aim of this study was to perform experiments, including bacterial cell growth and attachment, in a well-controlled environment.

Biofilm Formation Studies

Six strains, four different surfaces, two carbon sources, and one experimental control (AB10 medium with no carbon source) were analyzed in triplicate to assay biofilm formation for a total of 216 samples. In these experiments, reference polystyrene plates were prepacked and radiation-sterilized. The mineral-coated polystyrene plates were sterilized by immersion in a 70% ethanol medium for 1 h prior to incubation and dried under aseptic conditions in a laminar flow cabinet.

Noninvasive, in situ direct imaging using Syto9 stain (green fluorescent nucleic acid stain, supplied by Invitrogen) was used as the primary technique to assay biofilm. The reference polystyrene and metal-oxide coated polystyrene well-plates, each with 12 wells and a nominal culture area of 3.82 cm² for each well, were used as substrata for biofilm formation studies. Samples of bacteria suspension were prepared at an optical density (OD) of 0.01 at λ = 600 nm using AB10 medium, pH ≈ 6.5. Then, 2 ml of prepared medium was added to each micro-well. The 12 well-plates were incubated for 96 h at 20 °C (Fig. 7); then, 200 μl of each of the bacterial samples, from their planktonic phase, was transferred to a 96-micro well-plate and the OD was measured at λ = 630 nm to determine planktonic phase growth. To assess the planktonic phase of individual environmental isolates, the measured optical density (OD) at λ = 630 nm was calibrated against the number of colony-forming units (CFU) for each strain. This calibration was used to compare growth in the planktonic phase for each individual strain. The rest of the planktonic phase was discarded and each well was gently washed three times by adding 5 ml of 0.9% sterile NaCl solution that was slowly added to the well wall and bottom intersection, using a pipette tip, to remove cells in the planktonic phase and ensure that only bacterial cells which had attached to the surface were present.

Fig. 7

Schematic representation of incubating polystyrene and mineral-coated 12-well plates and directly imaging the strains attached to the studied surfaces. (a) Depicts confined lateral movements of the water-dipping objective due to the well’s sides. As seen, a circle of diameter 11 mm located at the centre of each well’s base was imaged for the studied substrata. (b) Shows direct imaging of the aluminium hydroxide-coated plates. (c) Illustrates the function of Z-height focusing, Z stacking, used in evaluating biofilm formation. This method was used for dense biofilms to better assess the numbers of cells attached to polystyrene and mineral surfaces

Each well of the reference polystyrene and coated plates was stained by adding 0.5 ml of Syto 9, which was diluted 500×. The thickness of the added stain layer that formed on the bottom of the well was approximately 1.25 mm (the surface area of each well was 3.82 cm²). The stained wells were directly imaged in situ using a 100× magnification Zeiss Achroplan water-dipping objective (Fig. 7). For imaging, a Zeiss AxioVision epifluorescence microscope with automated Z-height focusing (Z-stacking) was used for extended depth and field imaging. With this technique, a series of images are acquired at different focus positions, which allows imaging through a thick section or of a rough surface (Fig. 7). Images were captured with an Axiocam black & white camera using a 450–490 nm narrow-band pass filter. For each sample, 15 images were captured and then analyzed using AxioVision 4.6 and Image J software. From these digital images, direct cell counts were obtained and reported as cells/cm² (since each experiment was conducted in triplicate, each data point represents an average of 45 data points). The microscope water-dipping objective had restricted lateral motion, due to the well’s sides, which confined the imaging area. Images to study bacterial cell attachment on the substrate, at the bottom of each well, were taken from a circular accessible surface with a diameter of 11 mm located at the center of the wells. As mentioned earlier, microscope Z-stacking provided the option of acquiring images at different focus positions. This technique was used to determine biofilm depth when the cells had formed dense biofilms (Figs. 8 and 9) (Table 4).

Fig. 8

Total number of bacterial cells attached to mineral-coated polystyrene and polystyrene surfaces after 96 h of incubation in AB10 medium with a glucose carbon source

Fig. 9

Schematic representation of distribution of electric charges and position of zeta potential around a negatively charged bacterial cell

Table 4 AB10 medium and its ionic strength

Glycosphingolipids

These chemical structures are amphiphilic molecules and generally possess similarities with physicochemical and functional properties of the LPS (Gutman et al. 2014; Kawahara et al. 2001). Glycosphingolipids have a versatile chemical structure and can be found in the cell membranes of different organisms. Figure 10 shows the chemical structure of a glycosphingolipid; Gangiloside (GT1b) (Varki et al. 2008). Sph2 are unique among all Gram-negative bacteria as they have glycosphingolipid (GSL) instead of lipopolysaccharide (LPS) layer. Glycosphingolipid has a functional role in cell-cell recognition and signalling (Hakomori and Igarashi 1995); GSL molecular structure exhibits more than 200 variations in carbohydrate structure, which can be combined with at least ten common molecular species of ceramide (a composition of sphingosine and a fatty acid). This combination can create over 2000 possibilities of GSLs molecular species (Hakomori and Igarashi 1995). The presence of GSLs also can influence membrane proteins architecture as several classes of membrane associated proteins display a strong preference for the association of lipid-rich membrane domains (Prinetti et al. 2009).

Fig. 10

Chemical structure of a glycosphingolipid molecule, Gangiloside (GT1b)

Some research shows that by changing KCl concentration from 0 to 100 mM, Pseudomonas putida KT2442 cell wall biopolymer length changes from 440 nm to 160 nm (pH = 8) (Abu-Lail and Camesano 2003). Biopolymers can undergo a salt-induced conformational change from a soft, random structure in low ionic strength to a rigid structure in a highly ionic medium (Fig. 11). The effects of sodium chloride concentrations on Poly-L-glutamate (PGA) as a multifunctional biopolymer has been mentioned in the previous studies as a small amount of NaCl switches the preferred conformation of this polymer. Adding a concentration of 0.3 M NaCl to DI-water is sufficient to keep the dominant conformation of Poly-L-glutamate a complex α-helix rather than an extended structure, on a nanosecond time scale (Fedorov et al. 2009).

Fig. 11

Schematic representation of conformational changes of biopolymers due to changes in ionic strength. Low IS favors attachment, while high IS hinders it