X-Ray Photoelectron Spectroscopy on Microbial Cell Surfaces: A Forgotten Method for the Characterization of Microorganisms Encapsulated With Surface-Engineered Shells

Abstract

Encapsulation of single microbial cells by surface-engineered shells has great potential for the protection of yeasts and bacteria against harsh environmental conditions, such as elevated temperatures, UV light, extreme pH values, and antimicrobials. Encapsulation with functionalized shells can also alter the surface characteristics of cells in a way that can make them more suitable to perform their function in complex environments, including bio-reactors, bio-fuel production, biosensors, and the human body. Surface-engineered shells bear as an advantage above genetically-engineered microorganisms that the protection and functionalization added are temporary and disappear upon microbial growth, ultimately breaking a shell. Therewith, the danger of creating a "super-bug," resistant to all known antimicrobial measures does not exist for surface-engineered shells. Encapsulating shells around single microorganisms are predominantly characterized by electron microscopy, energy-dispersive X-ray spectroscopy, Fourier transform infrared spectroscopy, particulate micro-electrophoresis, nitrogen adsorption-desorption isotherms, and X-ray diffraction. It is amazing that X-ray Photoelectron Spectroscopy (XPS) is forgotten as a method to characterize encapsulated yeasts and bacteria. XPS was introduced several decades ago to characterize the elemental composition of microbial cell surfaces. Microbial sample preparation requires freeze-drying which leaves microorganisms intact. Freeze-dried microorganisms form a powder that can be easily pressed in small cups, suitable for insertion in the high vacuum of an XPS machine and obtaining high resolution spectra. Typically, XPS measures carbon, nitrogen, oxygen and phosphorus as the most common elements in microbial cell surfaces. Models exist to transform these compositions into well-known, biochemical cell surface components, including proteins, polysaccharides, chitin, glucan, teichoic acid, peptidoglycan, and hydrocarbon like components. Moreover, elemental surface compositions of many different microbial strains and species in freeze-dried conditions, related with zeta potentials of microbial cells, measured in a hydrated state. Relationships between elemental surface compositions measured using XPS in vacuum with characteristics measured in a hydrated state have been taken as a validation of microbial cell surface XPS. Despite the merits of microbial cell surface XPS, XPS has seldom been applied to characterize the many different types of surface-engineered shells around yeasts and bacteria currently described in the literature. In this review, we aim to advocate the use of XPS as a forgotten method for microbial cell surface characterization, for use on surface-engineered shells encapsulating microorganisms.

Keywords: beer brewing; biomineralization; biosorption; click chemistry; nanobiomaterials; self-assembly; yolk shell; zeta potentials.

Figure 1

Examples of characteristics of different organic, surface-engineered shells. (A) SEM micrograph of Escherichia coli encapsulated by layer-by-layer self-assembly of cationic polyallylamine and anionic humic acid for UV-protection (Eby et al., , copyright 2012, American Chemical Society). (B) Zeta potentials of E. coli upon self-assembly of different layers of cationic polyallylamine (layers 1 and 3) and different anionic polyelectrolytes (layers 2 and 4), including poly(vinyl sulfate) (solid line), poly(styrenesulfonate) (dashed line), and humic acid (dotted line) (Eby et al., , copyright 2012, American Chemical Society). (C) Layer-by-layer, polyelectrolyte encapsulated S. cerevisiae observed using CLSM. Red-fluorescence indicates metabolically active DAPI stained yeasts, while green-fluorescence represents the polyelectrolyte shell (Diaspro et al., , copyright 2002, American Chemical Society). (D) FTIR spectra of (PMAA-co-NH₂)₅ layer-by-layer self-assembled shells on S. cerevisiae surfaces before and after cross-linking to create a robust shell (Drachuk et al., , copyright 2012, American Chemical Society).

Figure 2

Examples of characteristics of different inorganic, surface-engineered shells. (A) SEM micrograph of B. cereus encapsulated by directly deposited CTAB terminated Au nanorods (upper image) and Au nanoparticles (bottom image) (Berry et al., , copyright 2005, American Chemical Society). (B) ¹¹B NMR spectra of 4-formylphenylboronic acid (black line), B(OH)₂ modified mesoporous SiO₂ nanoparticles (red line), demonstrating binding of B(OH)₂ modified SiO₂ mesoporous nanoparticles to S. cerevisae surfaces (blue line) (Geng et al., , copyright 2019, American Chemical Society). (C) Pore size distribution of the SiO₂ yolk-shell encapsulated cyanobacteria Synechocystis sp. PCC 7002, determined by analysis of nitrogen adsorption-desorption isotherms (Wang et al., , copyright 2020, Oxford University Press). (D) TEM micrograph of a cyanobacterium encapsulated by a SiO₂ yolk shell, showing a void between the shell and the bacterial cell surface characteristic to a yolk-shell (Wang et al., , copyright 2020, Oxford University Press). (E) XRD pattern of a calcium phosphate biomineralized shell encapsulating S. cerevisiae after layer-by-layer application of PDADMAC and PAA to initiate precipitation and mineralization (Wang et al., , copyright 2008, Wiley-VCH).

Figure 3

Examples of characteristics of different organic-inorganic, surface-engineered shells. (A) SEM micrograph and an Energy-dispersive X-ray line-scan of S. cerevisiae, encapsulated with a hybrid shell, composed of PDADMAC-PAA layers and silica nanoparticles (Wang et al., , copyright 2010, Wiley-VCH). (B) Magnetic Fe₃O₄ nanoparticles allow magnetic separation of encapsulated S. cerevisiae cells from suspension (Fakhrullin et al., , copyright 2010, The Royal Society of Chemistry). (C) Self-repair of biohybrid nanoshells composed of L-cysteine functionalized Au nanoparticles around S. cerevisiae upon division of an encapsulated mother cell (Jiang et al., , copyright 2015, The Royal Society of Chemistry). (D) Small-angle X-ray scattering (SAXS) diffraction pattern of ZIF-8 encapsulated S. cerevisiae, demonstrating a crystalline structure similar as observed in ZIF-8 crystals (Liang et al., , copyright 2016, WILEY-VCH).

Figure 4

(A) Preparation of microbial samples for XPS analyses (adapted from Van der Mei et al., 2000). (B) SEM image of the surface of a compressed powder of freeze-dried Streptococcus salivarius in a stainless steel cup, suitable for XPS analysis (Van der Mei and Busscher, , copyright 1990, Elsevier Inc).

Figure 5

Schematics and TEM micrographs of microbial cell walls, including yeasts, Gram-positive, and Gram-negative bacteria. Panel on Candida albicans taken from Hardison and Brown (2012) (copyright 2012, Nature Publishing Group) and Osumi (1998) (copyright 1998, Elsevier Science Ltd.), panel on Staphylococcus aureus taken from Boudjemaa et al. (2019) (copyright 2019, Elsevier B.V.) and E. coli panel taken from Beveridge (1999) (copyright 1999, American Society for Microbiology).

Figure 6

Wide scans of microbial cell surfaces of yeasts, Gram-positive, and Gram-negative bacteria. Panel on S. cerevisiae taken from Xia et al. (2015) (copyright 2014, Elsevier B.V.), panel B. subtilis taken from Leone et al. (2006) (copyright, 2006, John Wiley and Sons) and panel on Aquabacterium commune taken from Ojeda et al. (2008) (copyright 2008, American Chemical Society).

Figure 7

Validation of microbial cell surface XPS, based on a comparison of C_1s binding energy components and elemental surface compositions measured. (A) Decomposition of the C_1s binding energy peak of B. subtilis into four components (Ahimou et al., , copyright 2007 Elsevier Inc.). (B) The fraction of carbon in bacterial cell surfaces bound to oxygen or nitrogen measured by XPS on a variety of different Gram-positive and Gram-negative strains as a function of the elemental surface concentration of oxygen and nitrogen with respect to carbon (Van der Mei et al., , copyright 2000 Elsevier Science B.V.; Dufrêne et al., , copyright 1997 American Society for Microbiology; Van der Mei et al., , copyright 1991 S. Karger AG. Basel).

Figure 8

The elemental surface concentration ratios N/C and O/C of Lactobacillus species as a function of their isoelectric points (IEP, pH units). 1, Lactobacillus acidophilus (eight strains); 2, Lactobacillus casei (eight strains); 3, Lactobacillus fermentum (four strains); 4, Lactobacillus plantarum (seven strains) (Millsap et al., , copyright 1997 NRC, Canada).

Figure 9

Examples of the use of microbial XPS. (A) Zeta potentials of top, S. cerevisiae and bottom, S. carlsbergensis fermenting yeast strains as a function of the concentration of phosphorus, measured using XPS (Amory and Rouxhet, , copyright 1988 Elsevier Science Publishers B.V.; Amory and Rouxhet, , copyright 1999-2021, John Wiley and Sons, Inc.) (B) Determination of the valency of Cr adsorbed to O. anthropic from Cr_2p binding energy peaks in Tris-HCl buffer (Tris) or LB medium (LB) as compared with spectra of Cr (III) in Cr₂O₃ and Cr (VI) in K₂Cr₂O₇ (Li et al., , copyright 2008, American Chemical Society).

Figure 10

XPS photoelectron binding energy spectra of microorganisms before and after encapsulation. (A) Narrow-scan O_1s photoelectron binding energy spectra of unencapsulated B. breve and (B) B. breve encapsulated by protein-assisted SiO₂ nanoparticle packing (Yuan et al., , copyright 2021, American Chemical Society). (C) Wide-scan of the photoelectron binding energy spectra of unencapsulated S. cerevisiae (Note absence of a Mn_2p electron binding energy peak) and (D) S. cerevisiae encapsulated by a MnO₂ nanozyme shell (Li et al., , copyright 2017, Wiley–VCH).