Jesus J. Ojeda

Characterization of the Cell Surface and Cell Wall Chemistry of Drinking Water Bacteria by Combining XPS, FTIR Spectroscopy, Modeling, and Potentiometric Titrations

Aquabacterium commune, a predominant member of European drinking water biofilms, was chosen as a model bacterium to study the role of functional groups on the cell surface that control the changes in the chemical cell surface properties in aqueous electrolyte solutions at different pH values. Cell surface properties of A. commune were examined by potentiometric titrations, modeling, X-ray photoelectron spectroscopy (XPS), and Fourier transform infrared (FTIR) spectroscopy. By combining FTIR data at different pH values and potentiometric titration data with thermodynamic model optimization, the presence, concentration, and changes of organic functional groups on the cell surface (e.g., carboxyl, phosphoryl, and amine groups) were inferred. The pH of zero proton charge, pH(zpc) = 3.7, found from titrations of A. commune at different electrolyte concentrations and resulting from equilibrium speciation calculations suggests that the net surface charge is negative at drinking water pH in the absence of other charge determining ions. In situ FTIR was used to describe and monitor chemical interactions between bacteria and liquid solutions at different pH in real time. XPS analysis was performed to quantify the elemental surface composition, to assess the local chemical environment of carbon and oxygen at the cell wall, and to calculate the overall concentrations of polysaccharides, peptides, and hydrocarbon compounds of the cell surface. Thermodynamic parameters for proton adsorption are compared with parameters for other gram-negative bacteria. This work shows how the combination of potentiometric titrations, modeling, XPS, and FTIR spectroscopy allows a more comprehensive characterization of bacterial cell surfaces and cell wall reactivity as the initial step to understand the fundamental mechanisms involved in bacterial adhesion to solid surfaces and transport in aqueous systems.

Identification and Quantification of Microplastics in Wastewater Using Focal Plane Array-Based Reflectance Micro-FT-IR Imaging

Microplastics (<5 mm) have been documented in environmental samples on a global scale. While these pollutants may enter aquatic environments via wastewater treatment facilities, the abundance of microplastics in these matrices has not been investigated. Although efficient methods for the analysis of microplastics in sediment samples and marine organisms have been published, no methods have been developed for detecting these pollutants within organic-rich wastewater samples. In addition, there is no standardized method for analyzing microplastics isolated from environmental samples. In many cases, part of the identification protocol relies on visual selection before analysis, which is open to bias. In order to address this, a new method for the analysis of microplastics in wastewater was developed. A pretreatment step using 30% hydrogen peroxide (H2O2) was employed to remove biogenic material, and focal plane array (FPA)-based reflectance micro-Fourier-transform (FT-IR) imaging was shown to successfully image and identify different microplastic types (polyethylene, polypropylene, nylon-6, polyvinyl chloride, polystyrene). Microplastic-spiked wastewater samples were used to validate the methodology, resulting in a robust protocol which was nonselective and reproducible (the overall success identification rate was 98.33%). The use of FPA-based micro-FT-IR spectroscopy also provides a considerable reduction in analysis time compared with previous methods, since samples that could take several days to be mapped using a single-element detector can now be imaged in less than 9 h (circular filter with a diameter of 47 mm). This method for identifying and quantifying microplastics in wastewater is likely to provide an essential tool for further research into the pathways by which microplastics enter the environment.

The applicability of reflectance micro-Fourier-transform infrared spectroscopy for the detection of synthetic microplastics in marine sediments

Synthetic microplastics (≤5-mm fragments) are globally distributed contaminants within coastal sediments that may transport organic pollutants and additives into food webs. Although micro-Fourier-transform infrared (micro-FT-IR) spectroscopy represents an ideal method for detecting microplastics in sediments, this technique lacks a standardized operating protocol. Herein, an optimized method for the micro-FT-IR analysis of microplastics in vacuum-filtered sediment retentates was developed. Reflectance micro-FT-IR analyses of polyethylene (PE) were compared with attenuated total reflectance FT-IR (ATR-FT-IR) measurements. Molecular mapping as a precursor to the imaging of microplastics was explored in the presence and absence of 150-μm PE fragments, added to sediment at concentrations of 10, 100, 500 and 1000ppm. Subsequently, polymer spectra were assessed across plastic-spiked sediments from fifteen offshore sites. While all spectra obtained of evenly shaped plastics were typical to PE, reflectance micro-FT-IR measurements of irregularly shaped materials must account for refractive error. Additionally, we provide the first evidence that mapping successfully detects microplastics without their visual selection for characterization, despite this technique relying on spectra from small and spatially separated locations. Flotation of microplastics from sediments only enabled a fragment recovery rate of 61 (±31 S.D.) %. However, mapping 3-mm(2) areas (within 47-mm filters) detected PE at spiking concentrations of 100ppm and above, displaying 69 (±12 S.D.) % of the fragments in these locations. Additionally, mapping detected a potential PE fragment in a non-spiked retentate. These data have important implications for research into the imaging of microplastics. Specifically, the sensitivity and spatial resolution of the present protocol may be improved by visualizing the entire filter with high-throughput detection techniques (e.g., focal plane array-based imaging). Additionally, since micro-FT-IR analyses depend on methods of sample collection, our results emphasize the urgency of developing efficient and reproducible techniques to separate microplastics from sediments.

Bio-precipitation of uranium by two bacterial isolates recovered from extreme environments as estimated by potentiometric titration, TEM and X-ray absorption spectroscopic analyses.

This work describes the mechanisms of uranium biomineralization at acidic conditions by Bacillus sphaericus JG-7B and Sphingomonas sp. S15-S1 both recovered from extreme environments. The U-bacterial interaction experiments were performed at low pH values (2.0-4.5) where the uranium aqueous speciation is dominated by highly mobile uranyl ions. X-ray absorption spectroscopy (XAS) showed that the cells of the studied strains precipitated uranium at pH 3.0 and 4.5 as a uranium phosphate mineral phase belonging to the meta-autunite group. Transmission electron microscopic (TEM) analyses showed strain-specific localization of the uranium precipitates. In the case of B. sphaericus JG-7B, the U(VI) precipitate was bound to the cell wall. Whereas for Sphingomonas sp. S15-S1, the U(VI) precipitates were observed both on the cell surface and intracellularly. The observed U(VI) biomineralization was associated with the activity of indigenous acid phosphatase detected at these pH values in the absence of an organic phosphate substrate. The biomineralization of uranium was not observed at pH 2.0, and U(VI) formed complexes with organophosphate ligands from the cells. This study increases the number of bacterial strains that have been demonstrated to precipitate uranium phosphates at acidic conditions via the activity of acid phosphatase.

The polymer physics and chemistry of microbial cell attachment and adhesion

The attachment of microbial cells to solid substrata is a primary ecological strategy for the survival of species and the development of specific activity and function within communities. An hypothesis arising from a biological sciences perspective may be stated as follows: The attachment of microbes to interfaces is controlled by the macromolecular structure of the cell wall and the functional genes that are induced for its biological synthesis. Following logically from this is the view that diverse attached cell behaviour is mediated by the physical and chemical interactions of these macromolecules in the interfacial region and with other cells. This aspect can be reduced to its simplest form by treating physico-chemical interactions as colloidal forces acting between an isolated cell and a solid or pseudo solid substratum. These forces can be analysed by established methods rooted in DLVO (Derjaguin, Landau, Verwey and Overbeek) theory. Such a methodology provides little insight into what governs changes in the behaviour of the cell wall attached to surfaces, or indeed other cells. Nor does it shed any light on the expulsion of macromolecules that modify the interface such as formation of slime layers. These physical and chemical problems must be treated at the more fundamental level of the structure and behaviour of the individual components of the cell wall, for example biosurfactants and extracellular polysaccharides. This allows us to restate the above hypothesis in physical sciences terms: Cell attachment and related cell growth behaviour is mediated by macromolecular physics and chemistry in the interfacial environment. Ecological success depends on the genetic potential to favourably influence the interface through adaptation of the macromolecular structure, We present research that merges these two perspectives. This is achieved by quantifying attached cell growth for genetically diverse model organisms, building chemical models that capture the variations in interfacial structure and quantifying the resulting physical interactions. Experimental observations combine aqueous chemistry techniques with surface spectroscopy in order to elucidate the cell wall structure. Atomic force microscopy methods quantify the physical interactions between the solid substrata and key components of the cell wall such as macromolecular biosurfactants. Our current approach focuses on considering individually mycolic acids or longer chain polymers harvested from cells, as well as characterised whole cells. This approach allows us to use a multifactorial approach to address the relative impact of the individual components of the cell wall in contact with model surfaces. We then combine these components to increase complexity step-wise, while comparing with the behaviour of entire cells. Eventually, such an approach should allow us to estimate and understand the primary factors governing microbial cell adhesion. Although the work addresses the cell-mineral interface at a fundamental level, the research is driven by a range of technology needs. The initial rationale was improved prediction of contaminant degradation in natural environments (soils, sediments, aquifers) for environmental cleanup. However, this area of research addresses a wide range of biotechnology areas including improved understanding of pathogen survival (e.g., in surgical environments), better process intensification in biomanufacturing (biofilm technologies) and new product development.

Chemical Characterization of Latent Fingerprints by Matrix-Assisted Laser Desorption Ionization, Time-of-Flight Secondary Ion Mass Spectrometry, Mega Electron Volt Secondary Mass Spectrometry, Gas Chromatography/Mass Spectrometry, X-ray Photoelectron Spectroscopy, and Attenuated Total Reflection Fourier Transform Infrared Spectroscopic Imaging: An Intercomparison

The first analytical intercomparison of fingerprint residue using equivalent samples of latent fingerprint residue and characterized by a suite of relevant techniques is presented. This work has never been undertaken, presumably due to the perishable nature of fingerprint residue, the lack of fingerprint standards, and the intradonor variability, which impacts sample reproducibility. For the first time, time-of-flight secondary ion mass spectrometry, high-energy secondary ion mass spectrometry, and X-ray photoelectron spectroscopy are used to target endogenous compounds in fingerprints and a method is presented for establishing their relative abundance in fingerprint residue. Comparison of the newer techniques with the more established gas chromatography/mass spectrometry and attenuated total reflection Fourier transform infrared spectroscopic imaging shows good agreement between the methods, with each method detecting repeatable differences between the donors, with the exception of matrix-assisted laser desorption ionization, for which quantitative analysis has not yet been established. We further comment on the sensitivity, selectivity, and practicability of each of the methods for use in future police casework or academic research.

In situ monitoring of the biofilm formation of Pseudomonas putida on hematite using flow-cell ATR-FTIR spectroscopy to investigate the formation of inner-sphere bonds between the bacteria and the mineral

Abstract In situ flow-cell ATR-FTIR using a hematite-coated germanium crystal was used to investigate the chemical interactions between Pseudomonas putida and hematite in real time, when compared with cells not attached to the mineral surface. ATR-FTIR spectra of bacteria growing on hematite showed a shift in the carboxylate signal when compared to the samples obtained from free cells, indicating a chemical interaction between the carboxylate groups and the Fe metal ions of the hematite surface. Small differences in the polysaccharide and phosphoryl regions of the IR spectra of bacteria attached to hematite were also observed. This work shows how the use of in-situ flow-cell experiments with a mineral-coated germanium crystal allows a better description of the bacterial interactions with minerals in real time, as an initial step to understand the fundamental mechanisms involved in the relationship between bacteria and mineral surfaces.

Analysis of bacteria on steel surfaces using reflectance micro-Fourier transform infrared spectroscopy.

Reflectance micro-Fourier transform infrared (FT-IR) analysis has been applied to characterize biofilm formation of Aquabacterium commune, a common microorganism present on drinking water distribution systems, onto the increasingly popular pipe material stainless steel EN1.4307. The applicability of the reflectance micro-FT-IR technique for analyzing the bacterial functional groups is discussed, and the results are compared to spectra obtained using more conventional FT-IR techniques: transmission micro-FT-IR, attenuated transmitted reflectance (ATR), and KBr pellets. The differences between the infrared spectra of wet and dried bacteria, as well as free versus attached bacteria, are also discussed. The spectra obtained using reflectance micro-FT-IR spectroscopy were comparable to those obtained using other FT-IR techniques. The absence of sample preparation, the potential to analyze intact samples, and the ability to characterize opaque and thick samples without the need to transfer the bacterial samples to an infrared transparent medium or produce a pure culture were the main advantages of reflectance micro-FT-IR spectroscopy.

Towards a mechanistic understanding of carbon stabilization in manganese oxides.

Minerals stabilize organic carbon (OC) in sediments, thereby directly affecting global climate at multiple scales, but how they do it is far from understood. Here we show that manganese oxide (Mn oxide) in a water treatment works filter bed traps dissolved OC as coatings build up in layers around clean sand grains at 3%w/wC. Using spectroscopic and thermogravimetric methods, we identify two main OC fractions. One is thermally refractory (>550 °C) and the other is thermally more labile (<550 °C). We postulate that the thermal stability of the trapped OC is due to carboxylate groups within it bonding to Mn oxide surfaces coupled with physical entrapment within the layers. We identify a significant difference in the nature of the surface-bound OC and bulk OC . We speculate that polymerization reactions may be occurring at depth within the layers. We also propose that these processes must be considered in future studies of OC in natural systems.

Determining the order of deposition of natural latent fingerprints and laser printed ink using chemical mapping with secondary ion mass spectrometry

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) chemical mapping was used to investigate the order of deposition of natural latent fingerprints and laser printed ink on paper. This feasibility study shows that sodium, potassium and C(3)H(5) positive ions were particularly abundant endogenous components of the natural fingerprints and also present in the paper examined, but were mostly absent in the laser printed ink. Mapping of these ions enables the observation of friction ridges from latent prints on the ink surface, only when a fingerprint was deposited above the layer of ink. As a demonstration of proof of concept, blind testing of 21 samples from three donors resulted in a 100% success rate. The sensitivity of this technique was investigated within this trial through the examination of up to fifth depletion fingerprints and ageing of up to 28 days. Migration of fingerprint and paper components to the ink surface, although observed with increased ageing time, was not found to compromise determination of the deposition sequence.