Biochemical characterization and mercury methylation capacity of Geobacter sulfurreducens biofilms grown in media containing iron hydroxide or fumarate.

Geobacter species are common in iron-rich environments and can contribute to formation of methylmercury (MeHg), a neurotoxic compound with high bioaccumulation potential formed as a result of bacterial and archaeal physiological activity. Geobacter sulfurreducens can utilize various electron acceptors for growth including iron hydroxides or fumarate. However, it remains poorly understood how the growth on these compounds affects physiological properties of bacterial cells in biofilms, including the capacity to produce MeHg. The purpose of this study was to determine changes in the biochemical composition of G. sulfurreducens during biofilm cultivation in media containing iron hydroxide or fumarate, and to quantify mercury (Hg) methylation capacity of the formed biofilms. Biofilms were characterized by Fourier-transform infrared spectroscopy in the attenuated total reflection mode (ATR-FTIR), Resonance Raman spectroscopy and confocal laser scanning microscopy. MeHg formation was quantified by mass spectrometry after incubation of biofilms with 100 nM Hg. The results of ATR-FTIR experiments showed that in presence of fumarate, G. sulfurreducens biofilm formation was accompanied by variation in content of the energy-reserve polymer glycogen over time, which could be cancelled by the addition of supplementary nutrients (yeast extract). In contrast, biofilms cultivated on Fe(III) hydroxide did not accumulate glycogen. The ATR-FTIR results further suggested that Fe(III) hydroxide surfaces bind cells via phosphate and carboxylate groups of bacteria that form complexes with iron. Furthermore, biofilms grown on Fe(III) hydroxide had higher fraction of oxidized cytochromes and produced two to three times less biomass compared to conditions with fumarate. Normalized to biofilm volume, the content of MeHg was similar in assays with biofilms grown on Fe(III) hydroxide and on fumarate (with yeast extract and without). These results suggest that G. sulfurreducens biofilms produce MeHg irrespectively from glycogen content and cytochrome redox state in the cells, and warrant further investigation of the mechanisms controlling this process.

The Combined Effect of Hg(II) Speciation, Thiol Metabolism, and Cell Physiology on Methylmercury Formation by Geobacter sulfurreducens.

The chemical and biological factors controlling microbial formation of methylmercury (MeHg) are widely studied separately, but the combined effects of these factors are largely unknown. We examined how the chemical speciation of divalent, inorganic mercury (Hg(II)), as controlled by low-molecular-mass thiols, and cell physiology govern MeHg formation by Geobacter sulfurreducens. We compared MeHg formation with and without addition of exogenous cysteine (Cys) to experimental assays with varying nutrient and bacterial metabolite concentrations. Cysteine additions initially (0-2 h) enhanced MeHg formation by two mechanisms: (i) altering the Hg(II) partitioning from the cellular to the dissolved phase and/or (ii) shifting the chemical speciation of dissolved Hg(II) in favor of the Hg(Cys)2 complex. Nutrient additions increased MeHg formation by enhancing cell metabolism. These two effects were, however, not additive since cysteine was largely metabolized to penicillamine (PEN) over time at a rate that increased with nutrient addition. These processes shifted the speciation of dissolved Hg(II) from complexes with relatively high availability, Hg(Cys)2, to complexes with lower availability, Hg(PEN)2, for methylation. This thiol conversion by the cells thereby contributed to stalled MeHg formation after 2-6 h Hg(II) exposure. Overall, our results showed a complex influence of thiol metabolism on microbial MeHg formation and suggest that the conversion of cysteine to penicillamine may partly suppress MeHg formation in cysteine-rich environments like natural biofilms.

Methylmercury formation in biofilms of Geobacter sulfurreducens.

Introduction: Mercury (Hg) is a major environmental pollutant that accumulates in biota predominantly in the form of methylmercury (MeHg). Surface-associated microbial communities (biofilms) represent an important source of MeHg in natural aquatic systems. In this work, we report MeHg formation in biofilms of the iron-reducing bacterium Geobacter sulfurreducens. Methods: Biofilms were prepared in media with varied nutrient load for 3, 5, or 7 days, and their structural properties were characterized using confocal laser scanning microscopy, cryo-scanning electron microscopy and Fourier-transform infrared spectroscopy. Results: Biofilms cultivated for 3 days with vitamins in the medium had the highest surface coverage, and they also contained abundant extracellular matrix. Using 3 and 7-days-old biofilms, we demonstrate that G. sulfurreducens biofilms prepared in media with various nutrient load produce MeHg, of which a significant portion is released to the surrounding medium. The Hg methylation rate constant determined in 6-h assays in a low-nutrient assay medium with 3-days-old biofilms was 3.9 ± 2.0 ∙ 10-14 L ∙ cell-1 ∙ h-1, which is three to five times lower than the rates found in assays with planktonic cultures of G. sulfurreducens in this and previous studies. The fraction of MeHg of total Hg within the biofilms was, however, remarkably high (close to 50%), and medium/biofilm partitioning of inorganic Hg (Hg(II)) indicated low accumulation of Hg(II) in biofilms. Discussion: These findings suggest a high Hg(II) methylation capacity of G. sulfurreducens biofilms and that Hg(II) transfer to the biofilm is the rate-limiting step for MeHg formation in this systems.

Chemical Functionalization of the Zinc Selenide Surface and Its Impact on Lactobacillus rhamnosus GG Biofilms.

Bacteria grow on surfaces and form communities called biofilms. Bacterial adhesion and properties of the derived biofilms depend on, among others, the nature of the supporting substrate. Here, we report how the surface properties of the substrate affect the biofilm growth of probiotic Lactobacillus rhamnosus GG (LGG). Hydrophilic (OH), hydrophobic (CH3), and positively charged (NH3+) surfaces were obtained by the functionalization of a ZnSe crystal with alkanethiol self-assembled monolayers (SAM). The self-assembly of alkanethiols onto ZnSe was studied in situ using infrared spectroscopy in attenuated total reflection mode (ATR-FTIR). The organization of grafted SAMs was analyzed based on the results of ATR-FTIR, high-energy elastic backscattering spectrometry, and contact angle measurements. The kinetics and adhesion strength of LGG initial attachment as well as its physiological state on surfaces terminated by the different functional groups were assessed by the combination of ATR-FTIR, force measurements based on atomic force microscopy, and fluorescent staining of bacteria. The strength of interactions between LGG and the surface was strongly affected by the terminal group of the alkanethiol chain. The -NH3+ groups displayed the highest affinity with LGG at the first stage of interaction. The surface properties also played an important role when LGG biofilms were further grown in a nutritive medium for 24 h under flow conditions. Notably, the analysis of the infrared spectra recorded during the biofilm cultivation revealed differences in the kinetics of growth and in the polysaccharide features of the biofilm depending on the substrate functionality. LGG biofilm was stable only on the positively charged surface upon rinsing. Findings of this work clearly show that the adhesion features and the growth of LGG biofilms are substrate-dependent.

In situ spectroscopic analysis of Lactobacillus rhamnosus GG flow on an abiotic surface reveals a role for nutrients in biofilm development.

In this work, infrared spectroscopy was used to monitor the changes in the biochemical composition of biofilms of the probiotic bacterium Lactobacillus rhamnosus GG (LGG) in three nutritive media (10-fold diluted MRS, AOAC, and mTSB), in situ and under flow conditions. Epifluorescence microscopy was used to observe the shape of LGG cells and their distribution on the surface. Spectroscopic fingerprints recorded as a function of time revealed a medium-dependent content of nucleic acids, phospholipids and polysaccharides in the biofilms. In addition, time-dependent synthesis of lactic acid was observed in MRS/10 and AOAC/10. Polysaccharides were produced to the highest extent in mTSB/10, and the biofilms obtained were the densest in this medium. The rod shape of the cells was preserved in MRS/10, whereas acidic stress induced in AOAC/10 and the nutritional quality of mTSB/10 led to strong morphological changes. These alterations due to the nutritive environment are important to consider in research and use of LGG biofilms.

Difficulties and flaws in performing accurate determinations of zeta potentials of metal nanoparticles in complex solutions-Four case studies.

The zeta potential (ZP) is a parameter commonly used to characterize metal nanoparticles (NPs) in solution. Such determinations are for example performed in nanotoxicology since the ZP influences e.g. the interaction between cells and different biomolecules. Four case studies on different metal NPs (Cu and Zn NPs, and citrate capped Ag NPs) are presented in this study in order to provide guidance on how to accurately interpret and report ZP data. Solutions of high ionic strength (150 mM NaCl) induce a higher extent of particle agglomeration (elucidated with Ag NPs) when compared with conditions in 10 mM NaCl, which further complicates the prediction of the ZP due to e.g. sedimentation and broadening of the zeta potential distribution. The particle size is seldom included specifically in the standard ways of determining ZP (Hückel and Smoluchowski approximations). However corrections are possible when considering approximations of the Henry function. This was seen to improve the analysis of NPs, since there are cases when both the Hückel and the Smulochowski approximations are invalid. In biomolecule-containing cell media (BEGM), the signal from e.g. proteins may interfere with the measured ZP of the NPs. The intensity distribution of the ZP of both the blank solution and the solution containing NPs should hence be presented in addition to the mean value. Due to an increased ionic strength for dissolving of metal NPs (exemplified by Zn NPs), the released metal ions must be considered when interpreting the zeta potential measurements. In this work the effect was however negligible, as the particle size was several hundred nm, conditions that made the Smoluchowski approximation valid despite an increased ionic strength. However, at low ionic strengths (mM range) and small-sized NPs (tens of nm), the effect of released metal ions can influence the choice of model for determining the zeta potential. Sonication of particle dispersions influences not only the extent of metal release but also the outermost surface oxide composition, which often results in an increased ZP. Surface compositional changes were illustrated for sonicated and non-sonicated Cu NPs. In all, it can be concluded that accurate measurements and interpretations are possible in most cases by collecting and reporting complementary data on characteristics such as particle size, ZP distributions, blank sample information, and particle oxide composition.