Hung Duc Nguyen

pH, redox potential and local biofilm potential microenvironments within Geobacter sulfurreducens biofilms and their roles in electron transfer

The limitation of pH inside electrode‐respiring biofilms is a well‐known concept. However, little is known about how pH and redox potential are affected by increasing current inside biofilms respiring on electrodes. Quantifying the variations in pH and redox potential with increasing current is needed to determine how electron transfer is tied to proton transfer within the biofilm. In this research, we quantified pH and redox potential variations in electrode‐respiring Geobacter sulfurreducens biofilms as a function of respiration rates, measured as current. We also characterized pH and redox potential at the counter electrode. We concluded that (1) pH continued to decrease in the biofilm through different growth phases, showing that the pH is not always a limiting factor in a biofilm and (2) decreasing pH and increasing redox potential at the biofilm electrode were associated only with the biofilm, demonstrating that G. sulfurreducens biofilms respire in a unique internal environment. Redox potential inside the biofilm was also compared to the local biofilm potential measured by a graphite microelectrode, where the tip of the microelectrode was allowed to acclimatize inside the biofilm. Biotechnol. Bioeng. 2012; 109: 2651–2662.

Increased Transfer of a Multidrug Resistance Plasmid in Escherichia coli Biofilms at the Air-Liquid Interface

ABSTRACT Although biofilms represent a common bacterial lifestyle in clinically and environmentally important habitats, there is scant information on the extent of gene transfer in these spatially structured populations. The objective of this study was to gain insight into factors that affect transfer of the promiscuous multidrug resistance plasmid pB10 in Escherichia coli biofilms. Biofilms were grown in different experimental settings, and plasmid transfer was monitored using laser scanning confocal microscopy and plate counting. In closed flow cells, plasmid transfer in surface-attached submerged biofilms was negligible. In contrast, a high plasmid transfer efficiency was observed in a biofilm floating at the air-liquid interface in an open flow cell with low flow rates. A vertical flow cell and a batch culture biofilm reactor were then used to detect plasmid transfer at different depths away from the air-liquid interface. Extensive plasmid transfer occurred only in a narrow zone near that interface. The much lower transfer frequencies in the lower zones coincided with rapidly decreasing oxygen concentrations. However, when an E. coli csrA mutant was used as the recipient, a thick biofilm was obtained at all depths, and plasmid transfer occurred at similar frequencies throughout. These results and data from separate aerobic and anaerobic matings suggest that oxygen can affect IncP-1 plasmid transfer efficiency, not only directly but also indirectly, through influencing population densities and therefore colocalization of donors and recipients. In conclusion, the air-liquid interface can be a hot spot for plasmid-mediated gene transfer due to high densities of juxtaposed donor and recipient cells.

Redox and pH microenvironments within Shewanella oneidensis MR-1 biofilms reveal an electron transfer mechanism.

The goal of this research was to quantify the variations in redox potential and pH in Shewanella oneidensis MR-1 biofilms respiring on electrodes. We grew S. oneidensis MR-1 on a graphite electrode, which was used to accept electrons for microbial respiration. We modified well-known redox and pH microelectrodes with a built-in reference electrode so that they could operate near polarized surfaces and quantified the redox potential and pH profiles in these biofilms. In addition, we used a ferri-/ferrocyanide redox system in which electrons were only transferred by mediated electron transfer to explain the observed redox potential profiles in biofilms. We found that regardless of the polarization potential of the biofilm electrode, the redox potential decreased toward the bottom of the biofilm. In a fully redox-mediated control system (ferri-/ferrocyanide redox system), the redox potential increased toward the bottom when the electrode was the electron acceptor. The opposite behavior of redox profiles in biofilms and the redox-controlled system is explained by S. oneidensis MR-1 biofilms not being redox-controlled when they respire on electrodes. The lack of a significant variation in pH implies that there is no proton transfer limitation in S. oneidensis MR-1 biofilms and that redox potential profiles are not caused by pH.

Characterization of Mono- and Mixed-Culture Campylobacter jejuni Biofilms

ABSTRACT Campylobacter jejuni, one of the most common causes of human gastroenteritis, is a thermophilic and microaerophilic bacterium. These characteristics make it a fastidious organism, which limits its ability to survive outside animal hosts. Nevertheless, C. jejuni can be transmitted to both humans and animals via environmental pathways, especially through contaminated water. Biofilms may play a crucial role in the survival of the bacterium under unfavorable environmental conditions. The goal of this study was to investigate survival strategies of C. jejuni in mono- and mixed-culture biofilms. We grew monoculture biofilms of C. jejuni and mixed-culture biofilms of C. jejuni with Pseudomonas aeruginosa. We found that mono- and mixed-culture biofilms had significantly different structures and activities. Monoculture C. jejuni biofilms did not consume a measurable quantity of oxygen. Using a confocal laser scanning microscope (CLSM), we found that cells from monoculture biofilms were alive according to live/dead staining but that these cells were not culturable. In contrast, in mixed-culture biofilms, C. jejuni remained in a culturable physiological state. Monoculture C. jejuni biofilms could persist under lower flow rates (0.75 ml/min) but were unable to persist at higher flow rates (1 to 2.5 ml/min). In sharp contrast, mixed-culture biofilms were more robust and were unaffected by higher flow rates (2.5 ml/min). Our results indicate that biofilms provide an environmental refuge that is conducive to the survival of C. jejuni.

Microscale Gradients of Oxygen, Hydrogen Peroxide, and pH in Freshwater Cathodic Biofilms

Cathodic reactions in biofilms employed in sediment microbial fuel cells is generally studied in the bulk phase. However, the cathodic biofilms affected by these reactions exist in microscale conditions in the biofilm and near the electrode surface that differ from the bulk phase. Understanding these microscale conditions and relating them to cathodic biofilm performance is critical for better-performing cathodes. The goal of this research was to quantify the variation in oxygen, hydrogen peroxide, and the pH value near polarized surfaces in river water to simulate cathodic biofilms. We used laboratory river-water biofilms and pure culture biofilms of Leptothrix discophora SP-6 as two types of cathodic biofilms. Microelectrodes were used to quantify oxygen concentration, hydrogen peroxide concentration, and the pH value near the cathodes. We observed the correlation between cathodic current generation, oxygen consumption, and hydrogen peroxide accumulation. We found that the 2 e(-) pathway for oxygen reduction is the dominant pathway as opposed to the previously accepted 4 e(-) pathway quantified from bulk-phase data. Biofouling of initially non-polarized cathodes by oxygen scavengers reduced cathode performance. Continuously polarized cathodes could sustain a higher cathodic current longer despite contamination. The surface pH reached a value of 8.8 when a current of only -30 μA was passed through a polarized cathode, demonstrating that the pH value could also contribute to preventing biofouling. Over time, oxygen-producing cathodic biofilms (Leptothrix discophora SP-6) colonized on polarized cathodes, which decreased the overpotential for oxygen reduction and resulted in a large cathodic current attributed to manganese reduction. However, the cathodic current was not sustainable.

Electrochemical biofilm control: mechanism of action

Although it has been previously demonstrated that an electrical current can be used to control biofilm growth on metal surfaces, the literature results are conflicting and there is no accepted mechanism of action. One of the suggested mechanisms is the production of hydrogen peroxide (H2O2) on metal surfaces. However, there are literature studies in which H2O2 could not be detected in the bulk solution. This is most likely because H2O2 was produced at a low concentration near the surface and could not be detected in the bulk solution. The goals of this research were (1) to develop a well-controlled system to explain the mechanism of action of the bioelectrochemical effect on 316L stainless steel (SS) surfaces and (2) to test whether the produced H2O2 can reduce cell growth on metal surfaces. It was found that H2O2 was produced near 316L SS surfaces when a negative potential was applied. The H2O2 concentration increased towards the surface, while the dissolved oxygen decreased when the SS surface was polarized to −600 mVAg/AgCl. When polarized and non-polarized surfaces with identical Pseudomonas aeruginosa PAO1 biofilms were continuously fed with air-saturated growth medium, the polarized surfaces showed minimal biofilm growth while there was significant biofilm growth on the non-polarized surfaces. Although there was no detectable H2O2 in the bulk solution, it was found that the surface concentration of H2O2 was able to prevent biofilm growth.

Microscale geochemical gradients in Hanford 300 Area sediment biofilms and influence of uranium

The presence and importance of microenvironments in the subsurface at contaminated sites were suggested by previous geochemical studies. However, no direct quantitative characterization of the geochemical microenvironments had been reported. We quantitatively characterized microscale geochemical gradients (dissolved oxygen (DO), H(2), pH, and redox potential) in Hanford 300A subsurface sediment biofilms. Our results revealed significant differences in geochemical parameters across the sediment biofilm/water interface in the presence and absence of U(VI) under oxic and anoxic conditions. While the pH was relatively constant within the sediment biofilm, the redox potential and the DO and H(2) concentrations were heterogeneous at the microscale (<500-1000 μm). We found microenvironments with high DO levels (DO hotspots) when the sediment biofilm was exposed to U(VI). On the other hand, we found hotspots (high concentrations) of H(2) under anoxic conditions both in the presence and in the absence of U(VI). The presence of anoxic microenvironments inside the sediment biofilms suggests that U(VI) reduction proceeds under bulk oxic conditions. To test this, we operated our biofilm reactor under air-saturated conditions in the presence of U(VI) and characterized U speciation in the sediment biofilm. U L(III)-edge X-ray absorption spectroscopy (XANES and EXAFS) showed that 80-85% of the U was in the U(IV) valence state.