Cell Adhesion and Biofilm Formation Analysis.

Cell adhesion to surfaces and ulterior biofilm formation are critical processes in microbial development since living in biofilms is the preferred way of life within microorganisms. These processes are known to influence not only microorganisms development in the environment, but also their participation in biotechnological processes and have been the focus of intense research that as a matter of fact, was mainly directed to the bacterial domain. Archaea also adhere to surfaces and have been shown forming biofilms, but studies performed until present did not exploit the diversity of methods probed to be useful along bacterial biofilm research.An experimental setup is described here with the aim of stimulating archaeal biofilm research. It can be used for studying cell adhesion and biofilm formation under controlled flow conditions and allows performing in situ optical microscopy (phase contrast, fluorescence, or confocal) and/or spectroscopic techniques (UV-Vis, IR, or Raman) to determine structural and functional biofilm features and their evolution in time. Variants are described with specific aims as working in anaerobiosis and allow sampling of biological material along time.

Unraveling Anaerobic Metabolisms in a Hypersaline Sediment.

The knowledge on the microbial diversity inhabiting hypersaline sediments is still limited. In particular, existing data about anaerobic hypersaline archaea and bacteria are scarce and refer to a limited number of genera. The approach to obtain existing information has been almost exclusively attempting to grow every organism in axenic culture on the selected electron acceptor with a variety of electron donors. Here, a different approach has been used to interrogate the microbial community of submerged hypersaline sediment of Salitral Negro, Argentina, aiming at enriching consortia performing anaerobic respiration of different electron acceptor compounds, in which ecological associations can maximize the possibilities of successful growth. Growth of consortia was demonstrated on all offered electron acceptors, including fumarate, nitrate, sulfate, thiosulfate, dimethyl sulfoxide, and a polarized electrode. Halorubrum and Haloarcula representatives are here shown for the first time growing on lactate, using fumarate or a polarized electrode as the electron acceptor; in addition, they are shown also growing in sulfate-reducing consortia. Halorubrum representatives are for the first time shown to be growing in nitrate-reducing consortia, probably thanks to reduction of N2O produced by other consortium members. Fumarate respiration is indeed shown for the first time supporting growth of Halanaeroarchaeum and Halorhabdus belonging to the archaea, as well as growth of Halanaerobium, Halanaerobaculum, Sporohalobacter, and Acetohalobium belonging to the bacteria. Finally, evidence is presented suggesting growth of nanohaloarchaea in anaerobic conditions.

Energetics, electron uptake mechanisms and limitations of electroautotrophs growing on biocathodes - A review.

Electroautotrophs are microorganisms that can take the electrons needed for energy generation, CO2 fixation and other metabolic reactions from a polarized electrode. They have been the focus of intense research for its application in wastewater treatment, bioelectrosynthetic processes and hydrogen generation. As a general trend, current densities produced by the electron uptake of these microorganisms are low, limiting their applicability at large scale. In this work, the electron uptake mechanisms that may operate in electroautotrophs are reviewed, aiming at finding possible causes for this low performance. Biomass yields, growth rates and electron uptake rates observed when these microorganisms use chemical electron donors are compared with those typically obtained with electrodes, to explore limitations and advantages inherent to the electroautotrophic metabolism. Also, the factors affecting biofilm development are analysed to show how interfacial interactions condition bacterial adhesion, biofilm growth and electrons uptake. Finally, possible strategies to overcome these limitations are described.

Biofilms of Halobacterium salinarum as a tool for phenanthrene bioremediation.

The use of hyperhalophilic microorganisms is emerging as a sustainable alternative to clean hydrocarbon-polluted hypersaline water bodies. In line with this practice, this work reports on the ability of the archaeon Halobacterium salinarum to develop biofilms on a solid surface conditioned by the presence of phenanthrene crystals, which results in the removal of the contaminating compound. The cell surface hydrophobicity does not change during the removal process and this organism is shown to constitutively produce a surfactant molecule with specific action on aromatic hydrocarbons, both indicating that phenanthrene removal might proceed through a non-contact mechanism. A new approach is presented to follow the process in situ through epifluorescence microscopy by monitoring phenanthrene auto-fluorescence.

Hyperhalophilic archaeal biofilms: growth kinetics, structure, and antagonistic interaction in continuous culture.

Biofilms by the hyperhalophilic archaea Halorubrum sp. and Halobacterium sp. were analyzed, and for the first time the progression of structural features and the developmental parameters of these sessile populations are described. Optical slicing and digital analysis of sequential micrographs showed that their three dimensional structure was microorganism dependent. Biofilms of Halobacterium sp. developed in clusters that covered about 30% of the supporting surface at the interface level and expanded over about 86 ± 4 µm in thickness, while Halorubrum sp. biofilms covered less than 20% of the surface and reached a thickness of 41 ± 1 µm. The kinetics of growth was lower in biofilms, with generation times of 27 ± 1 and 36 ± 2 h for Halobacterium sp. and Halorubrum sp., respectively, as compared to 8.4 ± 0.3 and 14 ± 1 h in planktonic cultures. Differences between microorganisms were also observed at the cell morphology level. The interaction between the two microorganisms was also evaluated, showing that Halobacterium sp. can outcompete already established Halorubrum sp. biofilms by a mechanism that might include the combined action of tunnelling swimmers and antimicrobial compounds.

Physiological stratification in electricity-producing biofilms of Geobacter sulfurreducens.

The elucidation of mechanisms and limitations in electrode respiration by electroactive biofilms is significant for the development of rapidly emerging clean energy production and wastewater treatment technologies. In Geobacter sulfurreducens biofilms, the controlling steps in current production are thought to be the metabolic activity of cells, but still remain to be determined. By quantifying the DNA, RNA, and protein content during the long-term growth of biofilms on polarized graphite electrodes, we show in this work that current production becomes independent of DNA accumulation immediately after a maximal current is achieved. Indeed, the mean respiratory rate of biofilms rapidly decreases after this point, which indicates the progressive accumulation of cells that do not contribute to current production or contribute to a negligible extent. These results support the occurrence of physiological stratification within biofilms as a consequence of respiratory limitations imposed by limited biofilm conductivity.

Stepping stones in the electron transport from cells to electrodes in Geobacter sulfurreducens biofilms.

Geobacter sulfurreducens bacteria grow on biofilms and have the particular ability of using polarized electrodes as the final electron acceptor of their respiratory chain. In these biofilms, electrons are transported through distances of more than 50 µm before reaching the electrode. The way in which electrons are transported across the biofilm matrix through such large distances remains under intense discussion. None of the two mechanisms proposed for explaining the process, electron hopping through outer membrane cytochromes and metallic like conduction through conductive PilA filaments, can account for all the experimental evidence collected so far. Aiming at providing new elements for understanding the basis for electron transport, in this perspective article we present a modelled structure of Geobacter pilus. Its analysis in combination with already existing experimental evidence gives support to the proposal of the "stepping stone" mechanism, in which the combined action of pili and cytochromes allows long range electron transport through the biofilm.

Limitations for current production in Geobacter sulfurreducens biofilms.

Devices that exploit electricity produced by electroactive bacteria such as Geobacter sulfurreducens have not yet been demonstrated beyond the laboratory scale. The current densities are far from the maximum that the bacteria can produce because fundamental properties such as the mechanism of extracellular electron transport and factors limiting cell respiration remain unclear. In this work, a strategy for the investigation of electroactive biofilms is presented. Numerical modeling of the response of G. sulfurreducens biofilms cultured on a rotating disk electrode has allowed for the discrimination of different limiting steps in the process of current production within a biofilm. The model outputs reveal that extracellular electron transport limits the respiration rate of the cells furthest from the electrode to the extent that cell division is not possible. The mathematical model also demonstrates that recent findings such as the existence of a redox gradient in actively respiring biofilms can be explained by an electron hopping mechanism but not when considering metallic-like conductivities.

A long way to the electrode: how do Geobacter cells transport their electrons?

The mechanism of electron transport in Geobacter sulfurreducens biofilms is a topic under intense study and debate. Although some proteins were found to be essential for current production, the specific role that each one plays in electron transport to the electrode remains to be elucidated and a consensus on the mechanism of electron transport has not been reached. In the present paper, to understand the state of the art in the topic, electron transport from inside of the cell to the electrode in Geobacter sulfurreducens biofilms is analysed, reviewing genetic studies, biofilm conductivity assays and electrochemical and spectro-electrochemical experiments. Furthermore, crucial data still required to achieve a deeper understanding are highlighted.

Evaluation of potato-processing wastewater treatment in a microbial fuel cell.

Wastewaters from potato-processing industries have been traditionally treated by a sequence of steps that include the production of methane as the anaerobic one. This work explores the feasibility of replacing or supplementing methanogenesis with the emerging technology of microbial fuel cells (MFCs). Electricity producing biofilms have been enriched from a real anaerobic sludge, and the conversion of potato-processing wastewater into electricity has been studied. When tested as a single treatment step, MFCs were able to process the wastewater with high COD removal but with low energetic conversion efficiency. On the other hand, as a complimentary step for methanogenesis, they improved conversion efficiency and significantly reduced the organic matter load of the final effluent. These results point at the combination of the energetic yield of methanogenesis and the improved COD removal of the electricity producing treatment as the implementation choice.

Metabolic turnover and catalase activity of biofilms of Pseudomonas fluorescens (ATCC 17552) as related to copper corrosion.

In this work we report the results of a combined biochemical and electrochemical study aimed to analyze both the growth of biofilms of Pseudomonas fluorescens on copper samples and its possible role in the instability of the metal/electrolyte interface. DNA and RNA were quantified along the time for biofilms grown on copper and glass to estimate both the growth of the bacterial population and its metabolic state (through the RNA/DNA ratio). The expression and specific activity of catalase were also determined to gain insight into their possible role in corrosion acceleration. The electrochemical behavior of the biofilm/copper interface was monitored by Linear Polarization Resistance (Rp) and electrochemical impedance spectroscopy (EIS) along the experiments. Results showed a longer lag phase for biofilms developing on copper that included a period of high metabolic activity (as measured by the RNA/DNA ratio) without biomass growth. Biological activity introduced a new time constant at intermediate frequencies in EIS spectra whose capacitive behavior increased with the biofilm development. The increment in this biofilm-related signal was accompanied by a strong limitation to charge transfer through a diffusion controlled process probably due to oxygen exhaustion by cells respiration, while the resistance of the interface decreased presumably due to oxide dissolution by local acidification under the colonies. In addition, catalase activity was found to be high in mature copper-tolerant biofilms, which differentially express a catalase isoform not present in biofilms growing on glass.

ATR-SEIRAs characterization of surface redox processes in G. sulfurreducens.

In this work we report on the occurrence of at least two different redox pairs on the cell surface of the electrogenic bacteria Geobacter sulfurreducens adsorbed on gold that are expressed in response to the polarization potential. As previously reported on graphite (Environ. Sci. Technol. 42 (2008) 2445) a typical low potential redox pair is found centered at around -0.06 V when cells are polarized for a few hours at 0.2 V, while a new pair centered at around 0.38 V is expressed upon polarization at 0.6 V. Reversible changes in the IR band pattern of whole cells where obtained by Attenuated Total Reflection-Surface Enhanced Infrared Absorption Spectroscopy (ATR-SEIRAS) upon potential cycling around both redox pairs. Changes clearly resemble the electrochemical turnover of oxidized/reduced states in c-type cytochromes, thus evidencing the nature of the involved molecules. The expression of external cytochromes in response to the potential of the electron acceptor suggests the existence of alternative pathways of electron transport with different energy yield, though it remains to be demonstrated.

Whole cell electrochemistry of electricity-producing microorganisms evidence an adaptation for optimal exocellular electron transport.

The mechanism(s) by which electricity-producing microorganisms interact with an electrode is poorly understood. Outer membrane cytochromes and conductive pili are being considered as possible players, but the available information does not concur to a consensus mechanism yet. In this work we demonstrate that Geobacter sulfurreducens cells are able to change the way in which they exchange electrons with an electrode as a response to changes in the applied electrode potential. After several hours of polarization at 0.1 V Ag/AgCl-KCl (saturated), the voltammetric signature of the attached cells showed a single redox pair with a formal redox potential of about -0.08 V as calculated from chronopotentiometric analysis. A similar signal was obtained from cells adapted to 0.4 V. However, new redox couples were detected after conditioning at 0.6 V. A large oxidation process beyond 0.5 V transferring a higher current than that obtained at 0.1 V was found to be associated with two reduction waves at 0.23 and 0.50 V. The apparent equilibrium potential of these new processes was estimated to be at about 0.48 V from programmed current potentiometric results. Importantly, when polarization was lowered again to 0.1 V for 18 additional hours, the signals obtained at 0.6 V were found to greatly diminish in amplitude, whereas those previously found at the lower conditioning potential were recovered. Results clearly show the reversibility of cell adaptation to the electrode potential and pointto the polarization potential as a key variable to optimize energy production from an electricity producing population.

Spectroelectrochemical examination of the interaction between bacterial cells and gold electrodes.

The interaction between bacterial cells of Pseudomonas fluorescens (ATCC 17552) and gold electrodes was analyzed by cyclic voltammetry (CV) and attenuated total reflection-surface-enhanced infrared absorption spectroscopy (ATR-SEIRAS). The voltammetric evaluation of cell adsorption showed a decrease in the double-layer capacitance of polyoriented single-crystal gold electrodes with cell adhesion. As followed by IR spectroscopy in the ATR configuration, the adsorption of bacterial cells onto thin-film gold electrodes was mainly indicated by the increase in intensity with time of amide I and amide II protein-related bands at 1664 and 1549 cm(-1), respectively. Bands at 1448 and 2900 cm(-1) corresponding to the scissoring and the stretching bands of CH2 were also detected, together with a minor peak at 1407 cm(-1) due to the vs COO- stretching. Weak signals at 1237 cm(-1) were due to amide III, and a broad band between 1100 and 1200 cm(-1) indicated the presence of alcohol groups. Bacteria were found to displace water molecules and anions coadsorbed on the surface in order to interact with the electrode intimately. This fact was evidenced in the SEIRAS spectra by the negative features appearing at 3450 and 3575 cm-1, corresponding to interfacial water directly interacting with the electrode and water associated with chloride ions adsorbed on the electrode, respectively. Experiments in deuterated water confirmed these assignments and allowed a better estimation of amide absorption bands. In CV experiments, an oxidation process was observed at potentials higher than 0.4 V that was dependent on the exposure time of electrodes in concentrated bacterial suspensions. Adsorbed bacterial cells were found to get closer to the gold surface during oxidation, as indicated by the concomitant increment in the main IR bacterial signals including amide I, a sharp band at 1240 cm(-1), and a broad one at 1120 cm(-1) related to phosphate groups in the bacterial membranes. It is proposed to be due to the oxidation of lipopolysaccharides on the outermost bacterial surface.

Stainless steels can be cathodically protected using energy stored at the marine sediment/seawater interface.

Laboratory-scale experiments were performed in which the corrosion protection of stainless steels in seawater was afforded by cathodic protection. The method was implemented for the first time using the potential difference at the marine sediment/seawater interface as the only source of electric power. Graphite electrodes buried in marine sediment, developing a potential of -0.45 V versus a saturated calomel electrode (SCE), were used as anodes to cathodically polarize UNS S30403 stainless steel coupons that were exposed to seawater. The cathodic protection system was operated with low polarization of stainless steel, typically to -0.2 V (vs SCE) and was found to properly prevent material failure even in the presence of a well-developed biofilm. With voltammetry, the protection current was found to be related to the oxidation of reduced sulfur compounds in the sediments. Results demonstrate that this inexpensive and environmentally friendly method can, so far, extend the service life of stainless steels in seawater.

Electrochemical polarization-induced changes in the growth of individual cells and biofilms of Pseudomonas fluorescens (ATCC 17552).

The effect of surface electrochemical polarization on the growth of cells of Pseudomonas fluorescens (ATCC 17552) on gold electrodes has been examined. Potentials positive or negative to the potential of zero charge (PZC) of gold were applied, and these resulted in changes in cell morphology, size at cell division, time to division, and biofilm structure. At -0.2 V (Ag/AgCl-3 M NaCl), cells elongated at a rate of up to 0.19 microm min(-1), rendering daughter cells that reached up to 3.8 microm immediately after division. The doubling time for the entire population, estimated from the increment in the fraction of surface covered by bacteria, was 82 +/- 7 min. Eight-hour-old biofilms at -0.2 V were composed of large cells distributed in expanded mushroom-like microcolonies that protruded several micrometers in the solution. A different behavior was observed under positive polarization. At an applied potential of 0.5 V, the doubling time of the population was 103 +/- 8 min, cells elongated at a lower rate (up to 0.08 microm min(-1)), rendering shorter daughters (2.5 +/- 0.5 microm) after division, although the duplication times were virtually the same at all potentials. Biofilms grown under this positive potential were composed of short cells distributed in a large number of compact microcolonies. These were flatter than those grown at -0.2 V or at the PZC and were pyramidal in shape. Polarization effects on cell growth and biofilm structure resembled those previously reported as produced by changes in the nutritional level of the culture medium.

Changes in the electrochemical interface as a result of the growth of Pseudomonas fluorescens biofilms on gold.

In this study, variations in corrosion potential and polarization resistance of thin-film gold electrodes as a result of the growth of Pseudomonas fluorescens biofilms on them are presented. The growth of the volumetric cell fraction of biofilms, as determined by optical sectioning and digital image analysis of phase-contrast images, was found to be exponential during at least 10 hours of incubation. As a consequence of biofilm growth, an exponential decay of the corrosion potential of gold was observed. Most importantly, an increase in polarization resistance of the interface was observed following a strong linear dependence on the mean thickness of biofilms (r = 0.997), as a consequence of oxygen consumption and diffusion limitations. The results presented indicate that the measurement of polarization resistance may be a suitable technique that could be applied easily in industrial or biotechnological systems for monitoring the formation of biofilms.