Nondestructive, reagent-free, low-volume fluidic set-up to study biofilms by using a transparent electrode, allowing simultaneous electrochemical and optical measurements.

AIMS: The aim was to develop an electrochemical/optical set-up and correlate it (as validation) with other chemical and physical methods to obtain a simple and cost-effective system to study biofilm formation. METHODS AND RESULTS: A simple microfluidic cell and methods allowed continuous monitoring of the first, critical steps of microbial attachment. We monitored sulfate-reducing bacteria (SRB) at the early stages of biofilm formation. Herein, we studied the formation and adherence of SRB consortium biofilms over an indium tin oxide (ITO) conducting surface using microbiological and chemical methods, microscopic observations [scanning electron microscopy (SEM) and optical], and electrochemical impedance spectroscopy (EIS) measurements. The SRB biofilm formation was evaluated for 30 d by SEM and EIS. Charge transfer resistance decreased when the microbial population colonized the electrode. The monitoring of early-stage biofilm formation was performed using EIS at a single frequency of 1 Hz during the first 36 h. CONCLUSIONS: The simultaneous use of optical, analytical, and microbiological methods allowed us to connect the kinetics of the growth of the microbial consortium to the values obtained via the electrochemical technique. The simple setup we present here can help laboratories with limited resources to study biofilm attachment and facilitates the development of various strategies to control biofilm development in order to avoid damage to metallic structures (microbiologically influenced corrosion, MIC) or the colonization of other industrial structures and medical devices.

Sticky Bacteria: Understanding the Behavior of a D-Galactose Adapted Consortium of Acidophilic Chemolithotroph Bacteria and Their Attachment on a Concentrate of Polymetallic Mineral.

Various strategies to accelerate the formation of biofilms on minerals have been studied, and one of them is the use of D-galactose as an inducer of EPS production in planktonic cells of biooxidant bacteria. With the aim to evaluate the influence on the attachment and the effect over the solubilization of a polymetallic mineral concentrate, the behavior of a microbial consortium formed by Acidithiobacillus thiooxidans DSM 14887T and Leptospirillum ferrooxidans DSM 2705T previously induced with D-galactose for the early formation of EPS was studied. These microorganisms were previously adapted to 0.15 and 0.25% of D-galactose, respectively; afterward, different proportions of both strains were put in contact with the particle surface of a concentrate of polymetallic mineral. Also, to evaluate the affinity of each bacterium to the mineral, attachment tests were carried out with one of these species acting as a pre-colonizer. The same consortia were used to evaluate the solubilization of the polymetallic mineral. The results obtained show that the induction by D-galactose increases the microbial attachment percentage to the mineral by at least 10% with respect to the control of non-adapted consortia. On the other hand, the tests carried out with pre-colonization show that the order of inoculation also affects the microbial attachment percentage. From the different proportions tested, it was determined that the use of a consortium with a proportion of 50% of each species previously adapted to D-galactose and inoculated simultaneously, present a microbial attachment percentage to the mineral greater than 95% and better solubilization of a polymetallic mineral, reaching values of 9.7 and 11.7mgL-1 h-1 of Fe3+ and SO4 2-, respectively. Therefore, the use of D-galactose in small concentrations as inducer of EPS in acidophilic cells and the selection of an adequate strategy of inoculation can be beneficial to improve biooxidation since it would allow this process to develop in a shorter time by achieving a greater number of attached cells in a shorter time accelerating the solubilization of a sulfide mineral. Graphical AbstractEPS production using D-galactose as inducer and its influence in the attachment of consortia formed by differents proportions of A. thiooxidans and L. ferrooxidans inoculated at the same time and when one of them acting as a pre-colonizer.

Biooxidation of Iron by Acidithiobacillus ferrooxidans in the Presence of D-Galactose: Understanding Its Influence on the Production of EPS and Cell Tolerance to High Concentrations of Iron.

Acidithiobacillus ferrooxidans, together with other microorganisms, has an important role on biohydrometallurgical processes. Such bacterium gets its energy from the oxidation of ferrous ion and reduced sulfur; in the first case, the accumulation of ferric ion as a product can cause its inhibition. It is known that the extracellular polymeric substances (EPS) may have an important role in the adaptation and tolerance to diverse inhibiting conditions. In the present study, it was tested how D-galactose can influence the production of extracellular polymeric substances (EPS) on At. ferrooxidans by evaluating at the same time its biooxidant activity and capacity to tolerate high concentrations of ferric ion. The visualization and quantification of EPS was done through a confocal laser scanning microscope (CLSM). The results show that at low cellular concentrations, the D-galactose inhibits the microbial growth and the biooxidation of ferrous ion; however, when the quantity of microorganisms is high enough, the inhibition is not present. By means of chemostat tests, several concentrations of D-galactose (0; 0.15; 0.25; and 0.35%) were evaluated, thus reaching the highest production of EPS when using 0.35% of this sugar. In cultures with such concentration of D-galactose, the tolerance of the bacterium was tested at high concentrations of ferric ion and it was compared with cultures in which sugar was not added. The results show that cultures with D-galactose reached a higher tolerance to ferric ion (48.15 ± 1.9 g L-1) compare to cultures without adding D-galactose (38.7 ± 0.47 g L-1 ferric ion). Also it was observed a higher amount of EPS on cells growing in the presence of D-galactose suggesting its influence on the greater tolerance of At. ferrooxidans to ferric ion. Therefore, according to the results, the bases of a strategy are considered to overproduce EPS by means of At. ferrooxidans in planktonic state, so that, it can be used as a pre-treatment to increase its resistance and tolerance to high concentrations of ferric ion and improve the efficiency of At. ferrooxidans when acting in biohydrometallurgical processes.

An electrochemical sensing approach for scouting microbial chemolithotrophic metabolisms.

The present study was aimed to test an electrochemical sensing approach for the detection of an active chemolithotrophic metabolism (and therefore the presence of chemolithotrophic microorganisms) by using the corrosion of pyrite by Acidithiobacillus ferrooxidans as a model. Different electrochemical techniques were combined with adhesion studies and scanning electron microscopy (SEM). The experiments were performed in presence or absence of A. ferrooxidans and without or with ferrous iron in the culture medium (0 and 0.5 g L-1, respectively). Electrochemical parameters were in agreement with voltammetric studies and SEM showing that it is possible to distinguish between an abiotically-induced corrosion process (AIC) and a microbiologically-induced corrosion process (MIC). The results show that our approach not only allows the detection of chemolithotrophic activity of A. ferrooxidans but also can characterize the corrosion process. This may have different kind of applications, from those related to biomining to life searching missions in other planetary bodies.