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Biofuel production as a sustainable source of green energy is considered a promising complement to petroleum in order to prevent environmental problems such as global warming. In this regard, microalgae can be one of the best options, since other plant resources may be used for human consumption and utilizing them for producing biofuel may cause an increase in their price. However, there are several challenges to extract oil from microalgae, e.g., high energy consumption, chemical solvents, and algae culture destruction, which should be addressed by new approaches. In this paper, we suggest nanosecond pulse electric field as a physical method for hydrocarbon extraction from microalgae. Botryococcus braunii with high hydrocarbon production potential was used as the microalga model. To obtain an effective extraction, nsPEFs with 87.5-kV/cm electric field, 200-ns pulse duration, 0.3-J/pulse energy, 1-Hz pulse repetition frequency, and 1–50 pulses were applied. Microscopic observations with image processing and chemical assessments were performed for analyzing the samples, understanding the extraction mechanisms, and comparing the outcomes. According to the results, 50 pulses with 16.7-J/mL energy consumption were sufficient for oil extraction, showing that the pulsed power approach can be used as an appropriate physical method for extracting oil from Botryococcus braunii .

The green algae Botryococcus braunii produces a high amount of extracellular hydrocarbon, making it a promising algae in the field of bio-fuels production. As it mainly produces squalene like hydrocarbons, cosmetic industries are also interested in its milking. Pulsed electric fields (PEF) are an innovative method allowing oil extraction from micro-algae. In common algae accumulating hydrocarbon inside cytoplasm (Chlorella vulgaris, Nannochloropsis sp., etc), electric fields can destroy cell membranes, allowing the release of hydrocarbon. However, for B.braunii, hydrocarbons adhere to the cell wall outside of cells as a matrix. In a previous article we reported that electric fields can unstick cells from a matrix, allowing hydrocarbon harvesting. In this work, we deeper investigated this phenomenon of cell hatching by following 2 parameters: the conductivity of the medium and the cultivation duration of the culture. Cell hatching is accurately evaluated by both microscopic and macroscopic observations. For high conductivity and a short time of cultivation, almost no effect is observed even after up to 1000 PEF pulses are submitted to the cells. While lower conductivity and a longer cultivation period allow strong cell hatching after 200 PEF pulses are applied to the cells. We identify 2 new crucial parameters, able to turn the method from inefficient to very efficient. It might help companies to save energy and money in case of mass production.

Oil production by microalgae is investigated as a possible solution to sustain the petroleum shortage. Some microalgae such as Botryococcus braunii have the advantage of being able to produce a high amount of hydrocarbon without requiring arable lands to grow on. Also, hydrocarbons extracted from B. braunii are suitable for the cosmetic industry, as they are long-chain hydrocarbons similar to squalene. As such, B. braunii oil might generate a high profit. However, harvesting hydrocarbon from microalgae cultures is difficult. Here we show an innovative way of collecting hydrocarbon from algae culture using high voltage electric discharges (HVED). Botryococcus braunii form a matrix full of hydrocarbons allowing many cells to stick together as microcolonies. When the energy applied is too high, hydrocarbons are destroyed; and when the energy is to low, algae culture stays unchanged. But when energy applied is just sufficient (near 625 J mL−1), cells leave colonies and sink to the bottom of the samples, while hydrocarbons remain unaffected and float to the surface of the samples. Such a phenomenon allows us to harvest the matrices of colonies which are empty of cells, suitable as a biomass for biofuel production.

Pulsed electric fields (PEFs) are applied as physical stimuli for DNA/drug delivery, cancer therapy, gene transformation, and microorganism eradication. Meanwhile, calcium electrotransfer offers an interesting approach to treat cancer, as it induces cell death easier in malignant cells than in normal cells. Here, we study the spatial and temporal cellular responses to 10 μs duration PEFs; by observing real-time, the uptake of extracellular calcium through the cell membrane. The experimental setup consisted of an inverted fluorescence microscope equipped with a color high-speed framing camera and a specifically designed miniaturized pulsed power system. The setup allowed us to accurately observe the permeabilization of HeLa S3 cells during application of various levels of PEFs ranging from 0.27 to 1.80 kV/cm. The low electric field experiments confirmed the threshold value of transmembrane potential (TMP). The high electric field observations enabled us to retrieve the entire spatial variation of the permeabilization angle (θ). The temporal observations proved that after a minimal permeabilization of the cell membrane, the ionic diffusion was the prevailing mechanism of the delivery to the cell cytoplasm. The observations suggest 0.45 kV/cm and 100 pulses at 1 kHz as an optimal condition to achieve full calcium concentration in the cell cytoplasm. The results offer precise levels of electric fields to control release of the extracellular calcium to the cell cytoplasm for inducing minimally invasive cancer calcium electroporation, an interesting affordable method to treat cancer patients with minimum side effects.

Pulsed electric fields (PEFs), which are generated by pulsed power technologies, are being tested for their applicability in food processing through protein conformational change and the poration of cell membranes. In this article, enzyme activity change and the permeabilization of agricultural products using pulsed power technologies are reviewed as novel, nonthermal food processes. Compact pulsed power systems have been developed with repetitive operation and moderate output power for application in food processing. Firstly, the compact pulsed power systems for the enzyme activity change and permeabilization are outlined. Exposure to electric fields affects hydrogen bonds in the secondary and tertiary structures of proteins; as a result, the protein conformation is induced to be changed. The conformational change induces an activity change in enzymes such as α-amylase and peroxidase. Secondly, the conformational change in proteins and the induced protein functional change are reviewed. The permeabilization of agricultural products is caused through the poration of cell membranes by applying PEFs produced by pulsed discharges. The permeabilization of cell membranes can be used for the extraction of nutrients and health-promoting agents such as polyphenols and vitamins. The electrical poration can also be used as a pre-treatment for food drying and blanching processes. Finally, the permeabilization of cell membranes and its applications in food processing are reviewed.

Enzyme inactivation is a common industrial step of food preservation. Conventional method is based on heating, thus reduce organoleptic property and virtues of the products. We propose a non-thermal method of enzyme inactivation based on pulsed electric fields. By comparing the effect of electric fields, heating and oxidation on alpha-amylase, we have shown that the electric field targets the active site of the enzyme via the tryptophan, resulting in a conformational disruption. An electric pulse of 2.5–12.5 kV/cm, 10 μs and 0.02–0.6 J, repeated at 1–30 Hz for a total applied energy of 1440 J, partially denatured alpha-amylase, reducing activity by up to 70%; however, this was not followed by protein aggregation, as observed for heat treatment to 70 °C. Those results brought new understanding on the mechanism of pulsed electric fields action on proteins, and might lead in new application in food industry or in medicine.

Once pathogens form a biofilm, they become more tolerant to drugs and quicker to recover from physical removal than planktonic cells. Because such robustness of a biofilm is associated with the active metabolism of its constituent microbes, establishment of a direct assay quantifying biofilm's metabolic activity is important for developing antibiofilm substrates and techniques. Current production capability via extracellular electron transport (EET) was recently found in Gram-positive pathogens, which we hypothesized to correlate with the metabolic activity of their biofilm. Here, we identified current production from the biofilm of oral pathogen Streptococcus mutans that enables the electrochemical assessments of their metabolic activity in situ which conventionally require gene insertion for a fluorescent protein expression. Single-potential amperometry (SA) showed that S. mutans produced an anodic current and formed a biofilm within 8 h on a +0.4 V electrode vs a standard hydrogen electrode (SHE) in the presence of the electron donor glucose. Current production was significantly decreased by the addition of a metabolic inhibitor Triclosan. Furthermore, the anabolic activity of a single cell using high-resolution mass spectroscopy revealed that higher current production resulted in a higher metabolic fixation of an atomically labeled nitrogen 15N. These results demonstrate that current production in S. mutans reflects its metabolic activity. Given electrochemical impedance spectroscopy (EIS) helps quantifying the bacterial cell adhesion on the electrode, combination of EIS and SA could be a novel assay for EET capable pathogens for quantifying their time-dependent metabolic activity, cellular electrode coverage and physiological response to antibiofilm compounds.

Biofuel production as a sustainable source of green energy is considered as promising complements to petroleum in order to prevent environmental problems such as global warming. In this regard, microalgae can be one of the best options since other plant resources may be used for human consumption, using them for producing biofuel may cause an increase in their price. However, there are several challenges to extract oil from microalgae, e.g., high energy consumption, chemical solvents, and algae culture destruction; which should be addressed by new approaches. This study suggests nanosecond pulse electric fields (nsPEF) as a physical method for hydrocarbon extraction from microalgae. Botryococcus braunii with high hydrocarbon production potential was used as microalga model. For nanosecond pulsed electric fields (nsPEF) experiments, 20 to 65 kV/cm electric fields with 80 ns pulse duration, with different pulse repetition frequencies and pulse numbers were applied. Fluorescence microscopic observation and image and chemical assessments were performed for analyzing the samples, understanding the extraction mechanisms, and comparing the outcomes. According to the results, pulsed power approach can be used as high efficiency physical method for extracting oil from Botryococcus braunii.

Recent understanding that specific algae have high hydrocarbon production potential has attracted considerable attention. Botryococcus braunii is a microalga with an extracellular hydrocarbon matrix, which makes it an appropriate green energy source. This study focuses on extracting oil from the microalgae matrix rather than the cells, eliminating the need for an excessive electric field to create electro-permeabilization. In such a way, technical limitations due to high extraction energy and cost can be overcome. Here, nanosecond pulsed electric fields (nsPEF) with 80 ns duration and 20–65 kV/cm electric fields were applied. To understand the extraction mechanism, the structure of the algae was accurately studied under fluorescence microscope; extraction was quantified using image analysis; quality of extraction was examined by thin-layer chromatography (TLC); and the cell/matrix separation was observed real-time under a microscope during nsPEF application. Furthermore, optimization was carried out by screening values of electric fields, pulse repetition frequencies, and energy spent. The results offer a novel method applicable for fast and continues hydrocarbon extraction process at low energy cost.

Aims This article shows the effect of nanosecond pulsed electric field (nsPEF) on Escherichia coli, which could imply a durable change in protein expressions and then impacted the phenotype of surviving bacteria that might lead to increase pathogenicity. Methods and Results The effects of nsPEF on E. coli viability and membrane permeabilization were investigated. One log10 reduction in bacterial counts was achieved at field strength of 107 V m−1 with a train of 500 successive pulses of 60 × 10−9 s. Incubation of germs after treatment with propidium iodide showed that membrane permeabilization was reversible. Possible protein changes of surviving bacteria were checked to assess potential phenotypical changes using two-dimensional electrophoresis. In our study, after 40 generations, only UniProt #P39187 was up-regulated with P ≤ 0·05 compared with the control and corresponded to the uncharacterized protein YtfJ. Antibiograms were used to check whether or not the pattern of cultivable bacteria after nsPEF deliveries changed. Conclusions The results tend to show that nsPEFs are able to inactivate bacteria and have probably no serious impact in E. coli protein patterns. Significance and Impact of the Study The use of nsPEF is a safe promising new nonthermal method for bacterial inactivation in the food processing and environmental industry.

BACKGROUND: Bacillus Calmette-Guerin (BCG) is the most effective treatment for non-muscle invasive bladder cancer. However, a failure in the initial response or relapse within the first five years of treatment has been observed in 20% of patients. We have previously observed that in vivo administration of an inhibitor of nitric oxide improved the response to BCG of bladder tumor bearing mice. It was described that this effect was due to a replacement of tumor tissue by collagen depots. The aim of the present work was to clarify the mechanism involved in this process. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrated that BCG induces NIH-3T3 fibroblast proliferation by activating the MAPK and PI3K signaling pathways and also differentiation determined by alpha-smooth muscle actin (alpha-SMA) expression. In vivo, intratumoral inoculation of BCG also increased alpha-SMA and collagen expression. Oral administration of L-NAME enhanced the pro-fibrotic effect of BCG. Peritoneal macrophages obtained from MB49 tumor-bearing mice treated in vivo with combined treatment of BCG with L-NAME also enhanced fibroblast proliferation. We observed that FGF-2 is one of the factors released by BCG-activated macrophages that is able to induce fibroblast proliferation. The involvement of FGF-2 was evidenced using an anti-FGF2 antibody. At the same time, this macrophage population improved wound healing rate in normal mice and FGF-2 expression was also increased in these wounds. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that fibroblasts are targeted by BCG both directly and through activated macrophages in an immunotherapy context of a bladder murine model. We also described, for the first time, that FGF-2 is involved in a dialog between fibroblasts and macrophages induced after BCG treatment. The fact that L-NAME administration improves the BCG effect on fibroblasts, NO inhibition, might represent a new approach to add to the conventional BCG therapy.