Integration of Pulsed Electric Fields in the Biorefinery Concept to Extract Microalgae Components of Interest for Food Industry

The high cultivation costs for microalgae and the complex and costly downstream processing make the price per ton of raw materials from microalgae biomass, such as biofuel, uneconomic. To overcome this detriment, it is suggested to use the residual biomass for biofuel production after obtaining high- and middle-value products. Microalgae are an attractive food source, since they are rich in proteins, peptides, carbohydrates, lipids, and other essential nutrients with protective and detoxifying roles (vitamins, minerals, pigments). A major problem is that these valuable components cannot be accessed without prior and adequate cell disruption. This chapter discusses a new strategy to integrate PEF treatment into biorefinery concept by implementation of an incubation step which facilitates the release of intracellular components after PEF treatment. Based on the results obtained from the microalgae Chlorella vulgaris and the cyanobacteria Arthrospira platensis, the dependence of various influencing factors such as temperature, pH value, and biomass concentration on protein release during the incubation step is shown. Finally, the efficiency of this approach is discussed

Nanosecond pulsed electric fields modulate the expression of the astaxanthin biosynthesis genes psy, crtR-b and bkt 1 in Haematococcus pluvialis

Nanosecond pulsed electric fields (nsPEFs) have been extensively studied with respect to cellular responses. Whether nsPEFs can regulate gene expression and to modulate the synthesis of valuable compounds, has so far been only tested in the context of apoptosis in cancer cells. We used the unicellular algae Haematococcus pluvialis as system to test, whether nsPEFs could alter gene expression and to promote the biosynthesis of astaxanthin. We find that nsPEFs induce a mild, but significant increase of mortality up to about 20%, accompanied by a moderate increase of astaxanthin accumulation. Steady-state transcript levels of three key genes psy, crtR-b and bkt 1 were seen to increase with a maximum at 3 d after PEF treatment at 50 ns. Pulsing at 25 ns reduce the transcripts of psy, crtR-b from around day 2 after the pulse, while those of bkt 1 remain unchanged. By blocking the membrane-located NADPH oxidase RboH, diphenylene iodonium by itself increased both, the levels of astaxanthin and transcripts of all three biosynthetic genes, and this increase was added up to that produced by nsPEFs. Artificial calcium influx by an ionophore did not induce major changes in the accumulation of astaxanthin, nor in the transcript levels, but amplified the response of crtR-b to nsPEFs at 25 ns, while decreased in 50 ns treatment. When Ca2+ influx was inhibited by GdCl3, the transcript of psy and bkt 1 were decreased for both 25 ns and 50 ns treatments, while crtR-b exhibited an obvious increase for the 25 ns treatment. We interpret these data in a working model, where nsPEFs permeabilise plasma and chloroplast membrane depending on pulse duration leading to a differential release of plastid retrograde signaling to the nucleus

Impact of incubation conditions on protein and C-Phycocyanin recovery from Arthrospira platensis post- pulsed electric field treatment

Pulsed electric field (PEF) was conducted for the extraction of proteins/C-Phycocyanins from Arthrospira platensis. The cyanobacterial suspension was treated with 1 μs long pulses at an electric field strength of 40 kV·cm−1 and a treatment energy of 114 kJ·kgsus−1 and 56 kJ·kgsus−1. For benchmarking, additional biomass was processed by high pressure homogenization. Homogeneity of the suspension prior to PEF-treatment influenced the protein/C-phycocyanin extraction efficiency. Stability of C-Phycocyanin during post-PEF incubation time was affected by incubation temperature and pH of the external medium. Biomass concentration severely affect proteins/C-Phycocyanins extraction yield via PEF-treatment. The optimum conditions for extraction of proteins/ C-Phycocyanin was obtained at 23 °C while incubating in pH 8-buffer. The energy demand for PEF-Treatment amounts to 0.56 MJ·kgdw−1 when processing biomass at 100 gdw·kgsus−1. PEF treatment enhances the protein/CPhycocyanin extraction yield, thus, it can be suggested as preferential downstream processing method for the production of C-Phycocyanin from A. platensis biomass

Nanosecond pulsed electrical fields enhance product recovery in plant cell fermentation

The potential of pharmacologically active secondary plant metabolites is limited by the low yield from often rare plants, and the lack of economically feasible chemical synthesis of these complex compounds. Plant cell fermentation offers an alternative strategy to overcome these constraints. However, the efficiency of this approach is limited by intracellular sequestration of the products, such that continuous bioprocessing is not possible. As a precondition for such a, more attractive, continuous process, it is of great importance to stimulate the export of the product into the medium without impairing viability and, thus, the productivity of the cells. Using nicotine alkaloids of tobacco as a case study, an alternative strategy is explored, where nanosecond pulsed electric fields (nsPEFs) are applied for the efficient downstream recovery of the products. To maintain cell viability and allow for the further use of biomass, cells were exposed to strong (1–20 kV·cm^-1}), but very short (10–100 ns) electric pulses, which leads to a temporary permeabilisation of cell membranes. Using two transgenic cell lines, where two key genes involved in the metabolism of the anti-Alzheimer compound nornicotine were overexpressed, we could show that this nsPEF treatment improved the partitioning of some nicotine alkaloids to the culture medium without impairing viability, nor the synthesis of alkaloids. However, this release was only partial and did not work for nornicotine. Thus, nsPEFs produced a fractionation of alkaloids. We explain this electrofractionation by a working model considering the differential intracellular compartmentalization of nicotineic alkaloids

Impact of incubation conditions on protein and C-Phycocyanin recovery from Arthrospira platensis post- pulsed electric field treatment

Pulsed electric field (PEF) was conducted for the extraction of proteins/C-Phycocyanins from Arthrospira platensis. The cyanobacterial suspension was treated with 1 μs long pulses at an electric field strength of 40 kV·cm−1 and a treatment energy of 114 kJ·kgsus−1 and 56 kJ·kgsus−1. For benchmarking, additional biomass was processed by high pressure homogenization. Homogeneity of the suspension prior to PEF-treatment influenced the protein/C-phycocyanin extraction efficiency. Stability of C-Phycocyanin during post-PEF incubation time was affected by incubation temperature and pH of the external medium. Biomass concentration severely affect proteins/C-Phycocyanins extraction yield via PEF-treatment. The optimum conditions for extraction of proteins/ C-Phycocyanin was obtained at 23 °C while incubating in pH 8-buffer. The energy demand for PEF-Treatment amounts to 0.56 MJ·kgdw−1 when processing biomass at 100 gdw·kgsus−1. PEF treatment enhances the protein/CPhycocyanin extraction yield, thus, it can be suggested as preferential downstream processing method for the production of C-Phycocyanin from A. platensis biomass

Biological signalling supports biotechnology – Pulsed electric fields extract a cell-death inducing factor from Chlorella vulgaris

Compared to mechanical extraction methods, pulsed electric field (PEF) treatment provides an energy-efficient and gentle alternative. However, the biological processes involved are poorly understood. The unicellular green microalga Chlorella vulgaris was used as model organism to investigate the effect of PEF treatment on biological cells. A viability assay using fluorescein diacetate measured by flow cytometry was established. The influence of developmental stage on viability could be shown in synchronised cultures when applying PEF treatment with very low specific energies where one part of cells undergoes cell death, and the other part stays viable after treatment. Reactive oxygen species generation after similar low-energy PEF treatment could be shown, indicating that PEFs could act as abiotic stress signal. Most importantly, a cell-death inducing factor could be extracted. A water-soluble extract derived from microalgae suspensions incubated for 24 h after PEF treatment caused the recipient microalgae to die, even though the recipient cells had not been subjected to PEF treatment directly. The working model assumes that low-energy PEF treatment induces programmed cell death in C. vulgaris while specifically releasing a cell-death inducing factor. Low-energy PEF treatment with subsequent incubation period could be a novel biotechnological strategy to extract soluble proteins and lipids in cascade process