Katja Zocher

Phycocyanin as water-soluble, accessory pigment is commonly extracted from Limnospira platensis (L. platensis). The pigment is often used as natural food colourant and is a potential antioxidant. Usually, the pigment is extracted with several freeze–thawing cycles with subsequent purification steps. The cell disintegration with physical plasma (here spark discharges) is already described for microalgae and was tested for the extraction of phycocyanin from L. platensis. Moreover, calcium chloride was examined as a non-toxic and inexpensive precipitator in the purification step. Samples were treated with water as solvent alone or precipitated with calcium chloride. Also, samples were divided for immediate analysis after extraction and 24 h incubation after the respective cell wall rupture method with subsequent analysis. The extracts were tested for C-phycocyanin (C-PC) concentration, content, selectivity, and radical scavenging activity. Spark discharges were administered with 4 Hz, 100 ns high-voltage pulses of 35 kV, resulting in a pulse energy of ca. 0.2 J/pulse. For freeze–thawing, a maximum yield of 2.75 mg/mL was obtained, for spark discharges, max. 0.24 mg/mL. However, despite a lower concentration, a remarkable purity was found for plasma-treated samples. In addition, radical scavenging activity was not influenced by the spark discharge exposure.

In the field of bioeconomy, biological materials are used as resource for a wide field of application, such as manufacturing of pharmaceutical products or fine chemicals and processes, e.g., aqua cultures, or mussel farming. Resources may be, amongst others, microorganisms, land plants, but also agricultural residues. To access these resources, technologies are necessary. Many methods already exist, but are accompanied by drawbacks on several levels. High-energy input, detrimental effects caused by heat or chemical impact, generation of toxic by-products, or incomplete extraction/purification are often observed for these standard methods. With physical plasmas, an interesting alternative has been developed to address several aspects from those drawbacks. Plasmas are effective against cell walls of microorganisms, such as microalgae, but also in the valorisation of manure for biogas production, or in the recycling of (waste) water. The unique characteristics of a plasma help to extract compounds from these materials due to shockwaves, which are generated during plasma generation. Also, the production of reactive oxygen and nitrogen species (RONS) are the decisive agents for the degradation of pollutant in water. Here, we review possible fields of application for physical plasmas in the frame of bioeconomy.

Glyphosate (GLP) is one of the most widely applied herbicides, and is found ubiquitously in the environment. The removal of glyphosate from waste water and soil is challenging and can be achieved with chemical or biological methods, which, nevertheless, suffer from different disadvantages. The application of a physical plasma for the removal of GLP in water was examined by the application of surface corona discharges in a wire-to-cylinder setup filled with argon. The plasma was ignited at the liquid surface without any additives. By applying a photometric method, GLP was detected after derivatisation with fluorenyl methoxycarbonyl chloride, whereas phosphate was determined with ammonium molybdate. A GLP degradation rate of 90.8% could be achieved within a treatment time of 30 minutes with an estimated energy efficiency of 0.32 g/kWh.

Phycocyanin is a blue colored pigment, synthesized by several species of cyanobacteria and red algae. Besides the application as a food-colorant, the pigmented protein is of high interest as a pharmaceutically and nutritionally valuable compound. Since cyanobacteria-derived phycocyanin is thermolabile, red algae that are adapted to high temperatures are an interesting source for phycocyanin extraction. Still, the extraction of high quality phycocyanin from red algae is challenging due to the strong and rigid cell wall. Since standard techniques show low yields, alternative methods are needed. Recently, spark discharges have been shown to gently disintegrate microalgae and thereby enable the efficient extraction of susceptible proteins. In this study, the applicability of spark discharges for phycocyanin extraction from the red alga Cyanidium caldarium was investigated. The efficiency of 30 min spark discharges was compared with standard treatment protocols, such as three times repeated freeze-thaw cycles, sonication, and pulsed electric fields. Input energy for all physical methods were kept constant at 11,880 J to ensure comparability. The obtained extracts were evaluated by photometric and fluorescent spectroscopy. Highest extraction yields were achieved with sonication (53 mg/g dry weight (dw)) and disintegration by spark discharges (4 mg/g dw) while neither freeze-thawing nor pulsed electric field disintegration proved effective. The protein analysis via LC-MS of the former two extracts revealed a comparable composition of phycobiliproteins. Despite the lower total concentration of phycocyanin after application of spark discharges, the purity in the raw extract was higher in comparison to the extract attained by sonication.

Microalgae possess a cell wall with remarkable physical and chemical strength, which has proved to be a bottleneck for extraction techniques. With the application of spark discharges instigated by 100-ns high voltage pulses, a novel method was established that was found to be effective and yet gentle for the extraction of sensitive, especially heat-sensitive, compounds. The spark, which was ignited directly in the submerged algae suspension, is characterised by several physical and chemical processes that might promote a successful disintegration of the cells. Among other characteristics, the conceivably most destructive mechanism, i.e. strong shockwaves, was examined with schlieren diagnostics to evaluate associated pressures. The results were compared with the tensile strength, determined by atomic force microscopy, of Chlorella vulgaris , which was chosen as model organism. A shockwave pressure of 500 MPa in the close vicinity of the discharge by far exceeded the elasticity modulus of the algae with 13.95 MPa, confirming the potential of mechanically breaking the cell wall. At the same time, bulk temperatures could be maintained close to room temperature by adjusting the operating parameters for the spark-application, hence, especially encouraging the extraction of thermally unstable intracellular substances.

Medicinal mushrooms contain highly valuable substances with proven positive effects on human health. To extract these components, different methods are available. Most of them suffer from individual disadvantages, therefore making them economically unviable. Pulsed electric fields (PEFs) could provide an opportunity to improve these processes. PEFs cause pore formation of cell membranes, facilitating substance transport out of cells. Thus, the influence of this technique on the extraction yield of medicinal mushrooms was studied for the first time. Lentinus edodes was used as model case and PEF treatment was compared with standard Soxhlet extraction alone. A square pulse generator (Electro Square Porator^™ ECM 830) with a voltage of 3 kV and pulse length of 100 μs was used for PEF treatment. Extraction was studied for fresh and dried fruiting bodies, and dichloromethane and hot water extracts were analyzed. Extracts were quantified gravimetrically, and carbohydrate yields were also determined qualitatively with GC-MS and quantitatively with anthrone method. PEFs could increase in particular the yield of water-soluble compounds of fresh mushroom material. However, the lipid fraction was not affected by PEF in neither fresh nor dried material.

Microalgae have recently gained interest, not only as source for biofuel, but also as a resource for pharmaceutical and nutritional substances. The bottleneck for extracting these valuable compounds from microalgae is a thick cell wall of high physical and chemical strength. Several extraction techniques are available, but suffer from different disadvantages. Therefore, new technologies are needed, especially those based on processes that will not affect the chemical composition of ingredients. Among these, physical plasma and pulsed electric fields (PEF) might be promising. Three different standard methods, microwave, ultrasound, and homogenization, were compared with plasma treatment and PEF. The plasma sources investigated were corona discharges, a plasma jet, a dielectric barrier discharge, spark discharges, and pin-to-liquid discharges. Chlorella vulgaris was chosen as a model organism. To detect successful cell wall rupture, the protein content of the supernatant and pigment concentration after treatment were determined. Scanning electron microscope images were taken to visualize cell wall damage. Microwave and spark discharge treatment were the most successful methods with comparable extracted total protein content in the supernatant. However, spark discharges achieved higher pigment yield than microwave extraction without the thermal degradation of the pigments observed for microwave extraction.