Surface properties of mycoparasitic Pythium species and their interaction with model materials.

Pythium oligandrum, a soil-born oomycete, is an effective biological control agent exhibiting antagonistic and parasitic activity against pathogenic fungi. This study is the first attempt to characterize its surface properties and to apply models of physicochemical interactions (thermodynamic, DLVO and XDLVO) to quantify its adhesion properties to a model material, represented by magnetic beads (MB). The predictions of interaction models were based on experimental data (contact angles, zeta potentials, size). Adhesion intensities (AI) were determined experimentally taking advantage of MB with different surface properties. The role of weak physicochemical interactions was estimated by comparing experimental AI with model predictions. The results revealed that the surface properties of the three Pythium spp. studied were very similar and fell within the range for hydrophilic microorganisms (ΔGTOT > 0) with a predominantly negative surface charge. The most reliable description of AI was obtained using the DLVO model, including Lifshitz-van der Waals and electrostatic interactions. The highest AI between Pythium spp. and all three MB was observed at pH 3, which was supported by the DLVO prediction. The greater agreement between the sphere-sphere geometric version of the DLVO model and experiment suggests that the surface protrusions of the oospores increase the efficiency of adhesion. The surface properties of the pathogenic fungi, characterized in this work, fell within the range defined by MB and therefore it can be expected that their physicochemical interactions with Pythium spp. will also be favourable.

Comparative assessment of UV-C radiation and non-thermal plasma for inactivation of foodborne fungal spores suspension in vitro.

Fungal contamination poses a persistent challenge to industries, particularly in food, healthcare, and clinical sectors, due to the remarkable resilience of fungi in withstanding conventional control methods. In this context, our research delves into the comparative efficacy of UV radiation and non-thermal plasma (NTP) on key foodborne fungal contaminants - Alternaria alternata, Aspergillus niger, Fusarium culmorum, and Fusarium graminearum. The study examined the impact of varying doses of UV radiation on the asexual spores of all mentioned fungal strains. Simultaneously, the study compared the effects of UV radiation and NTP on the metabolic activity of cells after spore germination and their subsequent germination ability. The results revealed that UV-C radiation (254 nm) did not significantly suppress the metabolic activity of cells after spore germination. In contrast, NTP exhibited almost 100% effectiveness on both selected spores and their subsequent germination, except for A. niger. In the case of A. niger, the effectiveness of UV-C and NTP was nearly comparable, showing only a 35% decrease in metabolic activity after 48 hours of germination, while the other strains (A. alternata, F. culmorum, F. graminearum) exhibited a reduction of more than 95%. SEM images illustrate the morphological changes in structure of all tested spores after both treatments. This study addresses a crucial gap in existing literature, offering insights into the adaptation possibilities of treated cells and emphasizing the importance of considering exposure duration and nutrient conditions (introduction of fresh medium). The results highlighted the promising antimicrobial potential of NTP, especially for filamentous fungi, paving the way for enhanced sanitation processes with diverse applications.

Exploring Non-Thermal Plasma and UV Radiation as Biofilm Control Strategies against Foodborne Filamentous Fungal Contaminants.

In recent years, non-thermal plasma (NTP) has emerged as a promising tool for decontamination and disinfection within the food industry. Given the increasing resistance of microbial biofilms to conventional disinfectants and their adverse environmental effects, this method has significant potential for eliminating biofilm formation or mitigating the metabolic activity of grown biofilms. A comparative study was conducted evaluating the efficacy of UV radiation and NTP in eradicating mature biofilms of four common foodborne filamentous fungal contaminants: Alternaria alternata, Aspergillus niger, Fusarium culmorum, and Fusarium graminearum. The findings reveal that while UV radiation exhibits variable efficacy depending on the duration of exposure and fungal species, NTP induces substantial morphological alterations in biofilms, disrupting hyphae, and reducing extracellular polymeric substance production, particularly in A. alternata and F. culmorum. Notably, scanning electron microscopy analysis demonstrates significant disruption of the hyphae in NTP-treated biofilms, indicating its ability to penetrate the biofilm matrix, which is a promising outcome for biofilm eradication strategies. The use of NTP could offer a more environmentally friendly and potentially more effective alternative to traditional disinfection methods.

UHPLC-PAD Protocol for the Simultaneous Identification of Polyphenols and Bitter Acids: Organoleptic and Nutraceutical Fingerprinting of Bergamot-Flavored Craft Beer.

In this study, a UHPLC-PDA method for the simultaneous identification of polyphenols and bitter acids (alpha, beta, and isoalpha) in beer was developed. The resulting chemical profiles were leveraged to distinguish the characteristics of four (IPA, Lager, Blanche, ALE) bergamot-flavored beers, produced on a pilot-scale plant. In a streamlined 29 min analysis, thirty polyphenols and fourteen bitter acids were successfully identified under optimized separation conditions. Validation, encompassing parameters such as LOD (from 0.028 ppm for isorhamnetin to 0.106 for narirutin), LOQ (from 0.077 ppm for naringenin to 0.355 for narirutin), R2 (always more than 0.9992), repeatability (from 0.67% for tangeretin to 6.38% for myricetin), and reproducibility (from 0.99% for sinensetin to 6% for naringin), was conducted for polyphenol quantification using constructed calibration curves with seven levels. Exploring polyphenolic components as potential discriminators among different beer styles, a total of thirty-two polyphenolic compounds were identified and quantified, including characteristic bergamot peel polyphenols like neoeriocitrin (from 7.85 ppm for CBS2 to 11.95 ppm in CBS1); naringin (from 4.56 ppm for CBS4 to 10.96 in CBS1), and neohesperidin (from 5.93 in CBS3 to 15.95 for CBS2). The multivariate analysis provided additional insights into variations among specific beer styles, revealing discrepancies in the presence or relative concentrations of specific compounds linked to brewing ingredients and processes. This research enhances the fingerprinting of the chemistry governing beer quality through a straightforward and cost-effective analytical approach.

Xanthohumol-Enriched Beer Does Not Exert Antitumorigenic Effects on HeLa Cell Line In Vivo.

Xanthohumol is a hop-derived flavonoid that has been widely examined for its health-protecting and antitumorigenic properties, but not yet in a natural beer matrix. The aim of the study was to investigate the antitumorigenic potential of a xanthohumol-enriched beer in vivo. Four groups of 4 × 10 nude mice were formed. Following the injection of HeLa tumorigenic cell lines, the treatment groups were administered a xanthohumol supplementation for 100 days, either dissolved in beer or in an ethanolic solution with the same alcohol strength as beer. The control groups received un-supplemented material. The terminal tumor masses, liver weights, and plasma antioxidant capacities (FRAP and ABTS methods) were measured. For the statistical analysis, a two-way ANOVA test was performed (p < 0.05). There were no statistically significant differences in tumor size between the groups. Xanthohumol did not induce higher levels of plasma antioxidant capacity, neither in beer nor in the water-ethanol matrix. The terminal liver weights were significantly higher in the control group receiving the unsupplemented ethanol solution. Xanthohumol dissolved in beer or in the water-alcohol matrix did not have a protective effect on tumor growth, nor did it have a positive effect on plasma antioxidant capacity either. However, beer with added xanthohumol had a less harmful effect on the liver compared to the supplemented water-ethanol solution. Our results indicate the possible negative countereffect of ethanol; however, further investigations are needed.

Detection of microscopic filamentous fungal biofilms - Choosing the suitable methodology.

Microscopic filamentous fungi are ubiquitous microorganisms that adapt very easily to a variety of environmental conditions. Due to this adaptability, they can colonize a number of various surfaces where they are able to start forming biofilms. Life in the form of biofilms provides them with many benefits (increased resistance to desiccation, UV radiation, antimicrobial compounds, and host immune response). The aim of this study is to find a reliable and reproducible methodology to determine biofilm growth of selected microscopic filamentous fungi strains. Several methods (crystal violet staining, MTT assay, XTT assay, resazurin assay) for the determination of total biofilm biomass and its metabolic activity were tested on four fungi - Alternaria alternata, Aspergillus niger, Fusarium culmorum and Fusarium graminearum, and their biofilm was also imaged by spinning disc confocal microscopy using fluorescent dyes. A reproducible biofilm quantification method is essential for the subsequent testing of the biofilm growth suppression using antifungal agents or physical methods. Crystal violet staining was found to be a suitable method for the determination of total biofilm biomass of selected strains, and the MTT assay for the determination of metabolic activity of the biofilms. Calcofluor white and Nile red fluorescent stains successfully dyed the hyphae of microscopic fungi.

Functional Properties of Dunaliella salina and Its Positive Effect on Probiotics.

The unicellular green microalga Dunaliella is a potential source of a wide range of nutritionally important compounds applicable to the food industry. The aim of this study was to assess the effect of Dunaliella salina dried biomass on the growth and adherence of 10 strains of Lactobacillus, Lacticaseibacillus, and Bifidobacterium. The immunomodulatory, antioxidant, and cytotoxic effects of D. salina on human peripheral mononuclear cells and simulated intestinal epithelial cell lines Caco-2 and HT-29 were evaluated. Furthermore, the hypocholesterolemic effects of the microalgae on lipid metabolism in rats fed a high-fat diet were analyzed. The addition of D. salina biomass had a positive effect on the growth of nine out of 10 probiotics and promoted the adherence of three bifidobacteria strains to human cell lines. The antioxidant and immunomodulatory properties of D. salina were concentration-dependent. The inflammatory cytokines (TNF-α and IL-6) were significantly increased following Dunaliella stimulation at the lowest concentration (0.5% w/v). Eight week supplementation of D. salina to the diet of hypercholesteromic rats significantly decreased the serum concentrations of LDL-C, VLDL, IDL-B, and IDL-C. D. salina is not cytotoxic in intestinal cell models; it promotes adherence of selected bifidobacteria, it affords immunomodulatory and antioxidant effects, and its addition to diets may help decrease atherosclerosis risk factors.

Factors Influencing the Production of Extracellular Polysaccharides by the Green Algae Dictyosphaerium chlorelloides and Their Isolation, Purification, and Composition.

The freshwater green microalgae, Dictyosphaerium chlorelloides (CCALA 330), has the ability to produce extracellular polysaccharides (EPS). Conditions for optimum growth and EPS overproduction were determined in laboratory-scale tubular photobioreactors (PBR) with a working volume of 300 mL. Multiple limitations in nutrient supply were proven to be an effective method for EPS overproduction. Salinity stress was also applied to the culture, but no significant increase in EPS production was observed. The effects of different nitrogen sources were examined and the microalgae exhibited the fastest growth and EPS production in medium containing ammonium nitrate. Under determined optimal conditions, EPS concentration reached 10 g/L (71% of the total biomass) and a total biomass of 14 g/L at the end of 17 days cultivation. Pilot-scale cultivation was also carried out in a column type airlift photobioreactor (PBR) with a working volume of 60 L. A new and efficient methodology was developed for separating cells from the EPS-containing culture broth. Due to the strong attachment between cells and EPS, high-pressure homogenization was carried out before a centrifugation process. The EPS in the supernatant was subsequently purified using ultrafiltration. The green microalgae Dictyosphaerium chlorelloides may therefore be appropriate for the commercial production of EPS.

Continuous electrocoagulation of Chlorella vulgaris in a novel channel-flow reactor: A pilot-scale harvesting study.

The most frequently used method to harvest microalgae on an industrial scale is centrifugation, although this has very high energy costs. To reduce these costs, a continuous electrocoagulation process for harvesting Chlorella vulgaris was developed and tested using a pilot-scale 111 L working volume device consisting of an electrolyser with iron electrodes, aggregation channel and lamellar settler. The flow rate of the microalgal suspension through the device was 240 L/h. When using controlled cultivation and subsequent electrocoagulation, a high harvesting efficiency (above 85%), a low Fe contamination in the harvested biomass (<4 mg Fe/g dry biomass, a harvested biomass complied with legislative requirements for food) and significant energy savings were achieved. When comparing electrocoagulation and subsequent centrifugation with the use of centrifugation alone, energy savings were 80 % for a biomass harvesting concentration of 0.23 g/L. Electrocoagulation was thus proven to be a feasible pre-concentration method for harvesting microalgae.

Influence of Cultivation Conditions on the Sioxanthin Content and Antioxidative Protection Effect of a Crude Extract from the Vegetative Mycelium of Salinispora tropica.

Due to their bioavailability, glycosylated carotenoids may have interesting biological effects. Sioxanthin, as a representative of this type of carotenoid, has been identified in marine actinomycetes of the genus Salinispora. This study evaluates, for the first time, the effect of cultivation temperature (T) and light intensity (LI) on the total cellular carotenoid content (TC), antioxidant activity (AA) and sioxanthin content (SX) of a crude extract (CE) from Salinispora tropica biomass in its vegetative state. Treatment-related differences in TC and SX values were statistically significantly and positively affected by T and LI, while AA was most significantly affected by T. In the S. tropica CE, TC correlated well (R2 = 0.823) with SX and somewhat less with AA (R2 = 0.777). A correlation between AA and SX was found to be less significant (R2 = 0.731). The most significant protective effect against oxidative stress was identified in the CE extracted from S. tropica biomass grown at the highest T and LI (CE-C), as was demonstrated using LNCaP and KYSE-30 human cell lines. The CE showed no cytotoxicity against LNCaP and KYSE-30 cell lines.

Laboratory scale cultivation of Salinispora tropica in shake flasks and mechanically stirred bioreactors.

OBJECTIVE: Marine actinomycetes from the genus Salinispora have an unexploited biotechnological potential. To accurately estimate their application potential however, data on their cultivation, including biomass growth kinetics, are needed but only incomplete information is currently available. RESULTS: This work provides some insight into the effect of temperature, salinity, nitrogen source, glucose concentration and oxygen supply on growth rate, biomass productivity and yield of Salinispora tropica CBN-440T. The experiments were carried out in unbaffled shake flasks and agitated laboratory-scale bioreactors. The results show that the optimum growth temperature lies within the range 28-30 °C, salinity is close to sea water and the initial glucose concentration is around 10 g/L. Among tested nitrogen sources, yeast extract and soy peptone proved to be the most suitable. The change from unbaffled to baffled flasks increased the volumetric oxygen transfer coefficient (kLa) as did the use of agitated bioreactors. The highest specific growth rate (0.0986 h-1) and biomass productivity (1.11 g/L/day) were obtained at kLa = 28.3 h-1. A further increase in kLa was achieved by increasing stirrer speed, but this led to a deterioration in kinetic parameters. CONCLUSIONS: Improvement of S. tropica biomass growth kinetics of was achieved mainly by identifying the most suitable nitrogen sources and optimizing kLa in baffled flasks and agitated bioreactors.

Electrocoagulation reduces harvesting costs for microalgae.

Centrifugation is the most commonly used method for harvesting autotrophically produced microalgae, but it is expensive due to high energy demands. With the aim of reducing these costs, we tested electrocoagulation with iron electrodes for harvesting Chlorella vulgaris. During extensive lab-scale experiments, the following factors were studied to achieve a high harvesting efficiency and a low iron content in the harvested biomass: electric charge, initial biomass concentration, pH, temperature, agitation intensity, residual salt content and electrolysis time. A harvesting efficiency greater than 95% was achieved over a broad range of conditions and the residual iron content in the biomass complied with legislative requirements for food. Using electrocoagulation as the pre-concentration step prior to centrifugation, total energy costs were reduced to 0.136 kWh/kg of dry biomass, which is less than 14% of that for centrifugation alone. Our data show that electrocoagulation is a suitable and cost-effective method for harvesting microalgae.

Cooking oil-surfactant emulsion in water for harvesting Chlorella vulgaris by sedimentation or flotation.

In this study, a novel harvesting emulsion (HEM) consisting of cooking oil in an aqueous solution of cetyltrimethylammonium bromide (CTAB) was tested for the harvesting of a technologically important microalga, Chlorella vulgaris. The influence of HEM dose, biomass and bovine serum albumin (BSA) (model interferer compound) on harvesting efficiency (E) were studied. The HEM E was over 90% at pH 10 (0.33% (v/v) cooking oil, 6.7 mg/L of CTAB) and 12 (0.13% (v/v) cooking oil, 2.7 mg/L of CTAB). Harvesting efficiencies at pH 4 and 7 were < 73.5% due to the absence of precipitate formation. Bovine serum albumin (10 mg/L) increased the HEM dose necessary to achieve E ˃ 90% by 1.2 (pH 10), and 3 fold (pH 12). By manipulating the dose of HEM and pH, the method of harvesting (flocculation/sedimentation or flotation) was adjustable depending on the technological requirements.

Heterotrophic cultivation of Chlorella vulgaris using saline waste water from the demineralization of cheese whey.

OBJECTIVE: Desalination of cheese whey by electrodialysis yields saline wastewater (SWW). The goal was to test this as the basis of a culture medium and to prove experimentally the concept that it was a suitable resource for heterotrophic cultivation of the freshwater green microalga Chlorella vulgaris. RESULTS: Optimization of glucose concentration, nitrogen source and medium salinity for microalgal growth was first carried out in defined medium (DM) and shake flasks. These results were then adopted in shake flask cultivation experiments using pre-treated SWW medium (PSWW). Subsequently, microalgal growth under optimized conditions was tested in bioreactors. Various media such as DM, PSWW and diluted PSWW (DPSWW) were compared. Volumetric biomass productivities decreased in the order DM (0.371 g L-1 h-1, urea) > DPSWW (0.315 g L-1 h-1, soy peptone) > PSWW (0.152 g L-1 h-1, soy peptone). Although biomass productivities in DPSWW and PSWW media were significantly lower than in DM, these media required the addition of only 66 and 33% of DM N sources, respectively. No other added DM component was necessary in (D)PSWW to achieve microalgal growth. CONCLUSIONS: Although the optimized cultivation of freshwater microalgae on alternative medium based on SWW resulted in biomass productivities lower than those on DM, the required addition of N sources was also lower. Potentially lower production costs of Chlorella biomass and the meaningful use of SWW are the main outcomes of this work.

Interference of model wastewater components with flocculation of Chlorella sorokiniana induced by calcium phosphate precipitates.

Amongst harvesting processes, alkaline flocculation stands out as a technically feasible and low cost method. The interference of model wastewater components with alkaline flocculation of Chlorella sorokiniana (pH 8-12), induced by calcium phosphate (CaP) precipitates, was evaluated. Between the compounds tested, inorganic nitrogen, sodium alginate, salinity and algal organic matter had no effect on flocculation efficiency (FE). The negative effect of humic acids, sodium dodecyl sulphate and alkalinity on FE was partial. Bovine serum albumin and bacterial organic matter (BOM) of Escherichia coli showed the strongest disruption of FE. The impact of BOM can be explained by the high protein content (65% of total organic carbon). Proteins, negatively charged at alkaline pH, interrupt microalgae flocculation by preferentially interacting with positively charged CaP precipitates. The simultaneous effects of multiple substances were tested to simulate real wastewater. The results confirm the need to investigate the composition of wastewater prior to alkaline flocculation.

Physico-chemical approach to adhesion of Alicyclobacillus cells and spores to model solid materials.

Acidothermophilic bacteria of the genus Alicyclobacillus are frequent contaminants of fruit-based products. This study is the first attempt to characterize the physico-chemical surface properties of two Alicyclobacillus sp. and quantify their adhesion disposition to model materials [diethylaminoethyl (DEAE), carboxyl- and octyl-modified magnetic beads] representing materials with different surface properties used in the food industry. An insight into the mechanism of adhesion was gained through comparison of experimental adhesion intensities with predictions of a colloidal interaction model (XDLVO). Experimental data (contact angles, zeta potentials, size) on interacting surfaces (cells and materials) were used as inputs into the XDLVO model. The results revealed that the most significant adhesion occurred at pH 3. Adhesion of both vegetative cells and spores of two Alicyclobacillus sp. to all materials studied was the most pronounced under acidic conditions, and adhesion was influenced mostly by electrostatic attractions. The most intensive adhesion of vegetative cells and spores at pH 3 was observed for DEAE followed by hydrophobic octyl and hydrophilic carboxyl surfaces. Overall, the lowest rate of adhesion between cells and model materials was observed at an alkaline pH. Consequently, prevention of adhesion should be based on the use of alkaline sanitizers and/or alkaline rinse water.

Physicochemical approach to alkaline flocculation of Chlorella vulgaris induced by calcium phosphate precipitates.

Alkaline flocculation has been studied due to its potential as a low-cost harvesting method for microalgae. However, surface properties (zeta potential, contact angles) as inputs into physicochemical interaction models have not yet been applied systematically. In this work, forced alkaline flocculation of the freshwater microalgae Chlorella vulgaris induced by calcium phosphate precipitates was studied as a model system. Response surface methodology was used to quantify the effect of independent variables (concentration of Ca2+ (0.5-0.5 mM) and PO43- (0.05-0.35 mM), pH (8-12) and ionic strength (1-19 mM)) on the zeta potential (ZP) of microalgae, and the turbidity (T) of inorganic precipitates. Flocculation tests and their modified versions were carried out. The flocculation efficiencies obtained were interpreted with respect to predictions of physicochemical interaction models. It was found that flocculation was possible under conditions where appropriate precipitates were formed in the presence of cells. Under these conditions, flocculation of negatively charged Chlorella vulgaris was induced not only by positively charged, but also by negatively charged calcium phosphate precipitates at an early phase of nucleation. The driving force for interactions between oppositely charged cells and precipitate particles was electrostatic attraction, while the attraction between equally charged entities may have resulted from a negative total balance of apolar (Lifsitz-van der Waals) and polar (acid-base) interactions. Medium components did not interfere with flocculation, while cellular organic matter decreased flocculation efficiency only to a very limited extent.

An application of cellular organic matter to coagulation of cyanobacterial cells (Merismopedia tenuissima).

Algae affect the performance of drinking water treatment significantly when they decay and release considerable amounts of cellular organic matter (COM). The study describes the cyanobacterium Merismopedia tenuissima and its COM and investigates the effect of their simultaneous coagulation. As COM is highly complex mixture, we characterised it in terms of hydrophobicity, protein content and molecular weights (MWs). To describe the coagulation mechanisms and molecular interactions in the system, we determined both COM and cell surface charge by means of potentiometric titration and zeta potential analysis, respectively, and performed the jar tests with single components and their mixtures with and without a coagulant (ferric sulphate). The coagulation tests performed with the individual components or with their mixtures proved efficient cell removals (up to 99%) but relatively low COM removals (37 ÷ 57%). This disproportion can be attributed to the prevalence of hydrophilic compounds and to the high portion of low-MW organics in COM. Coagulation of COM/cell mixtures achieved comparable efficacy with single component tests, using even lower coagulant doses. Furthermore, COM presence substantially deviated the pH optimum for cell removal and thus altered coagulation mechanisms. While single cells interacted prevailingly through adsorption onto Fe-oxide-hydroxides at about neutral pH (6.0-7.7), the COM/cell mixtures succumbed to charge neutralisation by Fe-hydroxopolymers within moderately acidic pH range (5.0-6.5). Moreover, COM initiated cell flocculation also at acidic pH in both the presence (pH 3.4-3.9) and the absence of a coagulant (pH 3.6-4.6). This supportive effect is ascribed to relatively high-MW COM (>10 kDa), serving as a natural flocculant through inter-particle bridging mechanism and exhibiting nearly the same COM/cell removals as ferric sulphate.

Use of saline waste water from demineralization of cheese whey for cultivation of Schizochytrium limacinum PA-968 and Japonochytrium marinum AN-4.

Saline waste water from demineralization of cheese whey was used as the main component of waste saline medium (WSM) for cultivation of thraustochytrids. The suitability of WSM for cultivation of Schizochytrium limacinum PA-968 and Japonochytrium marinum AN-4 was evaluated by comparison with cultivation on nutrient medium (NM) in shake flask and fermenter cultures. Biomass productivities achieved in WSM for the thraustochytrids were comparable with those in NM for both shake flask and fermenter cultures. The maximum total lipid content (56.71% dry cell weight) and docosahexaenoic acid productivity (0.86 g/L/day) were achieved by J. marinum AN-4 grown on WSM in shake flask and fermenter cultures, respectively. A cost estimate of WSM suggests that this medium could result in lower production costs for thraustochytrid biomass and lipids and contribute to the effective reduction in saline diary process waste water.

Magnetically modified microalgae and their applications.

The majority of algal cells can interact with a wide range of nano- and microparticles. Upon interaction the modified cells usually maintain their viability and the presence of foreign material on their surfaces or in protoplasm can provide additional functionalities. Magnetic modification and labeling of microalgal biomass ensures a wide spectrum of biotechnological, bioanalytical and environmental applications. Different aspects of microalgal cell magnetic modification are covered in the review, followed by successful applications of magnetic algae. Modified cells can be employed during their harvesting and removal, applied in toxicity microscreening devices and also as efficient adsorbents of different types of xenobiotics.