Microalgae cultivation trials in a membrane bioreactor operated in heterotrophic, mixotrophic, and phototrophic modes using ammonium-rich wastewater: The study of fouling.

In this work, microalgae cultivation trials were carried out in a membrane bioreactor to investigate fouling when the cultures of Chlorellavulgaris were grown under mixotrophic, heterotrophic, and phototrophic cultivation regimes. The Chlorella cultures were cultivated in wastewater as a source of nutrients that contained a high concentration of ammonium. In mixotrophic cultivation trials, the results showed that the elevated contents of carbohydrates in the soluble microbial product and proteins in extracellular polymeric substances probably initiated membrane fouling. In this case, the highest protein content was also found in extracellular polymeric substances due to the high nitrogen removal rate. Consequently, transmembrane pressure significantly increased compared to the phototrophic and heterotrophic regimes. The data indicated that cake resistance was the main cause of fouling in all cultivations. Higher protein content in the cake layer made the membrane surface more hydrophobic, while carbohydrates had the opposite effect. Compared to a mixotrophic culture, a phototrophic culture had a larger cell size and higher hydrophobicity, leading to less membrane fouling. Based on our previous data, the highest ammonia removal rate was reached in the mixotrophic cultures; nevertheless, membrane fouling appeared to be the fundamental problem.

Poly-ß-hydroxybutyrate production by Synechocystis MT_a24 in a raceway pond using urban wastewater.

Poly-ß-hydroxybutyrate (PHB) is a potential source of biodegradable plastics that are environmentally friendly due to their complete degradation to water and carbon dioxide. This study aimed to investigate PHB production in the cyanobacterium Synechocystis sp. PCC6714 MT_a24 in an outdoor bioreactor using urban wastewater as a sole nutrient source. The culture was grown in a thin-layer raceway pond with a working volume of 100 L, reaching a biomass density of up to 3.5 g L-1 of cell dry weight (CDW). The maximum PHB content was found under nutrient-limiting conditions in the late stationary phase, reaching 23.7 ± 2.2% PHB per CDW. These data are one of the highest reported for photosynthetic production of PHB by cyanobacteria, moreover using urban wastewater in pilot-scale cultivation which multiplies the potential of sustainable cultivation approaches. Contamination by grazers (Poterioochromonas malhamensis) was managed by culturing Synechocystis in a highly alkaline environment (pH about 10.5) which did not significantly affect the culture growth. Furthermore, the strain MT_a24 showed significant wastewater nutrient remediation removing about 72% of nitrogen and 67% of phosphorus. These trials demonstrate that the photosynthetic production of PHB by Synechocystis sp. PCC6714 MT_a24 in the outdoor thin-layer bioreactor using urban wastewater and ambient carbon dioxide. It shows a promising approach for the cost-effective and sustainable production of biodegradable carbon-negative plastics. KEY POINTS: • High PHB production by cyanobacteria in outdoor raceway pond • Urban wastewater used as a sole source of nutrients for phototrophic growth • Potential for cost-effective and sustainable production of biodegradable plastics.

Solar bioreactors used for the industrial production of microalgae.

Microalgae are excellent sources of biomass containing several important compounds for human and animal nutrition-proteins, lipids, polysaccharides, pigments and antioxidants as well as bioactive secondary metabolites. In addition, they have a great biotechnological potential for nutraceuticals, and pharmaceuticals as well as for CO2 sequestration, wastewater treatment, and potentially also biofuel and biopolymer production. In this review, the industrial production of the most frequently used microalgae genera-Arthrospira, Chlorella, Dunaliella, Haematococcus, Nannochloropsis, Phaeodactylum, Porphyridium and several other species is discussed as concerns the applicability of the most widely used large-scale systems, solar bioreactors (SBRs)-open ponds, raceways, cascades, sleeves, columns, flat panels, tubular systems and others. Microalgae culturing is a complex process in which bioreactor operating parameters and physiological variables closely interact. The requirements of the biological system-microalgae culture are crucial to select the suitable type of SBR. When designing a cultivation process, the phototrophic production of microalgae biomass, it is necessary to employ SBRs that are adequately designed, built and operated to satisfy the physiological requirements of the selected microalgae species, considering also local climate. Moreover, scaling up microalgae cultures for commercial production requires qualified staff working out a suitable cultivation regime. KEY POINTS: • Large-scale solar bioreactors designed for microalgae culturing. • Most frequently used microalgae genera for commercial production. • Scale-up requires suitable cultivation conditions and well-elaborated protocols.

Photosynthesis and biochemical characterization of the green alga Chlamydopodium fusiforme (Chlorophyta) grown in a thin-layer cascade.

Photosynthesis, growth and biochemical composition of the biomass of the freshwater microalga Chlamydopodium fusiforme cultures outdoors in a thin-layer cascade were investigated. Gross oxygen production measured off-line in samples taken from the outdoor cultures was correlated with the electron transport rate estimated from chlorophyll a fluorescence measurements. According to photosynthesis measurements, a mean of 38.9 ± 10.3 mol of photons were required to release one mole of O2, which is 4.86 times higher than the theoretical value (8 photons per 1 O2). In contrast, according to the fluorescence measurements, a mean of 11.7 ± 0.74 mol of photons were required to release 1 mol of O2. These findings indicate that fluorescence-based photosynthesis rates may not be fully replace oxygen measurements to evaluate the performance of an outdoor culture. Daily gross biomass productivity was 0.3 g DW L-1 day-1 consistently for 4 days. Biomass productivity was strongly affected by the suboptimal concentration at which the culture was operated and by the respiration rate, as the substantial volume of culture was kept in the dark (about 45% of the total volume). As the cells were exposed to excessive light, the photosynthetic activity was mainly directed to the synthesis of carbohydrates in the biomass. In the morning, carbohydrate content decreased because of the dark respiration. Per contra, protein content in the biomass was lower at the end of the day and higher in the morning due to carbohydrate consumption by respiration. The data gathered in these trials are important for the future exploitation of Chlamydopodium fusiforme as a potential novel species in the field of microalgae for the production of bio-based compounds.

Photosynthetic performance of Chlamydopodium (Chlorophyta) cultures grown in outdoor bioreactors.

The microalga Chlamydopodium fusiforme MACC-430 was cultured in two types of outdoor pilot cultivation units-a thin-layer cascade (TLC) and a raceway pond (RWP) placed in a greenhouse. This case study aimed to test their potential suitability for cultivation scale-up to produce biomass for agriculture purposes (e.g., as biofertilizer or biostimulant). The culture response to the alteration of environmental conditions was evaluated in "exemplary" situations of good and bad weather conditions using several photosynthesis measuring techniques, namely oxygen production, and chlorophyll (Chl) fluorescence. Validation of their suitability for online monitoring in large-scale plants has been one of the objectives of the trials. Both techniques were found fast and robust reliable to monitor microalgae activity in large-scale cultivation units. In both bioreactors, Chlamydopodium cultures grew well in the semi-continuous regime using daily dilution (0.20-0.25 day-1). The biomass productivity calculated per volume was significantly (about 5 times) higher in the RWPs compared to the TLCs. The measured photosynthesis variables showed that the build-up of dissolved oxygen concentration in the TLC was higher, up to 125-150% of saturation (%sat) as compared to the RWP (102-104%sat). As only ambient CO2 was available, its shortage was indicated by a pH increase due to photosynthetic activity in the thin-layer bioreactor at higher irradiance intensities. In this setup, the RWP was considered more suitable for scale-up due to higher areal productivity, lower construction and maintenance costs, the smaller land area required to maintain large culture volumes, as well as lower carbon depletion and dissolved oxygen build-up. KEY POINTS: • Chlamydopodium was grown in both raceways and thin-layer cascades in pilot-scale. • Various photosynthesis techniques were validated for growth monitoring. • In general, raceway ponds were evaluated as more suitable for cultivation scale-up.

Impact of photobioreactor design on microalgae-bacteria communities grown on wastewater: Differences between thin-layer cascade and thin-layer raceway ponds.

Thin-layer (TL) photobioreactors (PBRs) are characterised by high productivity. However, their use is limited to lab/pilot-scale, and a deeper level of characterisation is needed to reach industrial scale and test the resistance of multiple microalgae. Here, the performance and composition of eight microalgal communities cultivated in the two main TLs design (thin-layer cascade (TLC) and thin-layer raceway pond (RW)) were investigated through Illumina sequencing. Chlorella vulgaris showed robustness in both designs and often acted as an "invasive" species. Inoculum and reactor type brought variability. Eukaryotic microalgae inocula led to a more robust and stable community (higher similarity), however, RWs were characterised by a higher variability and did not favour the eukaryotic microalgae. The only cyanobacterial inoculum, Nostoc piscinale, was maintained, however the community was variable between designs. The reactor design had an effect on the N cycle with the TLC and RW configurations, enhancing nitrification and denitrification respectively.

Photosynthesis Monitoring in Microalgae Cultures Grown on Municipal Wastewater as a Nutrient Source in Large-Scale Outdoor Bioreactors.

Microalgae cultures were used for a WW treatment to remediate nutrients while producing biomass and recycling water. In these trials, raceway ponds (RWPs; 1 and 0.5 ha) were located next to a municipal (WW) treatment plant in Mérida, Spain. The ponds were used for continuous, all-year-round microalgae production using WW as a source of nutrients. Neither CO2 nor air was supplied to cultures. The objective was to validate photosynthesis monitoring techniques in large-scale bioreactors. Various in-situ/ex-situ methods based on chlorophyll fluorescence and oxygen evolution measurements were used to follow culture performance. Photosynthesis variables gathered with these techniques were compared to the physiological behavior and growth of cultures. Good photosynthetic activity was indicated by the build-up of dissolved oxygen concentration up to 380% saturation, high photochemical yield (Fv/Fm = 0.62-0.71), and relative electron transport rate rETR between 200 and 450 µmol e- m-2 s-1 at midday, which resulted in biomass productivity of about 15-25 g DW m-2 day-1. The variables represent reliable markers reflecting the physiological status of microalgae cultures. Using waste nutrients, the biomass production cost can be significantly decreased for abundant biomass production in large-scale bioreactors, which can be exploited for agricultural purposes.

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.

Characterization of an aerated submerged hollow fiber ultrafiltration device for efficient microalgae harvesting.

The present work characterizes a submerged aerated hollow fiber polyvinylidene fluorid (PVDF) membrane (0.03 µm) device (Harvester) designed for the ultrafiltration (UF) of microalgae suspensions. Commercial baker's yeast served as model suspension to investigate the influence of the aeration rate of the hollow fibers on the critical flux (CF, J c) for different cell concentrations. An optimal aeration rate of 1.25 vvm was determined. Moreover, the CF was evaluated using two different Chlorella cultures (axenic and non-axenic) of various biomass densities (0.8-17.5 g DW/L). Comparably high CFs of 15.57 and 10.08 L/m/2/h were measured for microalgae concentrations of 4.8 and 10.0 g DW/L, respectively, applying very strict CF criteria. Furthermore, the J c-values correlated (negative) linearly with the biomass concentration (0.8-10.0 g DW/L). Concentration factors between 2.8 and 12.4 and volumetric reduction factors varying from 3.5 to 11.5 could be achieved in short-term filtration, whereat a stable filtration handling biomass concentrations up to 40.0 g DW/L was feasible. Measures for fouling control (aeration of membrane fibers, periodic backflushing) have thus been proven to be successful. Estimations on energy consumption revealed very low energy demand of 17.97 kJ/m3 treated microalgae feed suspension (4.99 × 10-3 kWh/m3) and 37.83 kJ/kg treated biomass (1.05 × 10-2 kWh/kg), respectively, for an up-concentration from 2 to 40 g DW/L of a microalgae suspension.

Impact of glgA1, glgA2 or glgC overexpression on growth and glycogen production in Synechocystis sp. PCC 6803.

Low production rates are still one limiting factor for the industrial climate-neutral production of biovaluable compounds in cyanobacteria. Next to optimized cultivation conditions, new production strategies are required. Hence, the use of established molecular tools could lead to increased product yields in the cyanobacterial model organism Synechocystis sp. PCC6803. Its main storage compound glycogen was chosen to be increased by the use of these tools. In this study, the three genes glgC, glgA1 and glgA2, which are part of the glycogen synthesis pathway, were combined with the Pcpc560 promoter and the neutral cloning site NSC1. The complete genome integration, protein formation, biomass production and glycogen accumulation were determined to select the most productive transformants. The overexpression of glgA2 did not increase the biomass or glycogen production in short-term trials compared to the other two genes but caused transformants death in long-term trials. The transformants glgA1_11 and glgC_2 showed significantly increased biomass (1.6-fold - 1.7-fold) and glycogen production (3.5-fold - 4-fold) compared to the wild type after 96 h making them a promising energy source for further applications. Those could include for example a two-stage production process, with first energy production (glycogen) and second increased product formation (e.g. ethanol).

Quorum-Sensing Signals from Epibiont Mediate the Induction of Novel Microviridins in the Mat-Forming Cyanobacterial Genus Nostoc.

The regulation of the production of oligopeptides is essential in understanding their ecological role in complex microbial communities, including harmful cyanobacterial blooms. The role of chemical communication between the cyanobacterium and the microbial community harbored as epibionts within its phycosphere is at an initial stage of research, and little is understood about its specificity. Here, we present insight into the role of a bacterial epibiont in regulating the production of novel microviridins isolated from Nostoc, an ecologically important cyanobacterial genus. Microviridins are well-known elastase inhibitors with presumed antigrazing effects. Heterologous expression and identification of specific signal molecules from the epibiont suggest the role of a quorum-sensing-based interaction. Furthermore, physiological experiments show an increase in microviridin production without affecting cyanobacterial growth and photosynthetic activity. Simultaneously, oligopeptides presenting a selective inhibition pattern provide support for their specific function in response to the presence of cohabitant epibionts. Thus, the chemical interaction revealed in our study provides an example of an interspecies signaling pathway monitoring the bacterial flora around the cyanobacterial filaments and the induction of intrinsic species-specific metabolic responses. IMPORTANCE The regulation of the production of cyanopeptides beyond microcystin is essential to understand their ecological role in complex microbial communities, e.g., harmful cyanobacterial blooms. The role of chemical communication between the cyanobacterium and the epibionts within its phycosphere is at an initial stage of research, and little is understood about its specificity. The frequency of cyanopeptide occurrence also demonstrates the need to understand the contribution of cyanobacterial peptides to the overall biological impact of cyanopeptides on aquatic organisms and vertebrates, including humans. Our results shed light on the epibiont control of microviridin production via quorum-sensing mechanisms, and we posit that such mechanisms may be widespread in natural cyanobacterial bloom community regulation.

Selenium Incorporation to Amino Acids in Chlorella Cultures Grown in Phototrophic and Heterotrophic Regimes.

Microalgae accumulate bioavailable selenium-containing amino acids (Se-AAs), and these are useful as a food supplement. While this accumulation has been studied in phototrophic algal cultures, little data exists for heterotrophic cultures. We have determined the Se-AAs content, selenium/sulfur (Se/S) substitution rates, and overall Se accumulation balance in photo- and heterotrophic Chlorella cultures. Laboratory trials revealed that heterotrophic cultures tolerate Se doses ∼8-fold higher compared to phototrophic cultures, resulting in a ∼2-3-fold higher Se-AAs content. In large-scale experiments, both cultivation regimes provided comparable Se-AAs content. Outdoor phototrophic cultures accumulated up to 400 µg g-1 of total Se-AAs and exhibited a high level of Se/S substitution (5-10%) with 30-60% organic/total Se embedded in the biomass. A slightly higher content of Se-AAs and ratio of Se/S substitution was obtained for a heterotrophic culture in pilot-scale fermentors. The data presented here shows that heterotrophic Chlorella cultures provide an alternative for Se-enriched biomass production and provides information on Se-AAs content and speciation in different cultivation regimes.

Rapid screening test to estimate temperature optima for microalgae growth using photosynthesis activity measurements.

We have worked out a rapid 1-day test based on photosynthesis measurements to estimate suitable growth temperature of microalgae cultures. To verify the proposed procedure, several microalgae-Chlorella, Nostoc, Synechocystis, Scenedesmus, and Cylindrospermum-were cultured under controlled laboratory conditions (irradiance, temperature, mixing, CO2, and nutrient supply) to find the optima of photosynthetic activity using the range between 15 and 35 °C. These activities were recorded at each temperature step after 2 h of acclimation which should be sufficient as oxygen production and the PQ cycle are regulated by fast processes. Photosynthetic activity was measured using three techniques-oxygen production/respiration, saturating pulse analysis of fluorescence quenching, and fast fluorescence induction kinetics-to estimate the temperature optima which should correspond to high growth rate. We measured all variables that might have been directly related to growth-photosynthetic oxygen evolution, maximum photochemical yield of PSII, Fv/Fm, relative electron transport rate rETRmax, and the transients Vj and Vi determined by fast fluorescence induction curves. When the temperature optima for photosynthetic activity were verified in growth tests, we found good correlation. For most of tested microalgae strains, temperature around 30 °C was found to be the most suitable at this setting. We concluded that the developed test can be used as a rapid 1-day pre-screening to estimate a suitable growth temperature of microalgae strains before they are cultured in a pilot scale.

Bioethanol production from microalgae polysaccharides.

The worldwide growing demand for energy permanently increases the pressure on industrial and scientific community to introduce new alternative biofuels on the global energy market. Besides the leading role of biodiesel and biogas, bioethanol receives more and more attention as first- and second-generation biofuel in the sustainable energy industry. Lately, microalgae (green algae and cyanobacteria) biomass has also remarkable potential as a feedstock for the third-generation biofuel production due to their high lipid and carbohydrate content. The third-generation bioethanol production technology can be divided into three major processing ways: (i) fermentation of pre-treated microalgae biomass, (ii) dark fermentation of reserved carbohydrates and (iii) direct "photo-fermentation" from carbon dioxide to bioethanol using light energy. All three technologies provide possible solutions, but from a practical point of view, traditional fermentation technology from microalgae biomass receives currently the most attention. This study mainly focusses on the latest advances in traditional fermentation processes including the steps of enhanced carbohydrate accumulation, biomass pre-treatment, starch and glycogen downstream processing and various fermentation approaches.

Development of thin-layer cascades for microalgae cultivation: milestones (review).

In this work, the key moments of the development of the so-called thin-layer cascades (TLC) for microalgae production are described. Development started at the end of the 1950s when the first generation of TLCs was set-up in former Czechoslovakia. Since, similar units for microalgae culturing, which are relatively simple, low-cost and highly productive, have been installed in a number of other countries worldwide. The TLCs are characterized by microalgae growth at a low depth (< 50 mm) and fast flow (0.4-0.5 m/s) of culture compared to mixed ponds or raceways. It guarantees a high ratio of exposed surface to total culture volume (> 100 1/m) and rapid light/dark cycling frequencies of cells which result in high biomass productivity (> 30 g/m2/day) and operating at high biomass density, > 10 g/L of dry mass (DW). In TLCs, microalgae culture is grown in the system of inclined platforms that combine the advantages of open systems-direct sun irradiance, easy heat derivation, simple cleaning and maintenance, and efficient degassing-with positive features of closed systems-operation at high biomass densities achieving high volumetric productivity. Among significant advantages of thin layer cascades compared to raceway ponds are the operation at much higher cell densities, very high daylight productivities, and the possibility to store the culture in retention tanks at night, or in unfavourable weather conditions. Concerning the limitations of TLCs, one has to consider contaminations by other microalgae that limit cultivation to robust, fast-growing strains, or those cultured in selective environments.

In vitro bioaccessibility of selenoamino acids from selenium (Se)-enriched Chlorella vulgaris biomass in comparison to selenized yeast; a Se-enriched food supplement; and Se-rich foods.

Selenium (Se) is an indispensable microelement in our diet and health issues resulting from deficiencies are well documented. Se-containing food supplements are available on the market including Se-enriched Chlorella vulgaris (Se-Chlorella) which accumulates Se in the form of Se-amino acids (Se-AAs). Despite its popular uses, data about the bioaccessibility of Se-AAs from Se-Chlorella are completely missing. In the present study, gastrointestinal digestion times were optimized and the in vitro bioaccessibility of Se-AAs in Se-Chlorella, Se-yeast, a commercially available Se-enriched food supplement (Se-supplement) and Se rich foods (Se-foods) were compared. Higher bioaccessibility was found in Se-Chlorella (∼49%) as compared to Se-yeast (∼21%), Se-supplement (∼32%) and Se-foods. The methods used in production of Se-Chlorella biomass were also investigated. We found that disintegration increased bioaccessibility whereas the drying process had no effect. Similarly, temperature treatment by microwave oven also increased bioaccessibility whereas boiling water did not.

The synergistic effect of Selenium (selenite, -SeO32-) dose and irradiance intensity in Chlorella cultures.

Microalgae are able to metabolize inorganic selenium (Se) to organic forms (e.g. Se-proteins); nevertheless at certain Se concentration culture growth is inhibited. The aim of this work was to confirm the hypothesis that the limit of Se tolerance in Chlorella cultures is related to photosynthetic performance, i.e. depends on light intensity. We studied the relation between the dose and irradiance to find the range of Se tolerance in laboratory and outdoor cultures. At low irradiance (250 µmol photons m-2 s-1), the daily dose of Se below 8.5 mg per g of biomass (<20 µM) partially stimulated the photosynthetic activity (relative electron transport rate) and growth of Chlorella cultures (biomass density of ~1.5 g DW L-1) compared to the control (no Se added). It was accompanied by substantial Se incorporation to microalgae biomass (~0.5 mg Se g-1 DW). When the Se daily dose and level of irradiance were doubled (16 mg Se g-1 DW; 500 µmol photons m-2 s-1), the photosynthetic activity and growth were stimulated for several days and ample incorporation of Se to biomass (7.1 mg g-1 DW) was observed. Yet, the same Se daily dose under increased irradiance (750 µmol photons m-2 s-1) caused the synergistic effect manifested by significant inhibition of photosynthesis, growth and lowered Se incorporation to biomass. In the present experiments Chl fluorescence techniques were used to monitor photosynthetic activity for determination of optimal Se doses in order to achieve efficient incorporation without substantial inhibition of microalgae growth when producing Se-enriched biomass.

Supplementation with sodium selenite and selenium-enriched microalgae biomass show varying effects on blood enzymes activities, antioxidant response, and accumulation in common barbel (Barbus barbus).

Yearling common barbel (Barbus barbus L.) were fed four purified casein-based diets for 6 weeks in outdoor cages. Besides control diet, these were supplemented with 0.3 mg kg(-1) dw selenium (Se) from sodium selenite, or 0.3 and 1.0 mg kg(-1) from Se-enriched microalgae biomass (Chlorella), a previously untested Se source for fish. Fish mortality, growth, Se accumulation in muscle and liver, and activity of selected enzymes in blood plasma, muscle, liver, and intestine were evaluated. There was no mortality, and no differences in fish growth, among groups. Se concentrations in muscle and liver, activity of alanine aminotransferase and creatine kinase in blood plasma, glutathione reductase (GR) in muscle, and GR and catalase in muscle and liver suggested that selenium from Se-enriched Chlorella is more readily accumulated and biologically active while being less toxic than sodium selenite.

Resistance of common carp (Cyprinus carpio L.) to oxidative stress after chloramine-T treatment is increased by microalgae carotenoid-rich diet.

OBJECTIVES: In fish aquaculture, disinfectants are used against bacterial and protozoal infections. These compounds cause oxidative stress that may stimulate the generation of reactive oxygen species, and subsequently the alteration in antioxidant systems of exposed organisms. Antioxidants like carotenoids present in microalgae increase carp resistance to oxidative stress after chemical treatment. DESIGN: The aim of these experiments was to prove increased resistance of common carp (Cyprinus carpio L.) juveniles fed on experimental diets with microalgae biomass supplement (Algadiets) to oxidative stress caused by a disinfectant chloramine-T. In indoor experiments fish were fed on laboratory-prepared extruded diets containing supplement of Chlorella spp. (cf. C. vulgaris Beijerinck) biomass which contains antioxidants (carotenoids) like lutein. The young-of-the-year-old fish were acclimatized and fed on basal diet (control group) and the on diets containing 1, 2, 5 and 10% (w/w) of spray-dried Chlorella biomass (Algadiet 1, 2, 5 and 10) for 14 days followed by 6 weeks. Consequently, fish were treated daily with chloramine-T (Chl-T) at concentration of 10 mg x l(-1) for 1 h in three consecutive days. After this treatment, the indices of oxidative stress and antioxidant enzyme activity were assayed in fish gill, muscle and hepatopancreas. RESULTS: The fish fed on different Algadiets had increased antioxidant enzyme activities of glutathione peroxidase, glutathione reductase and catalase in flesh after the exposure to Chl-T. Higher activities of superoxide dismutase, glutathione peroxidase and glutathione reductase were also observed in the hepatopancreas in all tested concentrations compared to the control group fed on the basal diet. The increased production and activity of antioxidant enzymes confirmed improved protection ability of fish tissues against oxidative damage when microalgae biomass was supplemented to the fish diet which was more pronounced by higher microalgae supplement in Algadiet 5 and 10 where the content of carotenoids was 105 mg and 214 mg per kilogram of fed, respectively. CONCLUSION: The results show the positive effect of carotenoids from microalgae biomass to maintain the antioxidant capacity which increases resistance of fish to oxidative stress.

Detection of photosynthetic herbicides: algal growth inhibition test vs. electrochemical photosystem II biosensor.

We compared a novel PSII-biosensor assay with a standard algal growth inhibition test for detection of photosynthetic herbicides--diuron, atrazine and isoproturon in liquid samples. To evaluate the convenience and sensitivity, values of the parameters EC50 and LOD and the duration of assays were compared. The biosensor assay was made with an electrochemical biosensor toxicity analyser with immobilised Photosystem II (PSII) complex. Using the PSII-biosensor assay, higher sensitivity (LOD) to herbicides (10(-8)-10(-9)M) was achieved as compared to standard algal growth inhibition tests (about 10(-7)M). The results of both assays showed a good correlation as concerns their EC50 values while the interval of detectable concentrations is about twice wider for PSII-biosensor. A proposed measurement protocol includes the reference standard of phytotoxicity (RSP). The main advantage of the PSII-biosensor assay is that it can be completed in about 1h and is by 1-2 orders more sensitive than standard algal growth inhibition test, which takes 72 h.