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).

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.

Quantification of methionine and selenomethionine in biological samples using multiple reaction monitoring high performance liquid chromatography tandem mass spectrometry (MRM-HPLC-MS/MS).

Quantification of selenated amino-acids currently relies on methods employing inductively coupled plasma mass spectrometry (ICP-MS). Although very accurate, these methods do not allow the simultaneous determination of standard amino-acids, hampering the comparison of the content of selenated versus non-selenated species such as methionine (Met) and selenomethionine (SeMet). This paper reports two approaches for the simultaneous quantification of Met and SeMet. In the first approach, standard enzymatic hydrolysis employing Protease XIV was applied for the preparation of samples. The second approach utilized methanesulfonic acid (MA) for the hydrolysis of samples, either in a reflux system or in a microwave oven, followed by derivatization with diethyl ethoxymethylenemalonate. The prepared samples were then analyzed by multiple reaction monitoring high performance liquid chromatography tandem mass spectrometry (MRM-HPLC-MS/MS). Both approaches provided platforms for the accurate determination of selenium/sulfur substitution rate in Met. Moreover the second approach also provided accurate simultaneous quantification of Met and SeMet with a low limit of detection, low limit of quantification and wide linearity range, comparable to the commonly used gas chromatography mass spectrometry (GC-MS) method or ICP-MS. The novel method was validated using certified reference material in conjunction with the GC-MS reference method.

Thin-layer chromatography combined with diode laser thermal vaporization inductively coupled plasma mass spectrometry for the determination of selenomethionine and selenocysteine in algae and yeast.

In this work we present a simple and cost-effective approach for the determination of selenium species in algae and yeast biomass, based on a combination of thin-layer chromatography (TLC) with diode laser thermal vaporization inductively coupled plasma mass spectrometry (DLTV ICP MS). Extraction of freeze-dried biomass was performed in 4M methanesulphonic acid and the selenium species were vaporized from cellulose TLC plates employing a continuous-wave infrared diode laser with power up to 4 W using a simple laboratory-built apparatus. Selenomethionine and selenocysteine were quantified with limits of detection 3 µg L-1 in a Se-enriched microalgae Chlorella vulgaris and yeast certified reference material SELM-1. Results delivered by TLC-DLTV ICP MS were consistent with those obtained by a routine coupling of high-performance liquid chromatography (HPLC) to ICP MS. In addition, the TLC approach is capable of analyzing extract containing even undiluted crude hydrolysates that could damage HPLC columns.

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.