Relation between light absorption measured by the quantitative filter technique and attenuation of <Emphasis Type="Italic">Chlorella fusca</Emphasis> cultures of different cell densities: application to estimate the absolute electron transport rate (ETR)

In order to estimate microalgal carbon assimilation or production of Chlorella fusca cultures based on electron transport rate (ETR) as in vivo chlorophyll a fluorescence, it is necessary to determine the photosynthetic yield and the absorbed quanta by measuring the incident irradiance and the fraction of absorbed light, i.e., absorptance or absorption coefficient in the photosynthetic active radiation (PAR) region of the spectra. Due to difficulties associated with the determination of light absorption, ETR is commonly expressed as relative units (rETR) although this is not a good estimator of the photosynthetic production since photobiological responses depend on the absorbed light. The quantitative filter technique (QFT) is commonly used to measure the absorbed quanta of cells retained on a filter (AbQ_f) as estimator of the absorbed quanta of cell suspensions (AbQ_s) determined by using integrating spheres. In this study, light attenuation of thin-layer cell suspensions is determined by using a measuring system designed to reduce the scattering. The light attenuation is related to the absorptance as the fraction of absorbed light by both indoor and outdoor C. fusca cultures of different cell densities. A linear relation between AbQ_f and AbQ_s (R ²?=?0.9902, p?<?0.01) was observed, AbQ_f?=?1.98?×?AbQ_s, being 1.98 an amplification factor to convert AbQ_s values into AbQ_f ones. On the other hand, depending on the culture system, the convenience of the use of the absorptance, light absorption or specific light absorption coefficient expressed per area (thin-layer cascade or flat panel cultivators), volume (cylindrical and tubular photobioreactors), or chlorophyll units (any type of cultivation system) is discussed. The procedure for the measurement of light absorption presented in this study for C. fusca could be applied in other phytoplankton groups. The absorbed quanta as determined in this study can be used to express absolute ETR instead of relative ETR, since the first one provides much more relevant photobiological information of microalgae culture systems.     

Effects of <Emphasis Type="Italic">Ascophyllum nodosum</Emphasis> extract on growth and antioxidant defense systems of two freshwater microalgae

Microalgae in genus Chlorella and Scenedesmus are common in aquatic ecosystems and are widely used for various studies on algal growth and applications. Macroalgae may play an important role for control of microalgal growth, attributable to their rich content of bioactive compounds. In this study, the brown seaweed Ascophyllum nodosum was extracted with 70% acetone and the extract was used to treat the green microalgae, Chlorella vulgaris and Scenedesmus sp. Cell density and chlorophyll a concentration were used as growth indexes to evaluate the effects of A. nodosum extract (ANE) on the microalgae. The ANE with concentrations > 1% exhibited significant capability of inhibition of the growth of microalgae by over 80%. On the contrary, 1% ANE caused varying degrees of acceleration of cell proliferation and chlorophyll a synthesis in C. vulgaris and Scenedesmus sp., respectively. Analysis of antioxidant activities of the enzymes superoxide dismutase (SOD) and catalase (CAT) revealed the impact of ANE on the antioxidant defense system of the microalgae. The SOD and CAT activities were significantly depressed by high concentrations (> 2%) ANE, while a slight increase of the enzyme activities was observed with 1% ANE at the early period, which could be correlated to the growth response. Therefore, the mechanism of microalgae control could be related to the interaction between the ANE and the antioxidant defense systems. Phlorotannins are proposed as the principal algistatic components in the ANE which could be utilized in controlling microalgae growth.     

Biochemical and physiological responses of <Emphasis Type="Italic">Selenastrum gracile</Emphasis> (Chlorophyceae) acclimated to different phosphorus concentrations

Algae are able to adjust their metabolism according to their environment, maximizing growth rate and production of biomolecules under adverse conditions such as pulses of excess of a contaminant or limitation of a nutrient. In order to evaluate the effects of phosphorus (P) availability on the biochemical composition of the freshwater microalga Selenastrum gracile, we acclimated the microalgae to different phosphorus concentrations. After acclimation, exponentially growing cells were inoculated and after 120 h, samples were processed for the determination of carbohydrate, lipid, fatty acid, chlorophyll, cell density, growth rate, and dry weight. Cell density, growth rate, and dry weight decreased with less P, while chlorophyll a, carbohydrates, lipids, and fatty acids per cell increased under P limitation. According to our lipid class and fatty acid results, algae alter their metabolism and membrane configuration to avoid more structural or metabolic damage under limitation, especially at 23 μmol P L^?1. The most sensitive parameters under P limitation were chlorophyll a, lipids, and poly- and monounsaturated fatty acids. The changes in fatty acids contributed to the fluorescence and photosynthesis changes under P limitation, and they occurred before changes were detected in other parameters, such as growth rate. Furthermore, we suggest that prior acclimation to different P affected microalgal physiology and metabolism.     

Development of an alcohol-inducible gene expression system for recombinant protein expression in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

Microalgae have been widely considered for the production of valuable products, such as lipid-based biofuel, value-added pigments, and anti-photo aging reagents. More recently, microalgae have been considered an alternative host for recombinant protein production because of their economic benefits and ecofriendly characteristics. Additionally, many microalgal strains identified to date are generally recognized as safe (GRAS); therefore, the use of microalgae-based technology is promising. However, basic studies on the genetic engineering of microalgae are rare, despite their importance. Particularly, inducible promoter systems that can be applied for strain engineering or recombinant protein production are rarely studied; hence, a number of challenging issues remain unsolved. Therefore, in this study, we focused on the development of a convenient and compact-inducible promoter system that can be used in microalgae. Based on previous success with plant systems, we employed the alcohol-inducible AlcR-P_alcA system, which originates from the filamentous fungus, Aspergillus nidulans. This system comprises only two components, a regulatory protein, AlcR, and an inducible promoter, P_alcA. Therefore, construction and transformation of the gene cassettes can be easily performed. Ethanol-dependent gene expression was observed in the transformants with no significant growth retardation or inducer consumption observed in the cells cultivated under optimized conditions.     

Microalgal industry in China: challenges and prospects

Over the past 15 years, China has become the major producer of microalgal biomass in the world. Spirulina (Arthrospira) is the largest microalgal product by tonnage and value, followed by Chlorella, Dunaliella, and Haematococcus, the four main microalgae grown commercially. China’s production is estimated at about two-thirds of global microalgae biomass of which roughly 90 % is sold for human consumption as human nutritional products (‘nutraceuticals’), with smaller markets in animal feeds mainly for marine aquaculture. Research is also ongoing in China, as in the rest of the world, for other high-value as well as commodity microalgal products, from pharmaceuticals to biofuels and CO₂ capture and utilization. This paper briefly reviews the main challenges and potential solutions for expanding commercial microalgae production in China and the markets for microalgae products. The Chinese Microalgae Industry Alliance (CMIA), a network founded by Chinese microalgae researchers and commercial enterprises, supports this industry by promoting improved safety and quality standards, and advancement of technologies that can innovate and increase the markets for microalgal products. Microalgae are a growing source of human nutritional products and could become a future source of sustainable commodities, from foods and feeds, to, possibly, fuels and fertilizers.     

Effects of outdoor cultures on the growth and lipid production of <Emphasis Type="Italic">Phaeodactylum tricornutum</Emphasis> using closed photobioreactors

One of the principal challenges for large scale production of microalgae is the high costs of biomass production. Aiming for minimize this problem, microalgal biodiesel production should focus on outdoors cultures, using available solar light and allowing lower energy cost process. Testing species that proved to be common and easy to culture may be a good approach in this process. The present work reports indoor-outdoor cultures of Phaeodactylum tricornutum using different bioreactors types, using cell growth, biochemical composition, and the profiles of the fatty acids produced as the parameters to test the optimization processes. The results show that the use of outdoor cultures is a good choice to obtain P. tricornutum biomass with a good potential for biodiesel production. The microalgae produced reached better growth efficiency, major lipid content and showed an increment in the percentage of saturated fatty acids (required on the biodiesel production) respect indoor cultures. These results are important to show the relevance of using outdoor cultures as a way to improve the efficiency and the energetic balance of the biodiesel production with P. tricornutum algae.     

High biomass production and CO<Subscript>2</Subscript> fixation from biogas by <Emphasis Type="Italic">Chlorella</Emphasis> and <Emphasis Type="Italic">Scenedesmus</Emphasis> microalgae using tequila vinasses as culture medium

Tequila vinasses (TVs) generated during Tequila production are brown liquid residues rich in nutrients. The nutrient content of agro-industrial effluents represents an excellent resource to support low-cost biomass production of microalgae; nonetheless, it is crucial to select the suitable microalgal strain to attain the highest biomass production in each residue used. In this study, biomass production, CO₂ fixation from biogas, and cell compound accumulation by Chlorella vulgaris U162, Chlorella sp., Scenedesmus obliquus U169, and Scenedesmus sp. using biodigested and filtered TVs as culture medium were evaluated and compared with the conventional microalgal culture media, C30, BG-11, Bold 3N, and Bristol. The four microalgae evaluated attained the highest biomass production and CO₂ fixation rate cultured in both residues, accumulating mainly carbohydrates and proteins although the most appropriate microalga to be cultured in TVs was Chlorella sp., recording 2.30 g L^?1. Moreover, the nutrient ratio of filtered TVs was ideal to support biomass production while biodigested TVs need to be supplemented with nitrogen. Overall, these results demonstrated that tequila vinasses are an excellent resource to support high and quick biomass production of microalgae, which can be used to obtain biofuels as ethanol, biogas, and supplement food depicting an extra benefit during the appropriate disposal of this residue.     

Comparative analysis of photosynthetic and respiratory parameters in the psychrophilic unicellular green alga <Emphasis Type="Italic">Koliella antarctica</Emphasis>, cultured in indoor and outdoor photo-bioreactors

Effect of temperatures and illumination of temperate winter on photosynthesis and respiration was studied in the psychrophilic microalgae, Koliella antarctica (Trebouxiophyceae). Outdoor and indoor algal cultures were compared. Photosynthetic as well as respiration rates increased as light and temperature increased, until 35 °C, more in outdoor than in indoor cells, in agreement with the calculated Q₁₀ values. K. antarctica showed important strategy mechanisms of adaption to the several temperature and light conditions. These significant photo-acclimation and thermo-acclimation abilities make it possible to cultivate Koliella for different uses, under less expensive outdoor conditions. Therefore, K. antarctica shows important strategy mechanisms of adaption to various temperature and light conditions; moreover, by varying the culture conditions, it is possible to modulate and optimize the growth and accordingly the biomass production. This is a very interesting point since it has been proved that this microalga is a promising potential source of functional ingredients, such as polyunsaturated fatty acids and carotenoids, suitable for industrial purposes.     

Kinetic characterization of a novel acid ectophosphatase from <Emphasis Type="Italic">Enterobacter asburiae</Emphasis>

Expression of acid ectophosphatase by Enterobacter asburiae, isolated from Cattleya walkeriana (Orchidaceae) roots and identified by the 16S rRNA gene sequencing analysis, was strictly regulated by phosphorus ions, with its optimal activity being observed at an inorganic phosphate concentration of 7 mM. At the optimum pH 3.5, intact cells released p-nitrophenol at a rate of 350.76 ± 13.53 nmol of p-nitrophenolate (pNP)/min/10⁸ cells. The membrane-bound enzyme was obtained by centrifugation at 100,000 × g for 1 h at 4°C. p-Nitrophenylphosphate (pNPP) hydrolysis by the enzyme follows “Michaelis-Menten” kinetics with V = 61.2 U/mg and K_0.5 = 60 μM, while ATP hydrolysis showed V = 19.7 U/mg, K_0.5 = 110 μM, and n_H = 1.6 and pyrophosphate hydrolysis showed V = 29.7 U/mg, K_0.5 = 84 μM, and n_H = 2.3. Arsenate and phosphate were competitive inhibitors with Ki = 0.6 mM and K_i = 1.8 mM, respectively. p-Nitrophenyl phosphatase (pNPPase) activity was inhibited by vanadate, while p-hydroxymercuribenzoate, EDTA, calcium, copper, and cobalt had no inhibitory effects. Magnesium ions were stimulatory (K_0.5 = 2.2 mM and n_H = 0.5). Production of an acid ectophosphatase can be a mechanism for the solubilization of mineral phosphates by microorganisms such as Enterobacter asburiae that are versatile in the solubilization of insoluble minerals, which, in turn, increases the availability of nutrients for plants, particularly in soils that are poor in phosphorus.     

Effect of high light intensity on the photosynthetic apparatus of two hybrid lines of <Emphasis Type="Italic">Paulownia</Emphasis> grown on soils with different salinity

The objective of this investigation was to evaluate the simultaneous action of light stress and salinity. Pulse amplitude modulated chlorophyll fluorescence, P₇₀₀ redox state, and pigment analysis were used to assess the impact of high light intensity on Paulownia tomentosa × fortunei and Paulownia elongata × elongata grown on soils with different salinity. It was found that light stress reduced the amount of pigments and the efficiency of photochemical energy conversion, inhibited the maximum and the effective quantum yields of PSII photochemistry, decreased photochemical quenching and photosynthetic rate. Data also showed influence on the primary quinone acceptor (Q_A) reoxidation, which led to the restriction of the electron flow from Q_A to plastoquinone and stimulation of the cyclic electron flow. The possible reasons for the increased effects of the light stress under conditions of high salt concentration in soil for Paulownia tomentosa × fortunei are discussed.     

Examination of chlorophyll fluorescence in sulfur-deprived cells of <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

Pulse amplitude modulation fluorimetry was used to assess chlorophyll fluorescence parameters in Chlamydomonas reinhardtii cells during sulfur deprivation. A significant (fourfold) increase in the chlorophyll fluorescence yield (parameters F ₀ and F _m) normalized to the chlorophyll concentration was shown for deprived cells. The chlorophyll content did not change during the deprivation experiments. An analysis of nonphotochemical quenching of chlorophyll fluorescence indicated a considerable modification of the energy deactivation pathways in photosystem II (PSII) of sulfur-deprived cells. For example, starved cells exhibited a less pronounced pH-dependent quenching of excited states and a higher thermal dissipation of excess light energy in the reaction centers of PSII. It was also shown that the photosynthetic apparatus of starved cells is primarily in state 2 and that back transition to state 1 is suppressed. However, these changes cannot cause the discovered elevation of chlorophyll fluorescence intensity (F ₀ and F _m) in the cells under sulfur limitation. The observed increase in the chlorophyll fluorescence intensity under sulfur deprivation may be due to partial dissociation of peripheral light-harvesting complexes from the reaction centers of PSII or a malfunction of the dissipative cycle in PSII, involving cytochrome b ₅₅₉.     

Truncated light-harvesting chlorophyll antenna size in <Emphasis Type="Italic">Chlorella vulgaris</Emphasis> improves biomass productivity

Microalgae have been proposed as eco-friendly feedstocks for biodiesel production, because they accumulate large amounts of lipids and increase their biomass through photosynthesis. However, the photosynthetic efficiency of microalgae is too low for this strategy to be economically feasible. In an effort to overcome this issue, random mutants with reduced chlorophyll antenna size were generated by ethyl methanesulfonate (EMS)-mediated mutagenesis of Chlorella vulgaris. The antenna size mutant, herein designated E5, exhibited 56.5 and 75.8 % decreases in chlorophyll a and b contents, respectively, with significant reductions in the expression levels of peripheral light-harvesting antenna proteins in photosystem II. The saturated photosynthetic activity and electron transport rate of the E5 mutant were significantly higher and also showed reduced non-photochemical quenching (NPQ), compared to those of the wild type. Consequentially, the E5 mutant cultures achieved 44.5 % improvement in biomass productivity under high light (200 μmol photons m^?2 s^?1). These results suggest that improving the photosynthetic efficiency of microalgae could greatly enhance their biomass production, and such mutant strains can be applicable for large-scale outdoor cultivation which is typically exposed to high light intensity.     

The combined effects of blue light and dilution rate on lipid class and fatty acid composition of <Emphasis Type="Italic">Tisochrysis lutea</Emphasis>

The lipid class and the fatty acid compositions of microalgae highly influence bivalve larval and post-larval development. Light is an essential environmental factor for microalgal culture, and quantity and quality of light may induce changes in the biochemical composition of the algae. The objective of this study was to investigate the effect of light spectrum (blue vs. white light) on lipid class and fatty acid compositions of Tisochrysis lutea cultured in a chemostat. Two different dilution rates (D) were assayed for each light spectrum: 0.2 and 0.7 day^?1. Triacylglycerol (TAG), sterol, and hydrocarbon (HC) content increased sharply at low D. The proportion of alkenones was significantly reduced under blue light. Polyunsaturated fatty acids (PUFA), and particularly n-3 PUFA, content in phospholipids (PL) increased under blue light compared to white light at low D. Thus, blue light raised 22:6(n-3) levels in total lipids of T. lutea at low D. The cultivation of T. lutea in a chemostat at low D under blue light may improve nutritional value as feed for bivalve larvae by modifying the PUFA profile, especially increasing 22:6(n-3).     

Diel oscillation in the optical activity of carotenoids in the absorption spectrum of <Emphasis Type="Italic">Nannochloropsis</Emphasis>

In this paper we show that the absorption spectrum of the microalgae Nannochloropsis oceanica exhibits changes in response to the modulation of incident light. A model was used to analyze the contribution of different active pigments to the total absorption in the photosynthetically active radiation region and suggested consistent diel oscillations in the optical activity of carotenoids.     

Detection and imaging of lipids of <Emphasis Type="Italic">Scenedesmus obliquus</Emphasis> based on confocal Raman microspectroscopy

In this study, confocal Raman microspectroscopy was used to detect lipids in microalgae rapidly and non-destructively. Microalgae cells were cultured under nitrogen deficiency. The accumulation of lipids in Scenedesmus obliquus was observed by Nile red staining, and the total amount of lipids accumulated in the cells was measured by gravimetric method. The signals from different microalgae cells were collected by confocal Raman microspectroscopy to establish a prediction model of intracellular lipid content, and surface scanning signals for drawing pseudo color images of lipids distribution. The images can show the location of pyrenoid and lipid accumulation in cells. Analyze Raman spectrum data and build PCA-LDA model using four different bands (full bands, pigments, lipids, and mixed features). Models of full bands or pigment characteristic bands were capable of identifying S. obliquus cells under different nitrogen stress culture time. The prediction accuracy of model of lipid characteristic bands is relatively low. The correlation between the fatty acid content measured by the gravimetric method and the integral Raman intensity of the oil characteristic peak (1445 cm^?1) measured by Raman spectroscopy was analyzed. There was significant correlation (R ² = 0.83), which means that Raman spectroscopy is applicable to semi-quantitative detection of microalgal lipid content.     

Chlorophylls <Emphasis Type="Italic">d</Emphasis> and <Emphasis Type="Italic">f</Emphasis> and their role in primary photosynthetic processes of cyanobacteria

The finding of unique Chl d- and Chl f-containing cyanobacteria in the last decade was a discovery in the area of biology of oxygenic photosynthetic organisms. Chl b, Chl c, and Chl f are considered to be accessory pigments found in antennae systems of photosynthetic organisms. They absorb energy and transfer it to the photosynthetic reaction center (RC), but do not participate in electron transport by the photosynthetic electron transport chain. However, Chl d as well as Chl a can operate not only in the light-harvesting complex, but also in the photosynthetic RC. The long-wavelength (Q_y) Chl d and Chl f absorption band is shifted to longer wavelength (to 750 nm) compared to Chl a, which suggests the possibility for oxygenic photosynthesis in this spectral range. Such expansion of the photosynthetically active light range is important for the survival of cyanobacteria when the intensity of light not exceeding 700 nm is attenuated due to absorption by Chl a and other pigments. At the same time, energy storage efficiency in photosystem 2 for cyanobacteria containing Chl d and Chl f is not lower than that of cyanobacteria containing Chl a. Despite great interest in these unique chlorophylls, many questions related to functioning of such pigments in primary photosynthetic processes are still not elucidated. This review describes the latest advances in the field of Chl d and Chl f research and their role in primary photosynthetic processes of cyanobacteria.     

Enrichment as a screening method for a high-growth-rate microalgal strain under continuous cultivation system

Microalgae are a promising feedstock for renewable biodiesel production. High productivity of biodiesel production from microalgae is directly related to growth rate as well as lipid content of cells. In the present study, an enrichment process in a continuous cultivation system was developed to screen a high-growth-rate microalga from a mixed culture of microalgal species; Chlorella vulgaris, Chlorella protothecoides, and Chlamydomonas reinhardtii were used as test organisms for our experiments. The time-dependent washout of mixed microalgal pool was executed to successfully enrich the C. reinhardtii, which exhibits the higher growth rate than C. vulgaris and C. protothecoides under turbidostat conditions within 75 h. The domination of C. reinhardtii in the mixed culture was validated by on-line monitoring of growth rate and flowcytometric analysis. For the time-efficient production of microalgal biomass, this screening process has a high potential to segregate the fast-growing microalgal strains from the pool of various uncharacterized microalgal species and random mutants.     

Quantify the response of purslane plant growth,photosynthesis pigments and photosystem II photochemistry to cadmium concentration gradients in the soil

The cadmium (Cd), being a widespread soils pollutant and one of the most toxic heavy metals in the environment, adversely affects sustainable crop production and food safety. Pot experiment was conducted to quantify and simulate the response of purslane (Portulaca oleracea L.) plants to Cd toxicity. The purslane germinated seeds were cultivated in twelve Cd concentrations (from 0 to 300 mg/kg of Cd in soil) for six weeks and then some growth characteristics, photosynthesis pigments, and chlorophyll a fluorescence parameters were measured. The influence of Cd gradients in the soil on all growth parameters, photosynthesis pigments and chlorophyll a fluorescence parameters (except F_m and carotenoid content) were described by a segmented model. Furthermore, F_m and carotenoid contents were fitted to a linear model. The growth characteristics, chlorophyll content, photosynthetic pigments and some parameters of chlorophyll a fluorescence such as F_v, F_v/F_m, Y(II) and ETR decreased when Cd concentration increased. In contrast, F₀, Y(NPQ) and Y(NO) increased and F_m was not significantly affected. In general, most variations in the studied parameters were recorded with low concentrations of cadmium, which ranged from 0 to 125 mg/kg. Also, the growth characteristics (especially stem, leaf, and shoot dry weights) were more sensitive to Cd contamination than other parameters. Moreover, among chlorophyll fluorescence parameters, Y(NPQ) was the most sensitive to Cd concentration gradients in the soil that can be due to disturbances of antennae complex of PSII.     

Effect of glycerol and PEGMA coating on the efficiency of cell holding in alginate immobilized <Emphasis Type="Italic">Synechococcus elongatus</Emphasis>

Synechococcus elongatus cells were immobilized in alginate beads, and the effects of increasing the cross-linker concentration from 2 to 4 % CaCl₂ were evaluated, as well as the effects of coating the beads with either glycerol or poly(ethylene glycol) methyl ether methacrylate (PEGMA)—not previously reported for immobilized microalgae—to improve the holding time of the immobilized cells. S. elongatus cells remain metabolically active after coating with glycerol or PEGMA. There is an inverse relation between the glycerol concentration and the chlorophyll a content for the alginate beads cross-linked with 2 % CaCl₂. PEGMA diminishes the rate of liberation of cells as its concentration increases, although results suggest the ability of S. elongatus to degrade PEGMA, which increases the growth rate of the liberated cells, because of PEGMA being used as carbon source.