Chlorella Vulgaris Growth Research Articles

The Use of Light Spectra to Improve the Growth and Lipid Content of Chlorella vulgaris for Biofuels Production

The effects of light spectra on cell concentration, cell size, biomass production, proximate composition, pigment content, and fatty acid content in Chlorella vulgaris during two growth phases were measured. Growth rates were higher with white (0.70 division day−1) and blue light (0.67 divisions day−1). Cell size was greater in the exponential growth with yellow light (16.29 μm) versus blue light (14.26 μm). Higher organic dry weight (ODW) values were observed during exponential growth under green light (70.58 pg cell−1) compared with white light (36.56 pg cell−1). Proximate composition differed significantly by effect of light spectra and growth phases. Protein content was significantly higher in the exponential growth with white light (34.42%) and green (31.64%) light. Carbohydrate levels were significantly higher during stationary growth under yellow light (27.05%). In the exponential growth, lipid content was significantly higher with blue light (18.74%). Biomass productivity was highest in exponential growth phase with blue light (0.064 gL-1 day-1), meanwhile, in stationary growth phase yellow light (0.031 gL-1 day-1). Chlorophyll a and carotenoid levels were significantly greater during exponential growth with green and yellow lights. Fatty acid content was significantly modified by the light spectra and growth phase. The predominant saturated fatty acid was C:16:0 (palmitic acid), ranging between 10.26 (green light and exponential growth) and 20.01% (white light and stationary growth). The content of C18:1n-9 was higher (28.34 %) with the white light in the stationary growth, and C18:3n-3 content was greater with white (41.41%) and blue light (40.82%) in the exponential growth. It was concluded that blue light is the most suitable condition to induce biomass productivity and lipid content on both growth phases. Blue light during the stationary growth phase induces the production of lipids and saturated fatty acids (SFAs) which are suitable for biodiesel production.

Selection of nitrogen source affects the growth and metabolic enzyme activities of Chlorella vulgaris (Beijerinck) strain R-06/2 (chlorophyta)

The choice of nitrogen source in a cultivation medium can specifically affect the physiology and biochemistry of microalgae. To increase the production of low-cost valuable biomass, the preferred nitrogen form for each alga should be determined. The aim of our study was to analyze the effects of different nitrogen sources and cultivation times on the growth, biochemical composition and the activities of glutamine synthetase, glutamate synthase, glutamate dehydrogenase, malate dehydrogenase, aspartate aminotransferase and proteases of Chlorella vulgaris R-06/2. Media supplemented with urea or ammonium nitrate provided similarly (p>0.05) high growth rates for a short cultivation time (4 days). The two nitrogen compounds applied simultaneously ensured better biomass yield but for prolonged cultivation. In the exponential growth phase, ammonium nitrate stimulated (p<0.05) protein production, whereas urea enhanced (p<0.05) the carbohydrate content in older cultures as compared to the other nitrogen sources. The activity of each of the studied metabolic enzymes of C. vulgaris R-06/2 varied specifically depending on the nitrogen source and the growth phase, ensuring the maintenance of efficient, balanced metabolism under all cultivation conditions. When using large-scale cultivation to produce biomass for various useful applications, the selection of nitrogen source should be based on algal metabolism.

The impact of microwave radiation in the processes of carbon dioxide absorption by chlorophyll-producing microalgae

The influence of microwave radiation on the rate of СО2 uptake by chlorophyll-producing microalgae of the Chlorella Vulgaris has been established. Experimental dependencies of greenhouse gases absorption by microalgae depending on the impact of microwave electromagnetic (MEM) field have been obtained. The mathematical model of the dynamics of biomass growth of Chlorella Vulgaris microalgae depending on the time of electromagnetic radiation has been constructed. Based on the mathematical model solution and the experimental data obtained, a chart of the dependence of CO2 absorption by microalgae of the Chlorella Vulgaris provided that microwave radiation is present, has been constructed. The optimal value of microwave electromagnetic radiation for the design of technological schemes for the industrial purification of gas emissions from carbon dioxide by biological method has been established.

The Growth of Chlorella vulgaris Cultured in Liquid Organic Fertilizer of Water Hyacint H (Eichhornia crassipes) at Different Salinities

This study aims to determine the effect of salinity on the growth of microalgae C. vulgaris cultured in 5% concentration of water hyacinth liquid organic fertilizer. The research used a completely randomized design with four salinity treatments at 30, 35, 40 and 45 ppt in triplicates. The volume of culture media was 150 mL that consisted of 142.5 ml of sterile sea water and 7.5 ml of water hyacinth organic fertilizer. The increase in microalgae cell density was observed every other day using haemocytometer under light microscope. The culture was harvested after they reached stationary phase at day 8. Culture age (p= 0,00 0.05) from other salinities. This study suggested that 40 ppt can be used to culture C. vulgaris in 5 % concentration of water hyacinth organic medium to obtain better growth and higher biomass production.

Assessment of the optimum growth medium and the effect of different light intensities on growth and photosynthetic pigments of Chlorella vulgaris and Scenedesmus arvernensis

CHLORELLA vulgaris and Scenedesmus arvernensis are common eukaryotic green microalgae that have a very wide use in various applications. They can be used as raw materials in bio-energy production, in aquaculture nutrition, in pharmaceuticals, cosmetics, biofertilizers, biodegradation of wastes and dyes. So, this required several studies to test some factors that can influence the growth rate and evaluate their yield. This study investigated the effects of different media compositions: Bold’s basal medium, BG-11 medium, modified Chu-10 medium, and Kuhl’s medium on growth of C. vulgaris and S. arvernensis. Then, study the effect of four different light intensities on the growth rate using the optimized medium. The growth was recorded by estimating optical density (OD), cell counting (CC) and total chlorophyll contents. The results showed that the maximum growth was recorded at Modified CHU’s 10 medium for C. vulgaris and BG-11 medium for S. arvernensis. The growth was compared with the initial record of C. vulgaris and S. arvernensis inocula; the OD increased by 6.85 and 5.15 times, respectively. C. vulgaris showed the optimum CC 9.60 times and total chlorophyll 7.96 times. CC recorded 10.42 times and total chlorophyll increased 5.90 times for S. arvernensis. Light intensities experiment showed that C. vulgaris gave the best growth rate at 60 μmol photons m-2s-1, on the 35th day of cultivation with increasing in OD, CC and total chlorophyll, (6.18, 5.27 and 3.58 times), of the initial record, respectively. Concerning S. arvernensis optimum growth rate was reached at 40μmol photons m-2s-1, on the 35th day accompanied by an increment in OD, CC and total chlorophyll, (6.20, 4.55 and 10.04 times), of the initial record, respectively.

Effect of Land Agricultural Fertilizer on Growth of Marine Single Cell Protein, <i>Spirulina platensis</i>, <i>Chlorella vulgaris</i> and <i>Nannochloropsis</i>

This study aimed to determine the growth rate and protein content in Nannochloropsis oculata, Chlorella vulgaris, Spirulina platensis were cultured using agricultural fertilizers. The agricultural fertilizers used were Urea, ZA and TSP. Each single cell protein was cultured using the three types of fertilizers with a ratio of Urea: ZA and TSP as follows (A) 1: 2: 1 [(10:20:10 g / L)], (B) 2: 2: 1 [(20:20:10 g / L)] and (C) 3: 2: 1 [(30:20:10 g / L)], respectively. The results showed that the best ratio of Urea, ZA and TSP fertilizers to growth of Spirulina, Nannochloropsis oculata and Chlorella vulgaris was C [30:20:10 (g / L)] treatment. However, phyto-protein content in Nannochlorophsis differed significantly between fertilization treatments, C fertilization yielded the highest protein content (28.75±0.05%), when compared with A (25.13±0.01%) and B (25.14±0.02%), respectively. In Chlorella vulgaris, all fertilization treatments showed very significant differences, B fertilization (28.24%±00.006) yielded the highest phyto-protein content, if compared to A (23.63% ± 0.003) and C (19.74% ±0.006 ), respectively. All fertilization treatments showed very significant differences (P <0.05) on Spirulina platensis. The highest content of phyto-protein (62.68 + 0.05%) was present in treatment C, when compared to A (52.18 + 0.05%) and B (62.37 + 0.01%) treatments, respectively.

Effect of Physical Factors on the Growth of Chlorella Vulgaris on Enriched Media Using the Methods of Orthogonal Analysis and Response Surface Methodology

In addition to chemical factors, physical conditions also play a key role in the growth of microalgae. In this study, solid sediment in rivers was simulated by pure quartz sand with different particle sizes and the physical effects of disturbance rate, solid–liquid ratio and particle size on the growth of Chlorella vulgaris (C. vulgaris) were investigated through orthogonal analysis and response surface methodology (RSM) during co-cultivation of C. vulgaris and sediment. The result of ANOVA in orthogonal analysis showed that the effect ability of a single factor on biomass can be ranked as disturbance rate > particle size > solid–liquid ratio, 100 r/min disturbance rate and 30–40 M particle size are the most significant at the 0.05 level. Furthermore, the specific growth rate can reach 0.25/d and 0.27/d, respectively. With the growth of C. vulgaris, the pH of the solution reached a maximum of 10.7 in a week. The results from the RSM showed that strong interactions are reflected in the combinations of disturbance rate and solid–liquid ratio, and disturbance rate and particle size. Ramp desirability of the biomass indicates that the optimum levels of the three variables are 105 r/min disturbance rate, 0.117 g/mL solid–liquid ratio and 30–40 M particle size. In this case, the biomass can grow seven times in a week with 0.27/d specific growth rate and a pH value of 7–10.4. This study shows that the growth of C. vulgaris can be regulated by changing physical conditions simultaneously, and the optimization of physical conditions can be applied to biomass production, algae prediction and acid water treatment in rivers, lakes and reservoirs.

Growth of Chlorella vulgaris using wastewater from Nile tilapia (Oreochromis niloticus) farming in a low-salinity biofloc system

The present study aimed to analyze the growth of Chlorella vulgaris in the effluent of a Biofloc Technology (BFT) system used in the Nile tilapia fingerlings farming. The conditions were cultivated by 10 days at 25 ± 2ºC, 90 μmol photons m−2 s−1 irradiance, under constant aeration (without addition of CO2), and with different BFT effluent proportions (0, 50, and 100%). After 24 hours of the inoculation that marked the beginning of the experiment, the development and multiplication of the cells were verified, demonstrating that the BFT effluent used in the Nile tilapia farming was favorable for the cultivation of the microalga Chlorella vulgaris. As expected, the Provasoli culture medium presented the best performance (1,520 ± 75 104 cells mL-1) in relation to the growth of the microalga. However, during the first four days of cultivation, C. vulgaris showed higher growth in treatments containing BFT effluent. C. vulgaris removed 93.6% of the nitrate contained in the BFT effluent. The results of the present study showed the potential use of C. vulgaris on Integrated Multi-Trophic Aquaculture with Nile tilapia fingerlings. In addition to removing nitrate and other nitrogen compounds, C. vulgaris biomass could be used to feed zooplankton or Nile tilapia larvae.

A droplet-based gradient microfluidic to monitor and evaluate the growth of Chlorella vulgaris under different levels of nitrogen and temperatures

Algae are potential sources for different industrial products. However, biofuel production from algae still needs to reach valuable economical approaches. Select algae and optimize culture conditions considered main challenges for biofuel production. Traditional batch cultures' techniques are time and labor consuming. PDMS-based Microfluidics is time-and labor-saving technique with high throughput single cell analysis. We present gradient-based microfluidics as tools for studying algal growth under different cultivation conditions. Applying our device provides the opportunity to generate droplets with different concentrations of medium nutrients' in 200s which keeps reagent and time for preparing such solutions using conventional techniques. Chlorella vulgaris was encapsulated inside uniformly droplets with different NO3−-N concentrations of 2.46, 6.69, 10.91, and 15.14 mΜ/L. separated devices were exposed to different temperatures of 20, 27, and 37°C. The photosynthetic energy conversion for each cell per droplet was calculated. It was observed that the higher the NO3− concentration, the higher the growth kinetics. According to the presented results, the optimum nitrate and temperature conditions were 15.14 mΜ/L and 37°C. Our device is considered the third effort for applying a gradient chip in algal biotechnology field. It is unique and simple in use with high competence results.

Lignocellulosic residue as bio-carrier for algal biofilm growth: Effects of carrier physicochemical proprieties and toxicity on algal biomass production and composition

Five types of lignocellulosic materials were applied as the bio-carriers for low-cost algal biofilm cultivation of three algal strains. The effects of bio-carrier physicochemical properties and toxicity on algal cells growth and attachment were investigated. Rougher and hydrophilic bio-carrier could yield more algal biomass than smoother and hydrophobic bio-carrier. Pine sawdust (diameter: 0.420–0.595 mm) performed the best when cultured Diplosphaera sp. (9.61 g·m−2·day−1) biofilm. Meanwhile, bio-carriers could be leached by the culture medium during cultivation, and their energy conversion proprieties could be improved due to the reduced ash contents and the decreased crystallinities. In addition, Chlorella vulgaris growth tests indicated that pine sawdust (15.45%) leachate promoted cell growth, whereas rick husk (15.48%) and sugarcane bagasse (13.19%) leachate inhibited cell growth. And bio-carriers leachates also modified the chemical compositions (lipid, protein and carbohydrate) of algal cells and increased the corresponding saturated fatty acids methyl ester content (from 48.71 to 55.58–57.08%).

High-Throughput Screening of Chlorella Vulgaris Growth Kinetics inside a Droplet-Based Microfluidic Device under Irradiance and Nitrate Stress Conditions.

Biodiesel is an eco-friendly renewable fuel that can be derived from microalgae. Maximization of biomass and lipid productivities are considered the main challenges for algal biodiesel production. Since conventional batch cultures are time-, space-, and reagent-consuming with many restrictions to apply many replicates, microfluidic technology has recently emerged as an alternative low-cost and efficient technology with high throughput repeatability and reproducibility. Different applications of microfluidic devices in algal biotechnology have been reported, including cell identification, sorting, trapping, and metabolic screening. In this work, Chlorella vulgaris was investigated by encapsulating in a simple droplet-based micro-array device at different light intensities of 20, 80, and 200 µmol/m2/s combined with different nitrate concentrations of 17.6, 8.8, and 4.4 mM. The growth results for C. vulgaris within microfluidic device were compared to the conventional batch culture method. In addition, the effect of combined stress of deficiencies in irradiance and nitrogen availability were studied to illustrate their impact on the metabolic profiling of microalgae. The results showed that the most optimum favorable culturing conditions for Chlorella vulgaris growth within the microfluidic channels were 17.6 mM and 80 µmol/m2/s.

Growth and nutrient removal of Chlorella vulgaris in ammonia-reduced raw and anaerobically-digested piggery wastewaters

Growth and nutrient removal of Chlorella vulgaris in ammonia-reduced raw and anaerobically-digested piggery wastewaters

Toxic effects of tetracycline and its degradation products on freshwater green algae

Tetracycline antibiotics are the most widely used antibiotics in the world and the most common veterinary drugs and feed additives used in livestock, poultry and aquaculture operations. Because antibiotics cannot be completely removed by currently existing sewage treatment facilities, these materials enter the environment directly via sewage treatment plant discharge, where they degrade. Accordingly, the metabolism and the ecological toxicity of tetracycline degradation products are worthy of attention. Herein, we investigated the effects of tetracycline and its degradation products (anhydrotetracycline and epitetracycline hydrochloride) on the growth, cell structure and algal cell oxidative stress of common Chlorella vulgaris. The results showed that the 96h-EC50 values of tetracycline (TC), anhydrotetracycline (ATC) and epitetracycline (ETC) on algal cells were 7.73, 5.96 and 8.42 mg/L, respectively. Moreover, the permeability of algal cells exposed to high concentrations of these three drugs was significantly enhanced. In addition, there were structural changes in the cells such as plasmolysis and starch granule deposition appeared, the thylakoid lamellae in the chloroplasts became blurred and deformed, and the vacuoles became larger. Exposure to higher concentrations (>5 mg/L) of TC and its degradation products ATC and ETC significantly upregulated the activity of ROS, as well as the antioxidants SOD and CAT. The levels of the lipid peroxidation product MDA also showed the same trend. Finally, ATC had the strongest toxicity toward algal cells, followed by TC and then ETC.

Effects of activated carbon on the growth of Chlorella vulgaris in an aqueous solution

Algal blooms, if left unmanaged, can negatively impact lake ecosystems. An unexplored method of removing excess nutrients from lakes, and therefore reducing algal blooms, is through the use of biochar. We hypothesize that due to the adsorptive characteristics of pyrolyzed material such as biochar and activated carbon, its presence would reduce the nutrient availability within aqueous solutions, therefore reducing algal growth. This experiment was conducted in an aqueous solution containing COMBO growth medium with and without the presence of activated carbon, studied under four conditions: 5 mg/L, 10 mg/L, 20 mg/L, and 50 mg/L phosphorous. We applied these treatments to an aqueous solution containing algae and measured fluorometer readings of the algae growth over a period of 12 days. An analysis of covariance followed by a Tukey’s HSD test demonstrated a significant difference between the means of samples containing activated carbon compared to samples without (p < 0.0001). Further, nutrient readings taken of each sample demonstrate a lower concentration of both phosphorus and nitrogen in samples containing activated carbon compared to those without. Our study demonstrates that activated carbon has the capacity to be used for the adsorption of phosphorous. This suggests that both activated carbon, as well as its more adsorptive counterpart, biochar, have the potential to be used in mitigating algal blooms and, more importantly, reducing the effects of anthropogenic eutrophication in aqueous environments.

Effects of crystalline nanocellulose on wastewater-cultivated microalgal separation and biomass composition

Microalgae are renewable and promising feedstock rich in biochemicals for biofuel and bioenergy production. The viability of this technology relies on the energy- and cost-efficient cultivation (culture medium used) and harvesting (coagulant applied) processes. The natural coagulant of crystalline nanocellulose modified with 1-(3-aminopropyl) imidazole (CNC-APIm) was demonstrated as a green and recyclable coagulant for microalgal harvesting. However, optimisation is still needed to ensure its applicability to microalgae cultivated on wastewaters, no effect on biomass composition, as well as cost-effective harvesting. In this study, microalgal growth and nutrient removal capacity of Chlorella vulgaris (C. vulgaris) were first investigated on two types of municipal wastewaters. C. vulgaris grew well on both primary and 30% (v/v) diluted centrate wastewaters with biomass productivities of 0.071 ± 0.005 and 0.062 ± 0.006 g/(L·d), respectively. High nitrogen and phosphorus removal efficiencies (91.1–100%) were obtained. Subsequently, the wastewater-cultivated C. vulgaris was harvested using a novel natural coagulant of crystalline nanocellulose modified with 1-(3-aminopropyl) imidazole (CNC-APIm). Based on the optimization results of the Design of Experiments driven response surface methodology approach, the optimal conditions for maximum HEs (86.5%) and RCs (38.5 g-algae/g-CNC) responses were determined for C. vulgaris under the following conditions: 0.02 g-CNC-APIm/g-algae of mass ratio, 5 s of CO2 sparging time, 8 min of air sparging time, and 50 ml/min of air flow rate. Moreover, no statistically significant differences were observed in the contents of carbohydrate, protein, lipid and fatty acids in the biomass harvested by centrifugation and CNC-APIm, respectively, suggesting that CNC-APIm would not impact the downstream microalgal application. According to the rough technical and economical estimation, CNC-APIm will be an alternative to conventional coagulants for commercial microalgal harvesting application.

Effects of Tryptophan Along with Sodium Pyruvate and Sodium Thiosulfate on Chlorella vulgaris Growth

Microalgae could serve as an ideal bioenergy resource for third-generation biofuels because of its endless features. However, in an effort to render it as a commercially viable bioenergy resource, certain improvements that enhance its productivity are required. In the present study, the role of three independent substrates, tryptophan, sodium thiosulfate, and sodium pyruvate, and their incorporation in modified BG-11 towards maximizing the Chlorella vulgaris FACHB-8 productivity was studied. These substrates could strongly impart a stimulating effect on microalgal productivity, firstly, either by promoting faster growth and chlorophyll content (tryptophan), or secondly by quenching the reactive oxygen species out of the culture medium (pyruvate), or thirdly by reducing the culture medium (thiosulfate). Response surface mediated optimal design model was effective in calculating the optimal concentration of these three independent substrates in modified BG-11. The observed actual values (1.54 g L⁻¹—biomass and 1.91 µg L⁻¹—chlorophyll) of the model were in close agreement with the predicted values (1.4 g L⁻¹—biomass and 1.82 µg L⁻¹—chlorophyll), thus, indicating the validity to the model. Tryptophan (2.2 times-chlorophyll and 2.5 times-biomass) was most effective in stimulating microalgal growth as compared with other substrates in modified BG-11. This study supported the concept that tryptophan after following one of the three pathways acted as the precursor for the production of indole acetic acid that in turn acted as a phytostimulator for microalgal growth. The outcomes of the present study would offer a step towards resolving the gaps present in producing viable, sustainable, high chlorophyll producing microalgal biomass. Effects of tryptophan along with sodium pyruvate and sodium thiosulfate on Chlorella vulgaris FACHB-8 growth

Effect of nutrients and gamma radiation on growth and lipid accumulation of Chlorella vulgaris for biodiesel production

ABSTRACTThe present work investigated the effects of nitrogen (N), phosphorus (P), magnesium (Mg), carbonate (CO3) concentrations and gamma radiation doses on growth and lipid production of Chlorel...

A semi-mechanistic model describing the influence of light and temperature on the respiration and photosynthetic growth of Chlorella vulgaris

The influence of light and temperature on microalgae kinetics has been assessed extensively and many models have been developed. However, limited attention has been paid to the influence of light and temperature on microalgae respiration and growth under conditions inducing (strong) photoinhibition and no clear consensus has been made on which model to use. Based on experimental data collected using a combined respirometer-titrimeter, a previously developed microalgae model (Decostere et al., 2016b) was first extended with a respiration process (r = rmin + δ μ). It was found that the dark respiration (rmin) was depending on both light and temperature and increased at conditions inducing photoinhibition and heat stress. Furthermore, out of five models describing the influence of light and temperature on the microalgae growth rate, the model of Dermoun et al. (1992) was determined to be the best suited model.

Impact of seconds to minutes photoperiods on Chlorella vulgaris growth rate and chlorophyll a and b content

In order to determine the impact of photoperiod duration, Chlorella vulgaris was studied in low cell density batch culture for seven days. Different photoperiod cycles ranging from minutes to seconds intervals were applied and their impact on microalgae cells growth and chlorophyll a and b contents were compared against a reference culture that was continuously illuminated. The selected photoperiods are typical of those due to alternance of dark and light zones along the algae trajectories inside photobioreactors. Throughout this study, all cultures received the same total amount of photons.When photoperiods of minutes duration were applied, the cells grew more slowly (0.052 ± 0.003 h−1) but contained higher average contents of chlorophyll pigments (0.27 ± 0.03 pg⋅cell−1) than those cells grown under continuous illumination (0.062 ± 0.003 h−1 and 0.42 ± 0.08 pg⋅cell−1, respectively). Cultures exposed to photoperiods of seconds duration grew the fastest (0.072 ± 0.002 h−1) with fewer photosynthetic units (0.61 ± 0.08 pg⋅cell−1) than the cells grown with continuous illumination. These results suggest that light harvesting systems have a higher efficiency with photoperiods of duration in the seconds range.

Effect of elevated benzophenone-4 (BP4) concentration on Chlorella vulgaris growth and cellular metabolisms.

Benzophenone-4 (BP4), as the raw material of common sunscreen products, usually shows strong eco-toxicity and endocrine-disrupting activity in aquatic animals. However, the potential adverse effect of BP4 on aquatic vegetation is still unclear. In order to evaluate the inhibitory effect of BP4 on phytoplankton, wild and acclimated Chlorella vulgaris was used as representative aquatic plant cells and experimental studies were conducted on the characteristics of its growth and cellular metabolisms upon exposure to elevated BP4 concentrations (1, 5, 10, 20, 50, and 100mgL-1). C. vulgaris basically appeared low sensitivity to BP4 exposure because the 96-h EC50 was measured as 65.16mgL-1 for its wild type. The 96-h EC50 of the acclimated type, which was pre-exposed to 10mgL-1 of BP4 and transferred twice, was 140.76mgL-1. By cellular response tests regarding non-enzymatic antioxidants carotenoid content, malondialdehyde (MDA), enzyme antioxidant superoxide dismutase (SOD) activity, and the photosynthetic efficiency, it was clarified that increasing exposure concentration elevated the hindrance to cellular metabolism. However, the rate of BP4 utilization as substrates for C. vulgaris growth showed a trend of decreasing with increasing BP4 concentration. The higher 96-h EC50 value of the acclimated C. vulgaris to BP4 inhibition than the wild C. vulgaris showed the enhanced tolerance capability; however, the continuous stress response of acclimated type should be taken into account when using microalgae species for toxicity assessment.