The growth and nutrient removal properties of heterotrophic microalgae Chlorella sorokiniana in simulated wastewater containing volatile fatty acids.

To reduce the high cost of organic carbon sources in waste resource utilization in the cultivation of microalgae, volatile fatty acids (VFAs) derived from activated sludge were used as the sole carbon source to culture Chlorella sorokiniana under the heterotrophic cultivation. The addition of VFAs in the heterotrophic condition enhanced the total nitrogen (TN) and phosphorus (TP) removal of C. sorokiniana, which proved the advantageous microalgae in using VFAs in the heterotrophic culture after screening in the previous study. To discover the possible mechanism of nitrogen and phosphorus adsorption in heterotrophic conditions by microalgae, the effect of different ratios of VFAs (acetic acid (AA): propionic acid (PA): butyric acid (BA)) on the nutrient removal and growth properties of C. sorokiniana was studied. In the 8:1:1 group, the highest efficiency (77.19%) of VFAs assimilation, the highest biomass (0.80 g L-1) and lipid content (31.35%) were achieved, with the highest TN and TP removal efficiencies of 97.44 % and 91.02 %, respectively. Moreover, an aerobic denitrifying bacterium, Pseudomonas, was determined to be the dominant genus under this heterotrophic condition. This suggested that besides nitrate uptake and utilization by C. sorokiniana under the heterotrophy, the conduct of the denitrification process was also the main reason for obtaining high nitrogen removal efficiency.

Effects of sulfamonomethoxine and trimethoprim co-exposures at different environmentally relevant concentrations on microalgal growth and nutrient assimilation.

In aquaculture around the world, sulfamonomethoxine (SMM), a long-acting antibiotic that harms microalgae, is widely employed in combination with trimethoprim (TMP), a synergist. However, their combined toxicity to microalgae under long-term exposures at environmentally relevant concentrations remains poorly understood. Therefore, we studied the effects of SMM single-exposures and co-exposures (SMM:TMP=5:1) at concentrations of 5 µg/L and 500 µg/L on Chlorella pyrenoidosa within one aquacultural drainage cycle (15 days). Photosynthetic activity and N assimilating enzyme activities were employed to evaluate microalgal nutrient assimilation. Oxidative stress and flow cytometry analysis for microalgal proliferation and death jointly revealed mechanisms of inhibition and subsequent self-adaptation. Results showed that exposures at 5 µg/L significantly inhibited microalgal nutrient assimilation and induced oxidative stress on day 7, with a recovery to levels comparable to the control by day 15. This self-adaptation and over 95 % removal of antibiotics jointly contributed to promoting microalgal growth and proliferation while reducing membrane-damaged cells. Under 500 µg/L SMM single-exposure, microalgae self-adapted to interferences on nutrient assimilation, maintaining unaffected growth and proliferation. However, over 60 % of SMM remained, leading to sustained oxidative stress and apoptosis. Remarkably, under 500 µg/L SMM-TMP co-exposure, the synergistic toxicity of SMM and TMP significantly impaired microalgal nutrient assimilation, reducing the degradation efficiency of SMM to about 20 %. Consequently, microalgal growth and proliferation were markedly inhibited, with rates of 9.15 % and 17.7 %, respectively, and a 1.36-fold increase in the proportion of cells with damaged membranes was observed. Sustained and severe oxidative stress was identified as the primary cause of these adverse effects. These findings shed light on the potential impacts of antibiotic mixtures at environmental concentrations on microalgae, facilitating responsible evaluation of the ecological risks of antibiotics in aquaculture ponds.

Growth, nutrient removal, and lipid productivity promotion of Chlorella sorokiniana by phosphate solubilizing bacteria Bacillus megatherium in swine wastewater: Performances and mechanisms.

Effects of a phosphorus-solubilizing bacteria (PSB) Bacillus megatherium on growth and lipid production of Chlorella sorokiniana were investigated in synthesized swine wastewater with dissolved inorganic phosphorus (DIP), insoluble inorganic phosphorus (IIP), and organic phosphorus (OP). The results showed that the PSB significantly promoted the algal growth in OP and IIP, by 1.10 and 1.78-fold, respectively. The algal lipid accumulation was also greatly triggered, respectively by 4.39, 1.68, and 1.38-fold in DIP, IIP, and OP. Moreover, compared with DIP, OP improved the oxidation stability of algal lipid by increasing the proportion of saturated fatty acids (43.8 % vs 27.9 %), while the PSB tended to adjust it to moderate ranges (30.2-41.6 %). Further, the transcriptome analysis verified the OP and/or PSB-induced up-regulated genes involving photosynthesis, lipid metabolism, signal transduction, etc. This study provided novel insights to enhance microalgae-based nutrient removal combined with biofuel production in practical wastewater, especially with complex forms of phosphorus.

Cultivation of the macrophyte Lemna minor and the microalgae Chlorella sorokiniana in thermal mineral waters: Biomass characteristics, radioisotopes and heavy metals content.

Microalgae and macrophytes are commonly used as human and animal food supplements. We examined the cultivation of the microalgae Chlorella sorokiniana and the duckweed Lemna minor in thermal waters under batch and sequencing batch conditions and we characterized the produced biomass for the presence of essential nutrients as well as for heavy metals and radioisotope content. The highest specific growth rate for the microalgae was observed when 5 or 15 mg/L N were supplemented while the optimal conditions for Lemna minor were observed in the co-presence of 5 mg/L N and 1.7 mg/L P. Lemna minor presented higher concentrations of proteins and lipids comparing to the studied microalgae. Both organisms contained high amounts of lutein (up to 1378 mg/kg for Lemna minor) and chlorophyll (up to 1518 mg/kg for Lemna minor) while ß-carotene and tocopherols were found at lower concentrations, not exceeding a few tens of mg/kg. The heavy metal content varied between the two species. Lemna minor accumulated more Cd, Cu, K, Mn, Na, Ni, and Zn whereas Al, Ca and Mg were higher in Chlorella sorokiniana. Both organisms could be a significant source of essential metals but the occasional exceedance of the statutory levels of toxic metals in food products raises concern for potential risk to either humans or animals. Application of gamma-spectroscopy to quantify the effective dose to humans from 228Ra, 226Ra and 40K showed that Chlorella sorokiniana was well under the radiological limits while the collected mass of Lemna minor was too small for radiological measurements with confidence.

Self-flocculation behaviour of cellulose-based bioflocculant synthesized from sewage water grown Chlorella sorokiniana and Scenedesmus abundans.

The global energy crisis has spurred a shift from conventional to clean and sustainable energy sources. Biomass derived from microalgae is emerging as an alternative energy source with diverse applications. Despite the numerous advantages of microalgae, large-scale biomass harvesting is not economical and convenient. Self-flocculation is considered an effective phenomenon facilitated by extracting the flocculating substances from microalgae that assist aggregation of algal cells into flocs. A novel cellulose-based bioflocculant has been synthesized from sewage water grown Chlorella sorokiniana and Scenedesmus abundans for harvesting application. The produced bioflocculant amounted to 38.5% and 19.38% of the dry weight of S. abundans and C. sorokiniana, respectively. Analysis via FTIR, XRD, and FESEM-EDX revealed the presence of cellulose hydroxyapatite (HA) in algae-derived cellulose. Harvesting efficiencies of 95.3% and 89.16% were attained for S. abundans and C. sorokiniana, respectively, at a dosage of 0.5 g/L. Furthermore, the bioflocculant was recovered, enabling its reuse with recovery efficiencies of 52% and 10% for S. abundans and C. sorokiniana, respectively. This simple and efficient approach has the potential to replace other harvesting methods, thereby contributing to the economic algal biofuel production.

Continuous microalgal culture module and method of culturing microalgae containing macular pigment.

This study established and investigated continuous macular pigment (MP) production with a lutein (L):zeaxanthin (Z) ratio of 4-5:1 by an MP-rich Chlorella sp. CN6 mutant strain in a continuous microalgal culture module. Chlorella sp. CN6 was cultured in a four-stage module for 10 days. The microalgal culture volume increased to 200 L in the first stage (6 days). Biomass productivity increased to 0.931 g/L/day with continuous indoor white light irradiation during the second stage (3 days). MP content effectively increased to 8.29 mg/g upon continuous, indoor white light and blue light-emitting diode irradiation in the third stage (1 day), and the microalgal biomass and MP concentrations were 8.88 g/L and 73.6 mg/L in the fourth stage, respectively. Using a two-step MP extraction process, 80 % of the MP was recovered with a high purity of 93 %, and its L:Z ratio was 4-5:1.

Developing a Chromochloris zofingiensis Mutant for Enhanced Production of Lutein under CO2 Aeration.

Microalgae are competitive and commercial sources for health-benefit carotenoids. In this study, a Chromochloris zofingiensis mutant (Cz-pkg), which does not shut off its photosystem and stays green upon glucose treatment, was generated and characterized. Cz-pkg was developed by treating the algal cells with a chemical mutagen as N-methyl-N'-nitro-N-nitrosoguanidine and followed by a color-based colony screening approach. Cz-pkg was found to contain a dysfunctional cGMP-dependent protein kinase (PKG). By cultivated with CO2 aeration under mixotrophy, the mutant accumulated lutein up to 31.93 ± 1.91 mg L-1 with a productivity of 10.57 ± 0.73 mg L-1 day-1, which were about 2.5- and 8.5-fold of its mother strain. Besides, the lutein content of Cz-pkg could reach 7.73 ± 0.52 mg g-1 of dry weight, which is much higher than that of marigold flower, the most common commercial source of lutein. Transcriptomic analysis revealed that in the mutant Cz-pkg, most of the genes involved in the biosynthesis of lutein and chlorophylls were not down-regulated upon glucose addition, suggesting that PKG may regulate the metabolisms of photosynthetic pigments. This study demonstrated that Cz-pkg could serve as a promising strain for both lutein production and glucose sensing study.

Bioremediation of hemotoxic and oxidative stress induced by polyethylene microplastic in Clarias gariepinus using lycopene, citric acid, and chlorella.

Despite extensive research on the toxic effects of microplastics (MPs), there is no obtainable data on the use of phytobioremediation against MPs toxicity in fish. This study aimed to investigate the protective role of lycopene, citric acid, and chlorella against the toxic effects of MPs in African catfish (Clarias gariepinus) using hematology, biochemical, antioxidants, erythron profiles (poikilocytosis and nuclear abnormalities) and the accumulation of MPs in tissues as biomarkers. Five groups of fish received: normal diet (control); MPs (500 mg/kg diet) (Group 2); MPs (500 mg/kg diet) + lycopene (500 mg/kg diet) (Group 3); MPs (500 mg/kg diet) + citric acid (30 g/kg diet) (Group 4); and MPs (500 mg/kg diet) + chlorella (50 g/kg diet) (Group 5) for 15 days. Group 2 had significantly higher amounts of MPs in the stomach, gills, and feces, electrolyte imbalances (HCO3, Fe, Na+, K+, Ca+2, Cl-, and anion gap, hematobiochemical alterations, and decreases in the activities of superoxide dismutase, catalase, total antioxidant capacity, and glutathione S-transferases compared to the control group. Additionally, Group 2 had significant increase in the percentage of poikilocytosis, and nuclear abnormalities in RBC's compared to the control group. The co-treatment of MPs-exposed fish with lycopene, citric acid, and chlorella-supplemented diets ameliorated the hematological, biochemical, and erythron profile alterations, but only slightly enhanced the antioxidant activity. Overall, lycopene, citric acid, and chlorella can be recommended as a feed supplement to improve hematobiochemical alterations and oxidative damage induced by MPs toxicity in the African catfish (C. gariepinus).

Ultrahigh-cell-density heterotrophic cultivation of the unicellular green alga Chlorella sorokiniana for biomass production.

Heterotrophic cultivation of Chlorella has achieved commercial success, but the application of Chlorella biomass is still limited due to the high cost of biomass production. In this study, an effective and industrially scalable heterotrphic cultivation technology has been developed for a production strain Chlorella sorokiniana GT-1. Under the optimized culturing conditions, the ultrahigh biomass concentration of 271 and 247 g L-1 was achieved in 7.5 L bench-scale and 1000 L pilot-scale fermenters, respectively. Technoeconomic (TE) analysis indicated that the production cost of C. sorokiniana GT-1 could be reduced to $1601.27 per ton of biomass if the biomass concentration reached 200 g L-1 , which is 24.2% lower than that of the reported highest Chlorella biomass production through fermentation with the same TE model. Under the same growth conditions, the maximum biomass concentration of a low-starch mutant SLM2 was reduced to 93 g L-1 , which was 54% lower than that of the wild type, indicating the capabilities of C. sorokiniana GT-1 cells in accumulating large amounts of starch are essential for achieving the ultrahigh-cell-density under the heterotrophic conditions. In addition, the ultrahigh-cell-density growth potential of C. sorokiniana GT-1 cells was inferred to be related to the intrinsic biological characteristics including the tolerance to low dissolved oxygen and a moderate doubling time under the heterotrophic conditions as well. The breakthrough in cultivation technology is promising for Chlorella industry and would expand its applications in food and feed.

Efficient isolation and cultivation of endosymbiotic Chlorella from Paramecium bursaria on agar plates by co-culture with yeast cells.

Paramecium bursaria is a ciliate that harbors Chlorella-like unicellular green algae as endosymbionts. The relationship between the host P. bursaria and the endosymbiotic Chlorella is facultative; therefore, both partners can be cultured independently and re-combined to re-establish symbiosis, making this system suitable for studying algal endosymbiosis. However, despite many previous studies, cultivation of endosymbiotic Chlorella remains difficult, particularly on agar plates. Here we describe a simple agar plate method for efficiently isolating and culturing cells of the endosymbiotic alga Chlorella variabilis from an individual P. bursaria cell, by co-culturing them with yeast Saccharomyces cerevisiae. The co-culture with the yeast significantly improved the colony-forming efficiency of the alga on agar. Growth assays suggest that the main role of the co-cultured yeast cells is not to provide nutrients for the algal cells, but to protect the algal cells from some environmental stresses on the agar surface. Using the algal cells grown on the plates and a set of specially designed primers, direct colony PCR can be performed for screening of multiple endosymbiont clones isolated from a single host ciliate. These methods may provide a useful tool for studying endosymbiotic Chlorella species within P. bursaria and various other protists.

Metabolomic Study of Heterotrophically Grown Chlorella sp. Isolated from Wastewater in Northern Sweden.

There are numerous strains of Chlorella with a corresponding variety of metabolic pathways. A strain we previously isolated from wastewater in northern Sweden can grow heterotrophically as well as autotrophically in light and has higher lipid contents under heterotrophic growth conditions. The aims of the present study were to characterize metabolic changes associated with the higher lipid contents in order to enhance our understanding of lipid production in microalgae and potentially identify new compounds with utility in sustainable development. Inter alia, the amino acids glutamine and lysine were 7-fold more abundant under heterotrophic conditions, the key metabolic intermediate alpha-ketoglutarate was more abundant under heterotrophic conditions with glucose, and maltose was more abundant under heterotrophic conditions with glycerol than under autotrophic conditions. The metabolite 3-hydroxy-butyric acid, the direct precursor of the biodegradable plastic PHB (poly-3-hydroxy-butyric acid), was also more abundant under heterotrophic conditions. Our metabolomic analysis has provided new insights into the alga's lipid production pathways and identified metabolites with potential use in sustainable development, such as the production of renewable, biodegradable plastics, cosmetics, and nutraceuticals, with reduced pollution and improvements in both ecological and human health.

Magnetic field as promoter of growth in outdoor and indoor assays of Chlorella fusca.

This study aimed to evaluate the influence of a magnetic field (MF) intensity of 25 mT on Chlorella fusca cultivation in outdoor and indoor conditions, and evaluate the changes in the macromolecules, pigment content and protein profile. C. fusca was cultivated for 15 d in raceway photobioreactor. MF was applied for 24 h d-1 and 1 h d-1. In outdoor cultivation, MF applied for 24 h d-1 increased 23% in the biomass concentration, while indoor assays resulted in an increase in both modes, with biomass production increasing between 70 and 85%. Biomass composition was altered when MF was applied for 1 h d-1 in indoor assays; the highest protein content was achieved (32.7%). Nitrate consumption was higher in outdoor assays, while MF application did not alter the protein profile. The results showed that combining the outdoor conditions with MF is advantageous, as higher biomass concentration can be achieved with lower energy expenditure.

Mixotrophic growth regime as a strategy to develop microalgal bioprocess from nutrimental composition of tequila vinasses.

The selection of a suitable growth regime can increase the physiological performance of microalgae and improve bioprocess based on these microorganisms from agro-industrial residues. Thus, this study assessed the biotechnology capacity-biomass production, biochemical composition, and nutrient uptake-from tequila vinasses (TVs) as the nutrient source of three indigenous microalgae-Chlorella sp., Scenedesmus sp., and Chlamydomonas sp.-cultured under heterotrophic and mixotrophic conditions. The results demonstrated that under the mixotrophic regime, the three microalgae evaluated reached the highest nitrogen uptake, biomass production, and cell compound accumulation. Under this condition, Chlorella sp. and Scenedesmus sp. showed the highest nutrient uptake and biomass production, 1.7 ± 0.3 and 1.9 ± 0.3 g L-1, respectively; however, the biochemical composition, mainly carbohydrates and proteins, varied depending on the microalgal strain and its growth regime. Overall, our results demonstrated the biotechnological capacity of native microalgae from TVs, which may vary not only depending on the microalgal strain but also the culture strategy implemented and the characteristics of the residue used, highlighting-from a perspective of circular bio-economy-the feasibility of implementing microalgal bioprocess to reuse and valorize the nutrimental composition of TVs through biomass and high-valuable metabolite production, depicting a sustainable strategy for tequila agro-industry in Mexico.

Growth Performance and Antioxidative Response of Chlorella pyrenoidesa, Dunaliella salina, and Anabaena cylindrica to Four Kinds of Ionic Liquids.

Ionic liquids are widely used for lipid and pigment extractions from microalgae. It is possible that ionic liquids are discharged into environments. The evaluation of growth performance and antioxidative response of ionic liquids to microalgae is helpful to explore the stress regulation mechanism and investigate possible environmental risk. Ionic liquids induce production of reactive oxygen species (ROS) to microalgae. These oxidative stresses are possible from cations, anions, and salinity. In this study, the growth inhibitions of [BMIM]Br, [BMIM]Cl, [EMIM]Cl, and [EMIM]EtOSO3 to Anabaena cylindrica, Chlorella pyrenoidesa, and Dunaliella salina were evaluated. It was interesting that Br- and two kinds of cations, [BMIM] and [EMIM], had significant effects on growth inhibitions of these microalgae. IC50 values of these ionic liquids for A. cylindrica, C. pyrenoidesa, and D. salina were also estimated based on the results of growth inhibitions. It was proved that [EMIM]Cl is relatively harmless to C. pyrenoidesa and D. salina, and [EMIM]EtOSO3 is relatively or practically harmless to C. pyrenoidesa. [BMIM]Br and [BMIM]Cl are practically harmless to A. cylindrica and C. pyrenoidesa, and relatively harmless to D. salina. More than 0.8 g/L [EMIM]EtOSO3 led to bleaching of both A. cylindrica and D. salina at 48 h which was shown that the anion, EtOSO3-, had higher inhibition to A. cylindrica and D. salina than Cl-. In addition, high concentration of ionic liquids led to reductions of chlorophyll content in these three kinds of microalgae, increase of ROS levels and malondialdehyde contents for most of the cases. High concentration of ionic liquids also increased the activities of superoxide dismutase in three kinds of microalgae. There were positive correlations between ROS levels or MDA content, and inhibitions ratios of these ionic liquids to microalgae except [EMIM]Cl to A. cylindrica. These antioxidant enzymes were beneficial for reducing the ROS induced by ionic liquids.

Using Phycocyanin as Spectral Converter on the Growth Parameters and Lipid Content of the Green Microalga Chlorella sp. in a Double Layer Flat Panel Photobioreactor.

The focus of this research is spectral shifting of light using phycocyanin solution extracted from the blue-green microalga Spirulina platensis in order to increase biomass productivity of the green microalga Chlorella sp. Also, lipid and chlorophyll content of the green alga was investigated. With regard to the shift of the spectrum with the phycocyanin solution, a double layer flat panel photobioreactor and two different spectral shifting strategies were used. In each strategy, the effect of two different concentrations of the solution was investigated. In the first strategy, the light passes through the chamber containing the solution and then enters the microalga culture chamber. In the second strategy, the light first enters the culture chamber and then enters the chamber containing phycocyanin pigment. The results showed that the use of phycocyanin pigment by both strategies increased the biomass productivity (P) and the specific growth rate (µmax) with a significant difference compared to the control system; the increase in P for first strategy was up to about 69%. Moreover, the use of phycocyanin solution with a lower concentration had a greater effect on the increase of total chlorophyll content; however, the solution with a higher concentration was more successful in the production of cell lipid content. Using the phycocyanin solution as spectral converter in a double layer flat panel photobioreactor increased the biomass productivity and chlorophyll content.

Facile Hydrothermal Synthesis of Chlorella-Derived Environmentally Friendly Fluorescent Carbon Dots for Differentiation of Living and Dead Chlorella.

The judgment of microalgae viability is a vital procedure in the process of microalgae culture and treatment, which also plays an important role in bioremediation, bioindication, and pharmacology fields. The current conventional methods for defining living/dead microalgal cells are complicated or laborious. Hence, developing a simple and reliable detection method for microalgae viability is still challenging. Here, we developed chlorella-based carbonized polymer dots (c-CPDs) by a hydrothermal method. Due to their small average size of 5.0 nm, obvious excitation-dependent emission, stable fluorescence properties, and low toxicity, c-CPDs could be used for distinguishing living or dead chlorella by testing different fluorescence characteristics of c-CPD-labeled chlorella. Compared with conventional cellular dyes used for differentiating living/dead microalgae, c-CPDs significantly reduced toxicity, showing good sensitivity and reliability. This work provided a method to prepare environmentally friendly carbon dots (CDs) using microalgae, which had potential to be prepared on a large scale and might be applied feasibly in the preparation of doped CDs by controlling the growth of chlorella.

Shear stress computation in a millimeter thin flat panel photobioreactor: Numerical design validated by experiments.

Flat panels are the most spread type of photobioreactors for studying light effects on a microalgae culture. Their low thickness, usually between 1 and 3 cm, aims at ensuring light homogeneity across the culture. Yet because optical density has to remain very low, studies are still limited to low cell density cultures. To alleviate this problem, even thinner photobioreactors can be designed. Nevertheless, thin flat panel reactors are very prone to induce high shear stress. This work aimed at designing a new millimeter thin panel photobioreactor to study light effects on Chlorella and Scenedesmus genera. We proposed a numerical workflow that is capable of assessing the shear stress intensity in such a reactor. The numerical predictions were validated at three different levels: 2D preliminary simulations were able to reproduce bubble commonly known behaviors; close to the nozzle, the predictions were successfully confronted to shadowgraphy experimental reference; at the mini bioreactor scale, experimental and numerical mixing were found to be close. After these throughout validations, shear stress intensity in the photobioreactor was calculated over 1000 Lagrangian tracers. The experienced shear stress was agglomerated at the population level. From the computed shear stress, it was possible to choose the minimal reactor thickness that would not hinder cell growth.

Toxic mechanism of three azole fungicides and their mixture to green alga Chlorella pyrenoidosa.

Currently, few studies have investigated the joint toxicity mechanism of azole fungicides at different exposure times and mixed at the relevant environmental concentrations. In this study, three common azole fungicides, namely, myclobutanil (MYC), propiconazole (PRO), and tebuconazole (TCZ), were used in studying the toxic mechanisms of a single substance and its ternary mixture exposed to ambient concentrations of Chlorella pyrenoidosa. Superoxide dismutase (SOD), catalase (CAT), chlorophyll a (Chla), and total protein (TP), were used as physiological indexes. Results showed that three azole fungicides and ternary mixture presented obvious time-dependent toxicities at high concentrations. MYC induced a hormetic effect on algal growth, whereas PRO and TCZ inhibit algal growth in the entire range of the tested concentrations. The toxicities of the three azole fungicides at 7 days followed the order PRO > TCZ > MYC. Three azole fungicides and their ternary mixture induced different levels of SOD and CAT activities in algae at high concentrations. The ternary mixture showed additive effects after 4 and 7 days exposure, but no effect was observed at actual environmental concentrations. The toxic mechanisms may be related to the continuous accumulation of reactive oxygen species, which not only affected protein structures and compositions but also damaged thylakoid membranes, hindered the synthesis of proteins and chlorophyll a, and eventually inhibited algal growth. These findings increase the understanding of the ecotoxicity of azole fungicides and use of azole fungicides in agricultural production.

Fast and easy quantitative characterization of methanotroph-photoautotroph cocultures.

Recent research has demonstrated that synthetic methanotroph-photoautotroph cocultures offer a highly promising route to convert biogas into value-added products. However, there is a lack of techniques for fast and accurate characterization of cocultures, such as determining the individual biomass concentration of each organism in real-time. To address this unsolved challenge, we propose an experimental-computational protocol for fast, easy, and accurate quantitative characterization of the methanotroph-photoautotroph cocultures. Besides determining the individual biomass concentration of each organism in the coculture, the protocol can also obtain the individual consumption and production rates of O2 and CO2 for the methanotroph and photoautotroph, respectively. The accuracy and effectiveness of the proposed protocol was demonstrated using two model coculture pairs, Methylomicrobium alcaliphilum 20ZR-Synechococcus sp. PCC7002 that prefers high pH high salt condition, and Methylococcus capsulatus-Chlorella sorokiniana that prefers low salt and neutral pH medium. The performance of the proposed protocol was compared with a flow cytometry-based cell counting approach. The experimental results show that the proposed protocol is much easier to carry out and delivers faster and more accurate results in measuring individual biomass concentration than the cell counting approach without requiring any special equipment.

Chemical- and species-specific toxicity of nonylphenol and octylphenol to microalgae Chlorella pyrenoidosa and Scenedesmus obliquus.

As typical endocrine disrupters, nonylphenol (NP) and octylphenol (OP) are emerging pollutants that have attracted wide attention. This study investigated the toxicity effects of NP and OP on microalgae Chlorella pyrenoidosa and Scenedesmus obliquus, particularly on their growth inhibition, photosynthetic pigment, chlorophyll fluorescence, and superoxide dismutase and malondialdehyde levels. Results showed that the 96 h EC50 of NP and OP was 2.89 and 5.21 mg/L on C. pyrenoidosa, respectively, and 1.54 and 8.48 mg/L on S. obliquus, respectively. NP exerted a stronger inhibitory effect on cell growth, photosynthesis, and PSII activity, and it contributed more oxidative stress on C. pyrenoidosa than on S. obliquus. By contrast, OP exerted a stronger inhibitory effect on S. obliquus than on C. pyrenoidosa. Furthermore, the toxicity of OP to the tested microalgae was lower than that of NP. Principal component analysis (PCA) and Pearson's correlation indicate that the accumulation of reactive oxygen species is the dominant mechanism of NP and OP cellular toxicity. The principal components of NP and OP affecting microalgae are distinct in the PCA plot, and different endocrine disrupters have varying chemical-specific influences on algal cells. This study confirmed that the toxicity of NP and OP to microalgae C. pyrenoidosa and S. obliquus is chemical- and species-specific. These findings should be considered when assessing the health risk of environmental pollution.