Methylmercury Effect and Distribution in Two Extremophile Microalgae Strains Dunaliella salina and Coccomyxa onubensis from Andalusia (Spain).

The main entrance point of highly toxic organic Hg forms, including methylmercury (MeHg), into the aquatic food web is phytoplankton, which is greatly represented by various natural microalgal species. Processes associated with MeHg fate in microalgae cells such as uptake, effects on cells and toxicity, Hg biotransformation, and intracellular stability are detrimental to the process of further biomagnification and, as a consequence, have great importance for human health. The study of MeHg uptake and distribution in cultures of marine halophile Dunaliella salina and freshwater acidophilic alga Coccomyxa onubensis demonstrated that most of the MeHg is imported inside the cell, while cell surface adhesion is insignificant. Almost all MeHg is removed from the culture medium after 72 h. Significant processes in rapid MeHg removal from liquid medium are its abiotic photodegradation and volatilization associated with algal enzymatic activity. The maximum intracellular accumulation for both species was in 80 nM MeHg-exposed cultures after 24 h of exposure for D. salina (from 27 to 34 µg/gDW) and at 48 h for C. onubensis (up to 138 µg/gDW). The different Hg intakes in these two strains could be explained by the lack of a rigid cell wall in D. salina and the higher chemical ability of MeHg to pass through complex cell wall structures in C. onubensis. Electron microscopy studies on the ultrastructure of both strains demonstrated obvious microvacuolization in the form of many very small vacuoles and partial cell membrane disruption in 80 nM MeHg-exposed cultures. Results further showed that Coccomyxa onubensis is a good candidate for MeHg-contaminated water reclamation due to its great robustness at nanomolar concentrations of MeHg coupled with its very high intake and almost complete Hg removal from liquid medium at the MeHg levels tested.

Interaction of Naturally Occurring Phytoplankton with the Biogeochemical Cycling of Mercury in Aquatic Environments and Its Effects on Global Hg Pollution and Public Health.

The biogeochemical cycling of mercury in aquatic environments is a complex process driven by various factors, such as ambient temperature, seasonal variations, methylating bacteria activity, dissolved oxygen levels, and Hg interaction with dissolved organic matter (DOM). As a consequence, part of the Hg contamination from anthropogenic activity that was buried in sediments is reinserted into water columns mainly in highly toxic organic Hg forms (methylmercury, dimethylmercury, etc.). This is especially prominent in the coastal shallow waters of industrial regions worldwide. The main entrance point of these highly toxic Hg forms in the aquatic food web is the naturally occurring phytoplankton. Hg availability, intake, effect on population size, cell toxicity, eventual biotransformation, and intracellular stability in phytoplankton are of the greatest importance for human health, having in mind that such Hg incorporated inside the phytoplankton cells due to biomagnification effects eventually ends up in aquatic wildlife, fish, seafood, and in the human diet. This review summarizes recent findings on the topic of organic Hg form interaction with natural phytoplankton and offers new insight into the matter with possible directions of future research for the prevention of Hg biomagnification in the scope of climate change and global pollution increase scenarios.

Fate of active pharmaceutical ingredients in a northern high-rate algal pond fed with municipal wastewater.

Active pharmaceutical ingredients (APIs) are vital to human health and welfare, but following therapeutic use, they may pose a potential ecological risk if discharged into the environment. Today's conventional municipal wastewater treatment plants are not designed to remove APIs specifically, and various techniques, preferably cost-effective and environmentally friendly, are being developed and evaluated. Microalgae-based treatment of wastewater is a sustainable and low-cost approach to remove nutrients and emerging contaminants. In this study, a North Sweden high-rate algal pond (HRAP) using municipal untreated wastewater as medium, was investigated in terms of API distribution and fate. Three six-day batches were prepared during 18 days and a total of 36 APIs were quantified within the HRAP of which 14 were removed from the aqueous phase above 50% and seven removed above 90% of their initial concentrations. Twelve APIs of a hydrophobic nature were mostly associated with the algal biomass that was harvested at the end of each batch. HRAPs treatment successfully removed 69% of studied APIs (25 of 36 studied) in six day time. The distribution of various APIs between the aqueous phase and biomass suggested that several removal mechanisms may occur, such as hydrophobicity driven removal, passive biosorption and active bioaccumulation.

Uptake Kinetics of Methylmercury in a Freshwater Alga Exposed to Methylmercury Complexes with Environmentally Relevant Thiols.

Cellular uptake of dissolved methylmercury (MeHg) by phytoplankton is the most important point of entry for MeHg into aquatic food webs. However, the process is not fully understood. In this study we investigated the influence of chemical speciation on rate constants for MeHg accumulation by the freshwater green microalga Selenastrum capricornutum. We used six MeHg-thiol complexes with moderate but important structural differences commonly found in the environment. Rate constants for MeHg interactions with cells were determined for the MeHg-thiol treatments and a control assay containing the thermodynamically less stable MeHgOH complex. We found both elevated amounts of MeHg associated with whole cells and higher MeHg association rate constants in the control compared to the thiol treatments. Furthermore, the association rate constants were lower when algae were exposed to MeHg complexes with thiols of larger size and more "branched" chemical structure compared to complexes with simpler structure. The results further demonstrated that the thermodynamic stability and chemical structure of MeHg complexes in the medium is an important controlling factor for the rate of MeHg interactions with the cell surface, but not for the MeHg exchange rate across the membrane. Our results are in line with uptake mechanisms involving formation of MeHg complexes with cell surface ligands prior to internalization.

Statistical Methods for Rapid Quantification of Proteins, Lipids, and Carbohydrates in Nordic Microalgal Species Using ATR-FTIR Spectroscopy.

Attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy is a simple, cheap, and fast method to collect chemical compositional information from microalgae. However, (semi)quantitative evaluation of the collected data can be daunting. In this work, ATR-FTIR spectroscopy was used to monitor changes of protein, lipid, and carbohydrate content in seven green microalgae grown under nitrogen starvation. Three statistical methods-univariate linear regression analysis (ULRA), orthogonal partial least squares (OPLS), and multivariate curve resolution-alternating least squares (MCR-ALS)-were compared in their ability to model and predict the concentration of these compounds in the biomass. OPLS was found superior, since it i) included all three compounds simultaneously; ii) explained variations in the data very well; iii) had excellent prediction accuracy for proteins and lipids, and acceptable for carbohydrates; and iv) was able to discriminate samples based on cultivation stage and type of storage compounds accumulated in the cells. ULRA models worked well for the determination of proteins and lipids, but carbohydrates could only be estimated if already determined protein contents were used for scaling. Results obtained by MCR-ALS were similar to ULRA, however, this method is considerably easier to perform and interpret than the more abstract statistical/chemometric methods. FTIR-spectroscopy-based models allow high-throughput, cost-effective, and rapid estimation of biomass composition of green microalgae.

Use of pulsed electric field permeabilization to extract astaxanthin from the Nordic microalga Haematococcus pluvialis.

The Nordic microalgal strain Haematococcus pluvialis was exposed to various stress conditions to induce astaxanthin accumulation. Highest carotenoid content (19.1 mg·g-1dw) was achieved in nitrogen-free culture medium at a high light intensity. The efficiency of Pulsed Electric Field (PEF) pre-treatment of stressed fresh biomass of H. pluvialis followed by incubation in the growth medium was compared to classical disruption methods (bead-beating, freezing-thawing, thermal treatment or ultrasound) for the subsequent extraction of astaxanthin in ethanol. N-starved cells treated with PEF followed by aqueous incubation for 6 h resulted in extraction of 96% (18.3 mgcar·gdw-1) of the total carotenoid content compared to 80% (15.3 mgcar·gdw-1) using other physical methods. The proportion of free forms of astaxanthin was higher in PEF-treated samples compared to mechanical disruption, suggesting PEF triggering an esterase activity. PEF pre-treatment of the cells followed by incubation in growth medium improved astaxanthin extraction in the eco-friendly solvent ethanol.

Northern green algae have the capacity to remove active pharmaceutical ingredients.

Eight recently isolated microalgal species from Northern Sweden and the culture collection strain Scenedesmus obliquus RISE (UTEX 417) were tested for their ability to remove 19 pharmaceuticals from growth medium upon cultivation in short light path, flat panel photobioreactors. While the growth of one algal species, Chlorella sorokiniana B1-1, was completely inhibited by the addition of pharmaceuticals, and the one of Scenedesmus sp. B2-2 was strongly inhibited, the other algal strains grew well and produced biomass. In general, lipophilic compounds were removed highly efficient from the culture medium by the microalgae (>70% in average within 2 days). The most lipophilic compounds Biperiden, Trihexyphenidyl, Clomipramine and Amitriptyline significantly accumulated in the biomass of most algal species, with a positive correlation between accumulation and their total biomass content. More persistent in the growth medium were hydrophilic compounds like Caffeine, Fluconazole, Trimetoprim, Codeine, Carbamazepin, Oxazepam and Tramadol, which were detected in amounts of above 60% in average after algal treatment. While Coelastrella sp. 3-4 and Coelastrum astroideum RW10 were most efficient to accumulate certain compounds in their biomass, two algae species, Chlorella vulgaris 13-1 and Chlorella saccharophila RNY, were not only highly efficient in removing all 19 pharmaceuticals from the growth medium within 12 days, at the same time only small amounts of these compounds accumulated in their biomass allowing its further use. Chlorella vulgaris 13-1 was able to remove most compounds within 6 days of growth, while Chlorella saccharophila RNY needed 8-10 days."Wild" Nordic microalgae therefore are able to remove active pharmaceutical ingredients, equally or more efficient than the investigated culture collection strain, thereby demonstrating their possible use in sustainable wastewater reclamation in Nordic conditions.

Effect of selenate on viability and selenomethionine accumulation of Chlorella sorokiniana grown in batch culture.

The aim of this work was to study the effect of Se(+VI) on viability, cell morphology, and selenomethionine accumulation of the green alga Chlorella sorokiniana grown in batch cultures. Culture exposed to sublethal Se concentrations of 40 mg · L(-1) (212 µM) decreased growth rates for about 25% compared to control. A selenate EC50 value of 45 mg · L(-1) (238.2 µM) was determined. Results showed that chlorophyll and carotenoids contents were not affected by Se exposure, while oxygen evolution decreased by half. Ultrastructural studies revealed granular stroma, fingerprint-like appearance of thylakoids which did not compromise cell activity. Unlike control cultures, SDS PAGE electrophoresis of crude extracts from selenate-exposed cell cultures revealed appearance of a protein band identified as 53 kDa Rubisco large subunit of Chlorella sorokiniana, suggesting that selenate affects expression of the corresponding chloroplast gene as this subunit is encoded in the chloroplast DNA. Results revealed that the microalga was able to accumulate up to 140 mg · kg(-1) of SeMet in 120 h of cultivation. This paper shows that Chlorella sorokiniana biomass can be enriched in the high value aminoacid SeMet in batch cultures, while keeping photochemical viability and carbon dioxide fixation activity intact, if exposed to suitable sublethal concentrations of Se.

Use of ultrasonic spectroscopy and viscosimetry for the characterization of chicken skin collagen in comparison with collagens from other animal tissues.

Use of traditional sources of collagen such as pork, bovine, and carp has some limitations. Chicken skin can be valuable alternative. In this work collagen was isolated from chicken skin using a modified procedure. Molecular properties of chicken collagen were analyzed and compared to collagen from other animal skins. Acid-soluble collagen type I was obtained with a yield of 25% and water content around 67%. Viscosimetry and ultrasonic spectroscopy were newly used for molecular characterization. By ultrasonic attenuation measurements, a pre-aggregation phase in the interval from 20°C to 27°C was observed, which is a proof of disaggregation and liquefaction. From 40°C upward, the liquefaction process finishes and aggregation continues. In a bovine sample this phenomenon starts at 40°C, in chicken at 50°C, and continues until 70°C. By viscosimetry, the denaturation temperature was confirmed as 40°C for bovine and 50°C for chicken collagen. Chicken collagen has a two times higher lysine level than bovine, which provides molecular stability side-chain interactions. With regard to higher thermal stability and favorable amino acid composition, waste chicken skin has the potential to be an excellent alternative source of raw collagen with applications in the food industry and biomedicine.