Extremophilic Microalga Research Articles

Extremophile Microalgae: the potential for biotechnological application.

Microalgae are photosynthetic microorganisms that use sunlight as an energy source, and convert water, carbon dioxide, and inorganic salts into algal biomass. The isolation and selection of microalgae, which allow one to obtain large amounts of biomass and valuable compounds, is a prerequisite for their successful industrial production. This work provides an overview of extremophile algae, where their ability to grow under harsh conditions and the corresponding accumulation of metabolites are addressed. Emphasis is placed on the high-value products of some prominent algae. Moreover, the most recent applications of these microorganisms and their potential exploitation in the context of astrobiology are taken into account.

Synergistic effects between autotrophy and heterotrophy in optimization of mixotrophic cultivation of Chlorella sorokiniana in bubble-column photobioreactors

Mixotrophic cultivation of microalgae enables inorganic carbon capture through photosynthesis along with simultaneous organic carbon uptake. Extremophilic microalga Chlorella sorokiniana, capable of tolerating high CO2 levels, was cultivated mixotrophically in 1 L bubble-column photobioreactors. This study shows how the biomass yield increases as the algal CO2 uptake process shifts from being driven by Carbon Concentrating Mechanism (CCM) to being driven by direct diffusion, as the CO2 concentration increases from very low (0%) and low (atmospheric: 0.04%) to high (1%, 2%, 3%, 5%). The biomass yield and productivity increase from 1.05 g/L and 0.33 g/L/day, respectively, at atmospheric CO2 to 1.85 g/L and 0.48 g/L/day, respectively, at 3% CO2, with a corresponding doubling of the lipid yield. Using acetic acid as the organic carbon source allows it to directly enter the metabolic pathways at the acetyl-CoA junction and enhance fatty acid synthesis, while enhanced reactor illumination (below photo-inhibition) synergizes the phototrophic and photo-heterotrophic pathways. These three reactor scale parameters, namely, organic (acetic acid) and inorganic (CO2) carbon loading and illumination, are co-optimized to activate the synergies between the metabolic pathways for autotrophy and heterotrophy in mixotrophic algal growth, which, in turn, significantly enhances the biomass and the macromolecular yields at the cellular scale. This multiscale synergy leads to a four-fold increase in the biomass yield (from 0.7 to 2.79 g/L), a five-fold increase in biomass productivity (from 0.124 to 0.593 g/L/day), and a six-fold increase in the total lipid yield (from 0.12 to 0.75 g/L), while eliminating night-time biomass losses. Such an approach of co-optimising reactor-scale parameters to synergize the autotrophic and the heterotrophic metabolic pathways can be implemented in mass cultivation of microalgae, resulting in faster carbon sequestration and higher yields of biomass, lipids and other value-added co-products.

Microalgae wastewater treatment: Biological and technological approaches.

Current global environmental issues raise unavoidable challenges for our use of natural resources. Supplying the human population with clean water is becoming a global problem. Numerous organic and inorganic impurities in municipal, industrial, and agricultural waters, ranging from microplastics to high nutrient loads and heavy metals, endanger our nutrition and health. The development of efficient wastewater treatment technologies and circular economic approaches is thus becoming increasingly important. The biomass production of microalgae using industrial wastewater offers the possibility of recycling industrial residues to create new sources of raw materials for energy and material use. This review discusses algae‐based wastewater treatment technologies with a special focus on industrial wastewater sources, the potential of non‐conventional extremophilic (thermophilic, acidophilic, and psychrophilic) microalgae, and industrial algae‐wastewater treatment concepts that have already been put into practice.

Biomolecules from extremophile microalgae: From genetics to bioprocessing of a new candidate for large-scale production

Abstract Extremophile microalgae are potential candidates for large-scale production because the extreme growth conditions help avoiding contamination, especially in open pond reactors. Considering the role of microalgae cultivation in integrated production systems, especially reusing wastewater and gaseous effluents, the search for new potential strains is of great value. The microalga Galdieria sulphuraria is able to grow both in acidic (pH 0–4) and high-temperature environments (45–56 °C). It is of great interest in biology and biotechnology because is considered one of the oldest eukaryotes on Earth and can be used to produce biomolecules. A bibliometric study identified 3 main areas of study about this microalga: genetics (genome sequencing, horizontal gene transfer), bioactive molecules (phycocyanin, specific branched glycogen, and antioxidants) and wastewater treatment (removal of carbon, nitrogen, phosphorus, and heavy metals). A critical analysis of the advances on the scientific knowledge about G. suplhuraria indicates that the combination of (i) polyextremophile conditions for growth, (ii) metabolic versatility, (iii) removal of nutrients/metals from wastewaters and (iv) production of high value-added products makes this microalga a potential candidate for large scale production in integrated and sustainable systems.

Microalgae for biofuel production.

Microalgae have been used commercially since the 1950s and 1960s, particularly in the Far East for human health foods and in the United States for wastewater treatment. Initial attempts to produce bulk chemicals such as biofuels from microalgae were not successful, despite commercially favorable conditions during the 1970s oil crisis. However, research initiatives at this time, many using extremophilic microalgae and cyanobacteria (e.g., Dunaliella and Spirulina), did solve many problems and clearly identified biomass productivity and harvesting as the two main constraints stopping microalgae producing bulk chemicals, such as biofuels, on a large scale. In response to the growing unease around global warming, induced by anthropogenic CO2 emissions, microalgae were again suggested as a carbon neutral process to produce biofuels. This recent phase of microalgae biofuels research can be thought to have started around 2007, when a very highly cited review by Chisti was published. Since 2007, a large body of scientific publications have appeared on all aspects of microalgae biotechnology, but with a clear emphasis on neutral lipid (triacylglycerol) synthesis and the use of neutral lipids as precursors for biodiesel production. In this review, the key research on microalgal biotechnology that took place prior to 2007 will be summarized and then the research trends post 2007 will be examined emphasizing the research into producing biodiesel from microalgae.

A novel investigation of the growth and lipid production of the extremophile microalga Coccomyxa melkonianii SCCA 048 under the effect of different cultivation conditions: Experiments and modeling

In the present study, the effect of nitrogen manipulation on the growth of the extremophile microalgal strain Coccomyxa melkonianii SCCA 048 was investigated for the first time to study its ability to produce marketable products, such as lipids. To identify the best strategies for cultivating this alga in large scale systems, the nitrate- and pH-dependent growth kinetics and the corresponding kinetic parameters were first evaluated in a multiwell screening. Subsequently, the effects of initial nitrogen concentration on the growth rate, nitrate uptake, as well as the synthesis of lipids and their corresponding quality, were investigated in batch photobioreactors. The experimental results were then successfully interpreted by a simple, albeit novel and exhaustive, mathematical model which takes into account the effects of light intensity, nitrate concentration and pH on the growth and lipid synthesis of this strain. By assessing the pH-dependent kinetics, it was confirmed that Coccomyxa melkonianii is extremophile as far as pH conditions are concerned. Moreover, the obtained growth rates and lipid productivities were high enough to permit sustainable cultivation at large scale. In particular, a quite interesting lipid content (about 40 %wt) was achieved under nitrogen starvation conditions. The fatty acids obtained by transesterification of the extracted lipids might be potentially exploited in different industrial sectors. In addition, the proposed model represents a first step towards the development of a useful tool for the optimization and the design of raceway-type bioreactor where Coccomyxa melkonianii would be cultivated.

Harvesting from the extreme and polluted: Extremophile microalgae living and recovering Uranium

Laminar flows are a common problem in high rate algal ponds (HRAP) due to their long channels and gentle mixing by a single paddlewheel. Sustained laminar flows may modify the amount of light microalgal cells are exposed to, increase the boundary layer between the cell and the environment and increase settling out of cells onto the pond bottom. To date, there has been little focus on the effects of the time between mixing events (frequency of mixing) on the performance of microalgae in wastewater treatment HRAPs. This paper investigates the performance of three morphologically distinct microalgae in wastewater treatment high rate algal mesocosms operated at four different mixing frequencies (continuous, mixed every 45 min, mixed every 90 min and no mixing). Microalgal performance was measured in terms of biomass concentration, nutrient removal efficiency, light utilisation and photosynthetic performance. Microalgal biomass increased significantly with increasing mixing frequency for the two colonial species but did not differ for the single celled species. All three species were more efficient at NH4–N uptake as the frequency of mixing increased. Increased frequency of mixing supported larger colonies with improved harvest-ability by gravity but at the expense of efficient light absorption and maximum rate of photosynthesis. However, maximum quantum yield was highest in the continuously mixed cultures due to higher efficiency of photosynthesis under light limited conditions. Based on these results, higher microalgal productivity, improved wastewater treatment and better gravity based harvest-ability can be achieved with the inclusion of more mixing points and reduced laminar flows in full-scale HRAP.

The effects of contemporary selection and dispersal limitation on the community assembly of acidophilic microalgae.

Extremophilic microalgae are primary producers in acidic habitats, such as volcanic sites and acid mine drainages, and play a central role in biogeochemical cycles. Yet, basic knowledge about their species composition and community assembly is lacking. Here, we begin to fill this knowledge gap by performing the first large-scale survey of microalgal diversity in acidic geothermal sites across the West Pacific Island Chain. We collected 72 environmental samples in 12 geothermal sites, measured temperature and pH, and performed rbcL amplicon-based 454 pyrosequencing. Using these data, we estimated the diversity of microalgal species, and then examined the relative contribution of contemporary selection (i.e., local environmental variables) and dispersal limitation on the assembly of these communities. A species delimitation analysis uncovered seven major microalgae (fourred, two green, and one diatom) and higher species diversity than previously appreciated. A distance-based redundancy analysis with variation partitioning revealed that dispersal limitation has a greater influence on the community assembly of microalgae than contemporary selection. Consistent with this finding, community similarity among the sampled sites decayed more quickly over geographical distance than differences in environmental factors. Our work paves the way for future studies to understand the ecology and biogeography of microalgae in extreme habitats.

Antioxidant and anti-proliferative properties of extracts from heterotrophic cultures of Galdieria sulphuraria

This study explores the possibility to use the extremophilic microalga Galdieria sulphuraria (strain 064) as a source of natural biomolecules with beneficial and protective effects on human health. Galdieria was cultivated in heterotrophy conditions and cells extracts for their antioxidant and anti-proliferative properties were tested. Galdieria extracts showed high antioxidant power tested through ABTS assay and revealed high glutathione and phycocyanin contents. Based on Annexin-V FITC/propidium iodide and MTT analysis, algae extracts inhibited the proliferation of human adenocarcinoma A549 cells (51.2% inhibition) through the induction of apoptosis without cell cycle arrest. Besides, cytotoxicity and cytometry assays showed a positive pro-apoptotic mechanism. On these bases, we suggest that G. sulphuraria from heterotrophic culture, for its therapeutic potential, could be considered a good candidate for further studies with the aim to isolate bioactive anti-cancer molecules.

Isolation and characterization of a stress-responsive gene encoding a CHRD domain-containing protein from a halotolerant green alga

The genetic basis of stress resistance in extremophilic microalgae is not well studied. In this study, a gene of unknown function, the cluster58 or CL58 gene, was identified from the halotolerant green alga Chlamydomonas W80 and characterized. The CL58 gene encodes a protein containing a domain of unknown function, the CHRD domain, and a putative secretory signaling sequence at its N-terminus. The levels of CL58 mRNA increased in response to high copper levels and low temperatures. When the CL58 gene was heterologously expressed as a fusion gene with the NanoLuc luciferase gene in Chlamydomonas reinhardtii, a majority of the NanoLuc activity was detected in the culture medium compared with that in the intracellular fraction. A mutagenic analysis revealed that the putative secretory signaling sequence was sufficient for the secretion of the CL58-NanoLuc fusion protein. In addition, we expressed the protein encoded by the CL58 gene in Escherichia coli; the recombinant, soluble protein was then purified. In summary, we identified a novel gene from C. W80 that appears to encode a stress-responsive, CHRD domain-containing secreted protein.

Determination of Reduced and Total Glutathione Content in Extremophilic Microalga Galdieria phlegrea.

Glutathione is an important molecule involved in the primary and secondary metabolism of all organisms. The Glutathione redox status is an indicator of the cellular redox state. Therefore, it is important to have precise methods on hand to determine the glutathione redox status in the cell. In this protocol, we describe an improved spectrophotometric method to estimate the content of reduced (GSH) and oxidized (GSSG) forms of glutathione in the extremophilic microalga Galdieria phlegrea.

Galdieria sulphuraria Relieves Oily and Seborrheic Skin By Inhibiting the 5-α Reductase Expression in Skin Cells and Reducing Sebum Production In Vivo

The sebum has important protective functions for the skin, although its overproduction may lead to undesired aesthetic problems and even more serious skin diseases. Sebum overproduction is caused by the androgen hormone testosterone, which is regulated by the enzyme 5-α Reductase, and can be associated with an over-proliferation of parasitic microbes that cause serious infections linked to skin inflammatory reactions. From the extremophile microalga Galdieria sulphuraria, we obtained a water-soluble extract and we found that it was capable of inhibiting the enzyme 5-α Reductase, inducing the expression of the β-defensins, which represents the first defense response mechanism triggered by the skin cells to fight against undesired microbes, and stimulating the wound healing process in skin cell cultures. The microalga extract was finally employed in clinical tests on human volunteers and it was confirmed its capacity to regulate sebum production. The results suggested that the Galdieria extract had interesting potentialities as active ingredient in cosmetic and dermatological formulas, in particular in those addressed to oily and seborrheic skins.

Maximizing recovery of energy and nutrients from urban wastewaters

Historically, UWWs (urban wastewaters) that contain high levels of organic carbon, N (nitrogen), and P (phosphorous) have been considered an environmental burden and have been treated at the expense of significant energy input. With the advent of new pollution abatement technologies, UWWs are now being regarded as a renewable resource from which, useful chemicals and energy could be harvested. This study proposes an integrated, algal-based system that has the potential to treat UWWs to the desired discharge standards in a sustainable manner while recovering high fraction of its energy content as well as its N- and P-contents for use as fertilizers. Key embodiments of the system being proposed are: i) cultivation of an extremophile microalga, Galdieria sulphuraria, in UWW for removal of carbon, N, and P via single-step by mixotrophic metabolism; ii) extraction of energy-rich biocrude and biochar from the cultivated biomass via hydrothermal processing; and, iii) enhancement of biomass productivity via partial recycling of the nutrient-rich AP (aqueous product) from hydrothermal-processed biomass to the cultivation step to optimize productivity, and formulation of fertilizers from the remaining AP. This paper presents a process model to simulate this integrated system, identify the optimal process conditions, and establish ranges for operational parameters.

Microalgae as a safe food source for animals: nutritional characteristics of the acidophilic microalga Coccomyxa onubensis

BackgroundEdible microalgae are marine or fresh water mesophilic species. Although the harvesting of microalgae offers an abundance of opportunities to the food and pharmaceutical industries, the possibility to use extremophilic microalgae as a food source for animals is not well-documented.ObjectiveWe studied the effects of dietary supplementation of a powdered form of the acidophilic microalga Coccomyxa onubensis on growth and health parameters of laboratory rats.MethodFour randomly organized groups of rats (n=6) were fed a standard diet (Diet 1, control) or with a diet in which 0.4% (Diet 2), 1.25% (Diet 3), or 6.25% (Diet 4) (w/w) of the standard diet weight was substituted with dried microalgae powder, respectively. The four groups of animals were provided ad libitum access to feed for 45 days.ResultsC. onubensis biomass is rich in protein (44.60% of dry weight) and dietary fiber (15.73%), and has a moderate carbohydrate content (24.8%) and a low lipid content (5.4%) in which polyunsaturated fatty acids represent 65% of the total fatty acid. Nucleic acids are present at 4.8%. No significant difference was found in growth rates or feed efficiency ratios of the four groups of rats. Histological studies of liver and kidney tissue revealed healthy organs in control and C. onubensis-fed animals, while plasma hematological and biochemical parameters were within healthy ranges for all animals. Furthermore, animals fed a microalgae-enriched diet exhibited a statistically significant decrease in both blood cholesterol and triglyceride levels. The blood triglyceride content and very low density lipoprotein-cholesterol levels decreased by about 50% in rats fed Diet 4.ConclusionsThese data suggest that C. onubensis may be useful as a food supplement for laboratory animals and may also serve as a nutraceutical in functional foods. In addition, microalgae powder-supplemented diets exerted a significant hypocholesterolemic and hypotriglyceridemic effect in animals.

Culture, growth, pigments and lipid content of Scenedesmus species, an extremophile microalga from Soda Dam, New Mexico in wastewater

We isolated an extremophile green alga from Jemez New Mexico; 18S rDNA sequencing confirmed it as Scenedesmus species Novo. Cultures in TAP medium (24±1°C, 132–148μmolphotonsm−2s−1) attained peak biomass (27.4×106cellsml−1, 49.11μgchl aml−1 and 24.93μgcaroteneml−1) and a division rate of 0.54day−1. Under outdoor harsh conditions (6524–7360μmolphotonsm−2s−1 and 40°C), biomass peaked at 10.41×106cells, 6.92μgchl a, and 4.49μgcarotene. Indoor wastewater cultures enriched with TAP yielded 63 to 94.3pg lipid cell−1 and 16.7 to 36.6pg lipid cell−1 in outdoor cultures which are amongst the highest for microalgae; lipid as dry cell weight reached peak values of 85% and 33%, respectively. Total carotenoids (pg cell−1) ranged from 1.52 to 3.58 in indoor cultures and 0.34 to 1.08 for outdoor cultures comparable to 0.24–4.75 to literature values. Because of its ability to produce high levels of biomass, lipids and carotenes in wastewater under harsh ambient climatic conditions, Scenedesmus species Novo has the potential to sustain biotechnological applications. We recommend continued exploration of extremophile algal species native to New Mexico and beyond, to develop strategies for a viable and sustainable bio-economy.

Transcriptional response to copper excess and identification of genes involved in heavy metal tolerance in the extremophilic microalga Chlamydomonas acidophila.

High concentrations of heavy metals are typical of acidic environments. Therefore, studies on acidophilic organisms in their natural environments improve our understanding on the evolution of heavy metal tolerance and detoxification in plants. Here we sequenced the transcriptome of the extremophilic microalga Chlamydomonas acidophila cultivated in control conditions and with 500 μM of copper for 24 h. High-throughput 454 sequencing was followed by de novo transcriptome assembly. The reference transcriptome was annotated and genes related to heavy metal tolerance and abiotic stress were identified. Analyses of differentially expressed transcripts were used to detect genes involved in metabolic pathways related to abiotic stress tolerance, focusing on effects caused by increased levels of copper. Both transcriptomic data and observations from PAM fluorometry analysis suggested that the photosynthetic activity of C. acidophila is not adversely affected by addition of high amounts of copper. Up-regulated transcripts include several transcripts related to photosynthesis and carbohydrate metabolism, transcripts coding for general stress response, and a transcript annotated as homologous to the oil-body-associated protein HOGP coding gene. The first de novo assembly of C. acidophila significantly increases transcriptomic data available on extremophiles and green algae and thus provides an important reference for further molecular genetic studies. The differences between differentially expressed transcripts detected in our study suggest that the response to heavy metal exposure in C. acidophila is different from other studied green algae.

Cyclobutane pyrimidine dimers photolyase from extremophilic microalga: Remarkable UVB resistance and efficient DNA damage repair

Bacteria living in the Antarctic region have developed several adaptive features for growth and survival under extreme conditions. Chlamydomonas sp. ICE-Lis well adapted to high levels of solar UV radiation. A putative photolyase was identified in the Chlamydomonas sp. ICE-L transcriptome. The complete cDNA sequence was obtained by RACE-PCR. This PHR encoding includes a polypeptide of 579 amino acids with clear photolyase signatures belonging to class II CPD-photolyases, sharing a high degree of homology with Chlamydomonas reinhardtii (68%). Real-time PCR was performed to investigate the potential DNA damage and responses following UVB exposure. CPD photolyase mRNA expression level increased over 50-fold in response to UVB radiation for 6h. Using photolyase complementation assay, we demonstrated that DNA photolyase increased photo-repair more than 116-fold in Escherichia coli strain SY2 under 100μw/cm2 UVB radiation. To determine whether photolyase is active in vitro, CPD photolyase was over-expressed. It was shown that pyrimidine dimers were split by the action of PHR2. This study reports the unique structure and high activity of the enzyme. These findings are relevant for further understanding of molecular mechanisms of photo-reactivation, and will accelerate the utilization of photolyase in the medical field.

Interactions between P-limitation and different C conditions on the fatty acid composition of an extremophile microalga

The extremophilic microalga Chlamydomonas acidophila inhabits very acidic waters (pH 2-3.5), where its growth is often limited by phosphorus (P) or colimited by P and inorganic carbon (CO(2)). Because this alga is a major food source for predators in acidic habitats, we studied its fatty acid content, which reflects their quality as food, grown under a combination of P-limited and different carbon conditions (either mixotrophically with light + glucose or at high or low CO(2), both without glucose). The fatty acid composition largely depended on the cellular P content: stringent P-limited cells had a higher total fatty acid concentration and had a lower percentage of polyunsaturated fatty acids. An additional limitation for CO(2) inhibited this decrease, especially reflected in enhanced concentrations of 18:3(9,12,15) and 16:4(3,7,10,13), resulting in cells relatively rich in polyunsaturated fatty acids under colimiting growth conditions. The percentage of polyunsaturated to total fatty acid content was positively related with maximum photosynthesis under all conditions applied. The two factors, P and CO(2), thus interact in their effect on the fatty acid composition in C. acidophila, and colimited cells P-limited algae can be considered a superior food source for herbivores because of the high total fatty acid content and relative richness in polyunsaturated fatty acids.

Productivity and selective accumulation of carotenoids of the novel extremophile microalga Chlamydomonas acidophila grown with different carbon sources in batch systems

Cultivation of extremophile microorganisms has attracted interest due to their ability to accumulate high-value compounds. Chlamydomonas acidophila is an acidophile green microalga isolated by our group from Tinto River, an acidic river that flows down from the mining area in Huelva, Spain. This microalga accumulates high concentrations of lutein, a very well-known natural antioxidant. The aim of this study is to assess use of different carbon sources (CO(2), glucose, glycerol, starch, urea, and glycine) for efficient growth of and carotenoid production by C. acidophila. Our results reveal that growth of the microalga on different carbon sources resulted in different algal biomass productivities, urea being as efficient as CO(2) when used as sole carbon source (~20 g dry biomass m(-2) day(-1)). Mixotrophic growth on glucose was also efficient in terms of biomass production (~14 g dry biomass m(-2) day(-1)). In terms of carotenoid accumulation, mixotrophic growth on urea resulted in even higher productivity of carotenoids (mainly lutein, probably via α-carotene) than obtained with photoautotrophic cultures (70% versus 65% relative abundance of lutein, respectively). The accumulated lutein concentrations of C. acidophila reported in this work (about 10 g/kg dry weight, produced in batch systems) are among the highest reported for a microalga. Glycerol and glycine seem to enhance β-carotene biosynthesis, and when glycine is used as carbon source, zeaxanthin becomes the most accumulated carotenoid in the microalga. Strategies for production of lutein and zeaxanthin are suggested based on the obtained results.

Osmotic adjustments in a psychrophilic alga, Xanthonema sp. (Xanthophyceae)

Extremophile microalgae show a remarkable ability to cope with harsh environments, yet still relatively little is known about the molecular basis for such tolerance. In this work the susceptibility of a psychrophylic alga isolated from alpine snowbanks, Xanthonema sp., to either water or salt stress conditions was assessed, and mechanisms for osmotic adjustment were investigated. Cultures were treated with increasing concentrations of either salts or non-permeant solutes, as polyethylene glycol, and the resulting effect on growth rate was measured. Both the accumulation of compatible osmolytes and the activity of cation transporters were studied in response to the exposure to hyperosmotic conditions. Xanthonema showed a differential sensitivity to osmotic and ionic stress, with a noteworthy tolerance to NaCl. No evidence was found supporting an osmo-induced intracellular accumulation of the most common osmoprotectants. Salt tolerance seems to rely upon the inducible expression of an amiloride-resistant Na +/H + antiporter. Since in snow fields osmotic unbalance due to freeze/thaw is more likely to occur than excess salts, results suggest an allochthonous origin of the strain.