Extremophilic microorganisms for the biotechnological production of added-value molecules

Extremophilic microorganisms are especially interesting from the biotechnological point of view. These microorganisms have developed mechanisms that lead to the production of valuable substances in order to survive in certain environments considered extreme for life. These substances can be used by the cosmetic, pharmaceutical industry and in human or animal feeding. Compounds obtained from extremophilic microorganisms have a great added-value since they can have unique properties and their nature can be very diverse. In this doctoral Thesis, substances such as carotenoid pigments, phycobiliproteins and carbohydrates (exopolysaccharides) are studied. Pigments are especially interesting since in recent years there has been a growing demand, by consumers, towards natural food dyes. Carotenoid pigments, and also phycobiliproteins, are of great interest for their use as feed additives, especially as color enhancers. These pigments also have great value as antioxidants and, like exopolysaccharides, have been found to display anti-cancer and anti-inflammatory activity. In addition to the currently known applications, there are microorganisms from extreme environment that are yet to be discovered and whose potential has not been studied. However, despite the biotechnological potential of extremophilic microorganisms, their use in biotechnology has certain limitations related to culture conditions, and the profitability of the compounds of interest produced. Therefore, it necessary to improve the production processes of these microorganisms in order to obtain competitive products in the market. Based on this, this Thesis had as a main objective the isolation and the study of the biotechnological potential of microorganisms from extreme environment, and improving the production strategies of target compounds, with special emphasis on reducing production costs. For this, two extremophilic microorganisms were studied: a halophilic archaea, Haloferax mediterranei and a cyanobacterium from extreme arid environment, Chroococcidiopsis sp. On the one hand, H. mediterranei stands out for its ability to produce a C50 carotenoid, particularly bacterioruberin, with high antioxidant capacity. Its possible medical application or in food industries, such as nutraceuticals, or obtaining functional foods makes bacterioruberin interesting. On the other hand, Chroococcidiopsis sp. stands out for its versatility, since it is found in very diverse extreme environments. Due to the extreme radiation that affects the rocks of the Atacama Desert, this cyanobacterium colonizes the inside of the rocks, so that the light received by a part of the endolithic colonies can be meager and diffuse irradiation. In order to be more efficient in capturing light, these cyanobacteria produce phycobiliproteins, a pigment that can be used as a food coloring and also stands out for its antioxidant capacity. Furthermore, these cyanobacteria respond to desiccation by producing a shell of exopolysaccharides, which provides a moisture environment to the cyanobacterium and has a potential use for its antibacterial and anti-inflammatory characteristics. Based on this, we optimized the growth and target metabolites production from both extremophiles. In the case of H. mediterranei, the effect of physicochemical parameters such as temperature, salinity and pH on the growth and carotenoids production was studied. In addition, the effect of nutritional factors such as glucose and yeast extract was analyzed. It allowed us to propose possible production strategies. After the isolation and identification of Chroococcidiopsis sp., the study of parameters such as the source and concentration of nitrogen or agitation was carried out to improve the cyanobacterial growth. Furthermore, the effect of other parameters such as the light irradiance on growth and the phycobiliproteins accumulation were studied. Finally, with the idea of reducing production costs, agricultural fertilizers were used as culture media, which means several advantages in large-scale cultures. The results obtained in this Thesis have contributed to improve the productivity of the biomass of these microorganisms and to the improvement in the environmental and nutritional conditions of cultivation for an optimum production of high-added value compounds. In addition, our results allow us to predict the best conditions that might contribute to address the biotechnological process on a large scale

Haloferax mediterranei Cells as C50 Carotenoid Factories

Haloarchaea produce C50 carotenoids such as bacterioruberin, which are of biotechnological in-terest. This study aimed to analyze the effect of different environmental and nutritional conditions on the cellular growth and dynamics of carotenoids accumulation in Haloferax mediterranei. The maximum production of carotenoids (40 µg·mL−1) was obtained during the stationary phase of growth, probably due to nutrient-limiting conditions (one-step culture). By seven days of culture, 1 mL culture produced 22.4 mg of dry weight biomass containing 0.18 % (w/w) of carotenoids. On the other hand, carbon-deficient cultures (low C/N ratio) were observed to be optimum for C50 bacterioruberin production by Hfx. mediterranei, but negatively affected the growth of cells. Thus, a two-steps process was evaluated for optimum carotenoids yield. In the first step, a nutri-ent-repleted culture medium enabled the haloarchaea to produce biomass, while in the second step, the biomass was incubated under osmotic stress and in a carbon-deficient medium. Under the conditions used, the obtained biomass contained 0.27% (w/w) of carotenoids after seven days, which accounts for 58.49 µg·mL−1 of carotenoids for a culture with turbidity 14.0.This work was funded by a research grant from MINECO Spain (RTI2018-099860-B-I00) and the University of Alicante (VIGROB-309). We are also indebted to the Andalusian Government (research project BIO-214). ZM was assisted by a pre-doctoral grant from “Plan Propio de Investigación” of the University of Huelva, Spain. MG was awarded with a pre-doctoral fellowship from the Valencian Community Government (ACIF/2019/043)

Impact of microalgae-bacteria interactions on the production of algal biomass and associated compounds

A greater insight on the control of the interactions between microalgae and other microorganisms, particularly bacteria, should be useful for enhancing the efficiency of microalgal biomass production and associated valuable compounds. Little attention has been paid to the controlled utilization of microalgae-bacteria consortia. However, the studies of microalgal-bacterial interactions have revealed a significant impact of the mutualistic or parasitic relationships on algal growth. The algal growth, for instance, has been shown to be enhanced by growth promoting factors produced by bacteria, such as indole-3-acetic acid. Vitamin B12 produced by bacteria in algal cultures and bacterial siderophores are also known to be involved in promoting faster microalgal growth. More interestingly, enhancement in the intracellular levels of carbohydrates, lipids and pigments of microalgae coupled with algal growth stimulation has also been reported. In this sense, massive algal production might occur in the presence of bacteria, and microalgae-bacteria interactions can be beneficial to the massive production of microalgae and algal products. This manuscript reviews the recent knowledge on the impact of the microalgae-bacteria interactions on the production of microalgae and accumulation of valuable compounds, with an emphasis on algal species having application in aquaculture

A combination of metallomics and metabolomics studies to evaluate the effects of metal interactions in mammals. Application to Mus musculus mice under arsenic/cadmium exposure

Metals interactionsArsenicCadmiumMetallomicsMetabolomicsMus musculusMass spectrometryArsenic and cadmium are toxic metals of environmental significance with harmful effects on man. To study the toxicological and biochemical effects of arsenic/cadmium in mammals a combined metallomic and metabolomic approach has been developed, complemented with the measurement of biochemical parameters in blood and histopathological evaluation of liver injury in mice Mus musculus under exposure to both xenobiotics. Size-exclusion chromatography (SEC) was combined with affinity chromatography (AF) and ICP-MS detection using species unspecific isotopic dilution analysis (SUID) to characterize the biological effects of As/Cd on selenium containing proteins in the bloodstream of exposed mice. On the other hand, both direct infusion mass spectrometry (DIMS) and gas chromatography–mass spectrometry (GC–MS) provided information about changes in metabolites caused by metals. The results show that As/Cd exposure produces interactions in the distribution of both toxics between organs and plasma of mice and antagonistic interactions with selenium containing proteins in the bloodstream. Interplay with essential metabolic pathways, such as energy metabolism and breakdown of membrane phospholipids were observed, which are more pronounced under As/Cd exposure. In addition, heavy metal and metalloid causes differential liver injury, manifested by steatosis (non-alcoholic fatty liver disease, NAFLD) and infiltration of blood cells into the space of Disse. Biological significance This work presents new contributions in the study of arsenic/cadmium interactions in mice Mus musculus under controlled exposure. With the combination of metallomic and metabolomic approaches the traffic of As and Cd from liver to kidney by means of blood was observed and excretion of As (as arsenic metabolites) or Cd (as MTCd) is inhibited with the simultaneous administration of As/Cd, and these toxic elements have important influence in the levels of seleno-proteins in the plasma. In addition, the metabolomic approach reveals inhibition of different metabolic cycles such as tricarboxylic acid and phospholipid degradation that causes membrane damage and apoptosis that is histopathologically confirmed. This article is part of a Special Issue entitled: Environmental and structural proteomics.The authors thank the projects CTM2012-38720-G03-01 (Ministerio de Economia y Competitividad-Spain), P08-FQM-03554 and P09-FQM-04659 (Consejeria de Innovacion, Andalusian government). Miguel Angel Garcia Sevillano thanks the Ministerio de Educacion for a predoctoral scholarship

Identification, biochemical composition and phycobiliproteins production of Chroococcidiopsis sp. from arid environment

Molecular and microscopic studies were performed to identify Chroococcidiopsis sp., an endolithic cyanobacterium, isolated from gypsum rocks of Atacama Desert (Chile). It was adapted to grow in mineral liquid medium, with 9 mM nitrate, bubbled with CO2-enriched air (2.5 % v/v), and continuously illuminated with a white light of 70 μmol photons m–2 s–1. The obtained biomass (productivity of 0.21 g L–1 d–1) had a C/N ratio of 6.67, and it contained carbohydrates (45.40 % of dry weight), proteins (36.72 %), lipids (5.60 %) nucleic acids (3.90 %) and ashes (8.28 %). The lipid fraction was particularly rich in palmitic (29.86 % of total fatty acids), linoleic (18.20 %), palmitoleic (12.75 %), linolenic (10.92 %), stearic (9.64 %) and capric acid (6.29 %). Chroococcidiopsis sp. accumulated phycobiliproteins in a light-dependent process and produced 204 mg g–1, under incident light of 10 μmol photons·m–2·s–1, with a relative abundance of 40.9 % for phycocyanin, 23.3 % for phycoerythrin, and 35.8 % for allophycocyanin. The biomass from this cyanobacterium can be a good source of these pigments, especially APC (maximum of 95 mg g dw−1), which are of interest for pharmacological, cosmetic, and food industries.Ministerio de Ciencia, Innovación y Universidades PGC2018–094076-B-I0

Chemically-Induced Production of Anti-Inflammatory Molecules in Microalgae

Microalgae have been widely recognized as a valuable source of natural, bioactive molecules that can benefit human health. Some molecules of commercial value synthesized by the microalgal metabolism have been proven to display anti-inflammatory activity, including the carotenoids lutein and astaxanthin, the fatty acids EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid), and sulphated polysaccharides. These molecules can accumulate to a certain extent in a diversity of microalgae species. A production process could become commercially feasible if the productivity is high and the overall production process costs are minimized. The productivity of anti-inflammatory molecules depends on each algal species and the cultivation conditions, the latter being mostly related to nutrient starvation and/or extremes of temperature and/or light intensity. Furthermore, novel bioprocess tools have been reported which might improve the biosynthesis yields and productivity of those target molecules and reduce production costs simultaneously. Such novel tools include the use of chemical triggers or enhancers to improve algal growth and/or accumulation of bioactive molecules, the algal growth in foam and the surfactant-mediated extraction of valuable compounds. Taken together, the recent findings suggest that the combined use of novel bioprocess strategies could improve the technical efficiency and commercial feasibility of valuable microalgal bioproducts production, particularly anti-inflammatory compounds, in large scale processes

Outdoor Large-Scale Cultivation of the Acidophilic Microalga Coccomyxa onubensis in a Vertical Close Photobioreactor for Lutein Production

The large-scale biomass production is an essential step in the biotechnological applications of microalgae. Coccomyxa onubensis is an acidophilic microalga isolated from the highly acidic waters of Río Tinto (province of Huelva, Spain) and has been shown to accumulate a high concentration of lutein (9.7 mg g-1dw), a valuable antioxidant, when grown at laboratory-scale. A productivity of 0.14 g L-1 d-1 was obtained by growing the microalga under outdoor conditions in an 800 L tubular photobioreactor. The results show a stable biomass production for at least one month and with a lutein content of 10 mg g-1dw, at pH values in the range 2.5–3.0 and temperature in the range 10–25 ºC. Culture density, temperature, and CO2 availability in highly acidic medium are rate-limiting conditions for the microalgal growth. These aspects are discussed in this paper in order to improve the outdoor culture conditions for competitive applications of C. onubensis.Authors want to thank the PhD-Grant (2015/7949) from CEIMAR (Marine International Campus of Excellence, Spain) to Juan Luis Fuentes