Outdoor cultivation of microalgae for carotenoid production: current state and perspectives

Microalgae are a major natural source for a vast array of valuable compounds, including a diversity of pigments, for which these photosynthetic microorganisms represent an almost exclusive biological resource. Yellow, orange, and red carotenoids have an industrial use in food products and cosmetics as vitamin supplements and health food products and as feed additives for poultry, livestock, fish, and crustaceans. The growing worldwide market value of carotenoids is projected to reach over US$1,000 million by the end of the decade. The nutraceutical boom has also integrated carotenoids mainly on the claim of their proven antioxidant properties. Recently established benefits in human health open new uses for some carotenoids, especially lutein, an effective agent for the prevention and treatment of a variety of degenerative diseases. Consumers’ demand for natural products favors development of pigments from biological sources, thus increasing opportunities for microalgae. The biotechnology of microalgae has gained considerable progress and relevance in recent decades, with carotenoid production representing one of its most successful domains. In this paper, we review the most relevant features of microalgal biotechnology related to the production of different carotenoids outdoors, with a main focus on β-carotene from Dunaliella, astaxanthin from Haematococcus, and lutein from chlorophycean strains. We compare the current state of the corresponding production technologies, based on either open-pond systems or closed photobioreactors. The potential of scientific and technological advances for improvements in yield and reduction in production costs for carotenoids from microalgae is also discussed.     

Outdoor cultivation of lutein-rich cells of <Emphasis Type="Italic">Muriellopsis</Emphasis> sp. in open ponds

The growth performance of the chlorophycean microalga Muriellopsis sp. outdoors in open tanks agitated with a paddlewheel and its ability to accumulate carotenoids have been evaluated throughout the year. The cells grown in the open system had free lutein as the main carotenoid, with violaxanthin, β-carotene, and neoxanthin also present. Lutein content of the dry biomass ranged from 0.4 to 0.6%, depending on the growth and environmental conditions. In addition, the biomass of Muriellopsis sp. had a high content in both protein and lipids with about half of the fatty acids being of the polyunsaturated type, with α-linolenic acid accounting for almost 30% of the total fatty acids. The effect of determinant parameters on the performance of the cultures in open tanks was evaluated. Operating conditions that allow the maintenance of productive cultures were established under semicontinuous regime for 9 months throughout the year. Biomass and lutein yields in the open system were not far from those in closed tubular photobioreactors, and reached productivity values of 20 g dry biomass, containing around 100 mg lutein m⁻² day⁻¹ in summer. The outdoor culture of Muriellopsis sp. in open ponds thus represents a real alternative to established systems for the production of lutein.     

Antioxidant activity of <Emphasis Type="Italic">Haematococcus pluvialis</Emphasis> cells grown in continuous culture as a function of their carotenoid and fatty acid content

The influence of culture conditions on the quality of Haematococcus pluvialis biomass is assessed. Continuously grown cells have been characterised with respect to their astaxanthin, fatty acid content, and antioxidant activity and compared with those of non-growing haematocysts. Moderate limitation of nitrate availability (1.7 mM) under continuous growth conditions favoured the production of reddish palmelloid cells whose extracts possessed antioxidant activity equivalent to that of haematocyst extracts, despite the lower astaxanthin content (0.6%d.wt.), which is compensated by a higher fatty acid level (7.6%d.wt.). Green cells produced under nitrate saturation conditions (>4.7 mM) exhibit only 40% antioxidant activity than palmelloid. In addition, the major fatty acid present in palmelloid cells was oleic acid (40%f.a.), whereas, in both green cells and haematocysts, the main fatty acids were myristic, palmitic, and oleic acid (20–30%f.a. each). Biomass extracts were fractionated and analysed. The antioxidant capacity was a function of both the carotenoid and the fatty acid profiles, the antioxidant capacity of astaxanthin diesters fraction being 60% higher than astaxanthin monoesters fraction and twice than free astaxanthin. In such a way, the evaluation of the quality of H. pluvialis biomass must take into account both variables. When considering the production of H. pluvialis biomass for human consumption, special attention should be paid to the one-step continuous system approach for the generation of cells rich in both astaxanthin and fatty acids, as they have high antioxidant activity but without thick hard cell wall.     

Changes in photosynthetic electron transfer and state transitions in an herbicide-resistant D1 mutant from soybean cell cultures

Anomalies in photosynthetic activity of the soybean cell line STR7, carrying a single mutation (S268P) in the chloroplastic gene psbA that codes for the D1 protein of the photosystem II, have been examined using different spectroscopic techniques. Thermoluminescence emission experiments have shown important differences between STR7 mutant and wild type cells. The afterglow band induced by both white light flashes and far-red continuous illumination was downshifted by about 4 °C and the Q band was upshifted by 5 °C. High temperature thermoluminescence measurements suggested a higher level of lipid peroxidation in mutant thylakoid membranes. In addition, the reduction rate of P700⁺ was significantly accelerated in STR7 suggesting that the mutation led to an activation of the photosystem I cyclic electron flow. Modulated fluorescence measurements performed at room temperature as well as fluorescence emission spectra at 77 K revealed that the STR7 mutant is defective in state transitions. Here, we discuss the hypothesis that activation of the cyclic electron flow in STR7 cells may be a mechanism to compensate the reduced activity of photosystem II caused by the mutation. We also propose that the impaired state transitions in the STR7 cells may be due to alterations in thylakoid membrane properties induced by a low content of unsaturated lipids.     

Differential protein expression in ovaries of uninfected and Babesia-infected southern cattle ticks, Rhipicephalus (Boophilus) microplus

We used gel electrophoresis and mass spectrometry to investigate differences in protein expression in ovarian tissues from Babesia bovis-infected and uninfected southern cattle tick, Rhipicephalus (Boophilus) microplus. Soluble and membrane proteins were extracted from ovaries of adult female ticks, and analyzed by isoelectric focusing (IEF) and one-dimensional or two-dimensional (2-D) gel electrophoresis. Protein patterns were analyzed for differences in expression between infected and uninfected ticks. 2-D separation of proteins revealed a number of proteins that appeared to be up- or down-regulated in response to infection with Babesia, in particular membrane/membrane-associated proteins and proteins in a low molecular mass range between 6 and 36 kDa. A selection of differentially expressed proteins was subjected to analysis by capillary-HPLC-electrospray tandem mass spectrometry (HPLC-ESI-MS/MS). Among the ovarian proteins that were up-regulated in infected ticks were calreticulin, two myosin subunits, an endoplasmic reticulum protein, a peptidyl-prolyl cis–trans isomerase (PPIase), a cytochrome c oxidase subunit, a glutamine synthetase, and a family of Kunitz-type serine protease inhibitors. Among the down-regulated ovarian proteins were another PPIase, a hemoglobin subunit, and a lysozyme. This study is part of an ongoing effort to establish a proteome database that can be utilized to investigate specific proteins involved in successful pathogen transmission.     

Microbial biomass and activity of an agricultural soil amended with the solid phase of pig slurries

Information about the mineralisation rates and effects on soil microorganisms must be obtained prior to the rational use of organic wastes in agriculture or forestry. The objective of this work was to study the mineralisation of two manures derived from the solid phase of pig slurries and the effects on the soil microbial biomass of an agricultural soil. Samples of this soil were mixed at two different rates with two manures derived from the solid phase of pig slurry (composted, CSP, and non-composted, NSP), and then were incubated during 163 days. Carbon mineralised from manures was fitted to first-order kinetic model, and small differences were found between manures despite the composting of one of them. Approximately 45% of the C added was mineralised in the experimental period. The soil microbial biomass C (C(mic)) was increased by the amendments according to the application rate. The sudden increases of the qCO(2) in the treated samples were ephemeral. The most appreciable differences between these manures were those related with net N mineralisation, being greater in the NSP-treated samples. The application of the solid phase of pig slurries, composted or not, could be a feasible practice to enhance in a short-term the microbial biomass of agricultural soils. In order to avoid an excessive release of inorganic N, the use of composted materials is preferred.     

Assessment of bacterial diversity in the cattle tick Rhipicephalus (Boophilus) microplus through tag-encoded pyrosequencing

Background  

Ticks are regarded as the most relevant vectors of disease-causing pathogens in domestic and wild animals. The cattle tick, Rhipicephalus (Boophilus) microplus, hinders livestock production in tropical and subtropical parts of the world where it is endemic. Tick microbiomes remain largely unexplored. The objective of this study was to explore the R. microplus microbiome by applying the bacterial 16S tag-encoded FLX-titanium amplicon pyrosequencing (bTEFAP) technique to characterize its bacterial diversity. Pyrosequencing was performed on adult males and females, eggs, and gut and ovary tissues from adult females derived from samples of R. microplus collected during outbreaks in southern Texas.     

High sero-prevalence of caseous lymphadenitis identified in slaughterhouse samples as a consequence of deficiencies in sheep farm management in the state of Minas Gerais, Brazil

Background

Multiple congenital ocular anomalies (MCOA) syndrome is a hereditary congenital eye defect that was first described in Silver colored Rocky Mountain horses. The mutation causing this disease is located within a defined chromosomal interval, which also contains the gene and mutation that is associated with the Silver coat color (PMEL17, exon 11). Horses that are homozygous for the disease-causing allele have multiple defects (MCOA-phenotype), whilst the heterozygous horses predominantly have cysts of the iris, ciliary body or retina (Cyst-phenotype). It has been argued that these ocular defects are caused by a recent mutation that is restricted to horses that are related to the Rocky Mountain Horse breed. For that reason we have examined another horse breed, the Icelandic horse, which is historically quite divergent from Rocky Mountain horses.

Results

We examined 24 Icelandic horses and established that the MCOA syndrome is present in this breed. Four of these horses were categorised as having the MCOA-phenotype and were genotyped as being homozygous for the PMEL17 mutation. The most common clinical signs included megaloglobus, iris stromal hypoplasia, abnormal pectinate ligaments, iridociliary cysts occasionally extending into the peripheral retina and cataracts. The cysts and pectinate ligament abnormalities were observed in the temporal quadrant of the eyes. Fourteen horses were heterozygous for the PMEL17 mutation and were characterized as having the Cyst-phenotype with cysts and occasionally curvilinear streaks in the peripheral retina. Three additional horses were genotyped as PMEL17 heterozygotes, but in these horses we were unable to detect cysts or other forms of anomalies. One eye of a severely vision-impaired 18 month-old stallion, homozygous for the PMEL17 mutation was examined by light microscopy. Redundant duplication of non-pigmented ciliary body epithelium, sometimes forming cysts bulging into the posterior chamber and localized areas of atrophy in the peripheral retina were seen.

Conclusions

The MCOA syndrome is segregating with the PMEL17 mutation in the Icelandic Horse population. This needs to be taken into consideration in breeding decisions and highlights the fact that MCOA syndrome is present in a breed that are more ancient and not closely related to the Rocky Mountain Horse breed.