Effect of initial biomass density on growth and astaxanthin production of Haematococcus pluvialis in an outdoor photobioreactor

Initial biomass density (IBD) is an important factor that affects the viability and productivity of microalgae particularly when sunlight is used for photosynthesis. In this paper, the effect of IBD on photosynthesis, growth, and astaxanthin production of the green microalga Haematococcus pluvialis during the astaxanthin induction stage was studied in a glass column photobioreactor during different seasons. Of seven IBDs, i.e., 0.1, 0.5, 0.8, 1.5, 2.7, 3.5, and 5.0 g L^?1 tested, 0.8 g L^?1 IBD was optimal and resulted in the highest astaxanthin productivity of 17.1 mg L^?1 day^?1. Severe photoinhibition of photosynthesis occurred at low IBD (e.g., 0.1 g L^?1) cultures, especially in the winter, and severe light limitation to individual cells in high IBD cultures (>2.7 g L^?1) were responsible for reduced astaxanthin production. This was the first report quantitatively assessing IBD as the key limiting factor for astaxanthin production in H. pluvialis outdoor cultivation.     

Enhanced astaxanthin production from microalga,Haematococcus pluvialis by two-stage perfusion culture with stepwise light irradiation

For efficient astaxanthin production from the culture of green microalga, Haematococcus pluvialis, a two-stage mixotrophic culture system was established with stepwise increased light irradiance. By perfusion process, high density biomass (2.47 g/L) was achieved during the vegetative stage due to no detrimental effect of inhibitory metabolites, which was 3.09 and 1.67 times higher than batch and fed-batch processes, respectively. During the induction stage, biomass and astaxanthin were subsequently produced to the very high level 12.3 g/L and 602 mg/L, under stepwise increased light irradiance (150–450 μE/m²/s), respectively. These results indicate that the combinatorial approach of perfusion culture during the vegetative stage and stepwise light irradiation during the induction stage is a promising strategy for the simultaneous production of high concentration of biomass and astaxanthin in microalgae including H. pluvialis.     

Optimal design of scalable photo-bioreactor for phototropic culturing of Haematococcus pluvialis

The unicellular green microalgae, Haematococcus pluvialis, has been examined as a microbial source for the production of astaxanthin, which has been suggested as a food supplement for humans and is also prescribed as an ingredient in eye drops because of its powerful anti-oxidant properties. In this study, we estimated the effects of the slope of a V-shaped bottom design, the volumetric flow rate of air, height/diameter (H/D) ratio, and diameter of an air sparger on the performance of a photo-bioreactor. These parameters were selected because they are recognized as important factors effecting the mixing that produces increased cell density in the reactor. The mixing effect can be measured by changes in optical density in the bioreactor over a period of time. A 6 L indoor photo-bioreactor was prepared in a short time period of 24 h for the performance study. A bioreactor designed with a V-shaped bottom with a slope of 60° showed an optical density change of 0.052 at 680 nm, which was sixfold less than the change in a photo-bioreactor designed with a flat bottom. Studies exploring the effects of bioreactor configuration and a porous metal sparger with a 10 μm pore size showed the best performance at an H/D ratio of 6:1 and a sparger diameter of 1.3 cm, respectively. The optimal rate of air flow was 0.2 vvm. The indoor culture of microalgae in the photo-bioreactor was subsequently carried for an application study using the optimal values established for the important factors. The indoor culture system was composed of a light source controlled according to cell phase, a carbon dioxide feeder, a bag-type reactor with an H/D ratio of 6:1, and a temperature controller. Results demonstrated the efficient production of microalgal cells and astaxanthin in the amounts of 2.62 g/L and 78.37 mg/L, respectively, when using adequate hydrodynamic mixing. Furthermore, the optimal design of a photo-bioreactor can be applied for the phototropic culturing of other microalgae for mass production.     

Tagging of biomolecules with deuterated water (D2O) in commercially important microalgae

To understand the effect of any biomolecules in specific metabolic pathways in humans, bioavailability and for other basic understanding, stable isotopically-labelled biomolecules (preferably deuterated) is the fundamental pre-requisite. Production of deuterated biomolecules such as, astaxanthin, β-carotene, lutein, chlorophyll-a, and eicosapentaenoic acid (EPA, 20:5n-3) by metabolic tagging have been shown in commercially important microalgae, Haematococcus pluvialis and Phaeodactylum tricornutum. These microalgae were grown in appropriate optimized medium supplemented with 25 % (v/v) deuterated water. LC–MS analysis showed a maximum of 20, 25, 23, 24, and 27 % replacement of hydrogen by deuterium atoms respectively in astaxanthin, β-carotene, lutein, chlorophyll-a, and EPA. To our knowledge, this is the first report on the production of deuterated astaxanthin, chlorophyll-a and EPA by these microalgae.     

A semi-continuous cultivation method for <Emphasis Type="Italic">Haematococcus pluvialis</Emphasis> from non-motile cells to motile cells

Non-motile cells of Haematococcus pluvialis grow slowly, whereas motile cells grow fast and divide frequently. Cultivation from non-motile cells to motile cells of H. pluvialis was implemented to promote semi-continuous production. When old cultures which consist of non-motile cells were inoculated in fresh medium with an inoculation amount less than 15%, zoospores were produced in the non-motile cells and developed into motile cells, as the concentration of astaxanthin inducer in the medium was below the threshold value. This process was accomplished within 3 days after inoculation. Furthermore, enhancing KNO₃ content to 1200 mg L^?1 or reducing light intensity to 20 μmol photons m^?2?s^?1 could increase growth during the late culturing period of H. pluvialis and postpone the next round of transformation from motile cells to non-motile cells. A semi-continuous cultivation method for H. pluvialis from non-motile cells to motile cells is proposed in order to regulate the life cycle and promote industrial production. This cultivation mode shortens the inoculum cultivation stage and simplifies the production process of H. pluvialis, showing considerable commercial potential.     

Commercial production of astaxanthin from Haematococcus pluvialis using 25,000-liter outdoor photobioreactors

Recent developments in photobioreactor technology havemade the production of astaxanthin from Haematococcus pluvialis commercially viable. The coreof our astaxanthin production chain is the AquasearchGrowth Module (AGM), a 25,000 L enclosed andcomputerized outdoor photobioreactor.At Aquasearch's newly expanded facility (dedicatedJanuary 1999), three AGMs (total volume 75,000 L) areused to produce large amounts of clean, fast growing,H. pluvialis. The H. pluvialis biomassproduced in the AGMs is transferred daily to a pondculture system, where carotenogenesis and astaxanthinaccumulation are induced. Following a 5-dayinduction period, the reddened H. pluvialiscells are harvested by gravitational settling. Theharvested biomass, which averages > 2.5 astaxanthinas percent of the dry weight, is transferred to aprocessing building where a high pressure homogenizeris used to rupture the cells' walls. Once the biomasshas been homogenized, it is dried to less than 5%moisture utilizing proprietary drying technology. Thedried product is then ready to be packaged accordingto customer needs.The photobioreactor research program has almostdoubled the performance of the AGMs in the first ninemonths of operations: standing biomass concentrationincreased from 50 to 90 g m^-2 and productionincreased from 9 to 13 g m^-2 d^-1 during thisperiod. Here, we discuss the significance of thesechanges in production parameters to the viability ofcommercial production of astaxanthin and other highvalue products from microalgae.     

Extraction of astaxanthin from Haematococcus pluvialis by supercritical carbon dioxide fluid with ethanol modifier

Conventional solvent extraction methods cannot attain high‐quality antioxidant extracts from microalgae and also require solvent recovery and posttreatment. In this study, we utilized environmental friendly supercritical carbon dioxide fluid extraction (SFE‐CO₂) techniques to obtain pigment (i.e. astaxanthin) from Haematococcus pluvialis. The effects of key operating parameters on the extraction efficiency of astaxanthin were investigated, giving an optimal condition of H. pluvialis weight, 6.5 g; CO₂‐flow rate, 6.0 NL/min; extraction time, 20 min; extraction pressure, 4500 psi; volume of ethanol modifier added, 9.23 mL/g; extraction temperature, 50°C; modifier composition, 99.5%. Under these optimum conditions, the astaxanthin yield was 73.9% (10.92 mg/g dry H. pluvialis powder) after eight cycle of extraction cycles. The saponification index (C_S/C₀, representing the ratio of astaxanthin concentration after and before the saponification procedures) of the extract could be increased from 1 to 12.78 by saponification with 3.5 M NaOH.     

Role of media composition in biomass and astaxanthin production of Haematococcus pluvialis under two-stage cultivation

In the present study, the effects of four different culture media on the growth, astaxanthin production and morphology of Haematococcus pluvialis LUGU were studied under two-step cultivation. The interactions between astaxanthin synthesis and secondary messengers, reactive oxygen species (ROS) and mitogen-activated protein kinases (MAPK) were also investigated. In the first green vegetative cell stage, maximal biomass productivity (86.54 mg L⁻¹ day⁻¹) was obtained in BBM medium. In the induction stage, the highest astaxanthin content (21.5 mg g⁻¹) occurred in BG-11 medium, which was higher than in any other media. The expressions of MAPK and astaxanthin biosynthetic genes in BG-11 were higher than in any other media, whereas the ROS content was lower. Biochemical and physiological analyses suggested that the ROS, MAPK and astaxanthin biosynthetic gene expression was involved in astaxanthin biosynthesis in H. pluvialis under different culture media conditions. This study proposes a two-step cultivation strategy to efficiently produce astaxanthin using microalgae.

     

Effects of iron on fatty acid and astaxanthin accumulation in mixotrophic Chromochloris zofingiensis

The integration of oleaginous microalgae cultivation with high-value products is considered a low-cost approach for manufacturing algae-based biodiesel. The objective of this study was to investigate the potential of using Fe(II) to produce fatty acids and astaxanthin in mixotrophic Chromochloris zofingiensis. Fatty acid biosynthesis was less sensitive than astaxanthin formation to the changes in Fe²⁺ concentrations. However, the enhancement and inhibition of fatty acids formation were concomitant with an increase and a decrease in the production of astaxanthin, respectively. The highest contents of astaxanthin and total fatty acids were simultaneously obtained at 0.2 mM Fe²⁺ with the corresponding values of 2.2 mg g^?1 (i.e., 25.8 mg l^?1) and 41.8 % dry weight (i.e., 5 g l^?1).     

Construction of transplastomic lettuce (Lactuca sativa) dominantly producing astaxanthin fatty acid esters and detailed chemical analysis of generated carotenoids

The plastid genome of lettuce (Lactuca sativa L.) cv. Berkeley was site-specifically modified with the addition of three transgenes, which encoded β,β-carotenoid 3,3′-hydroxylase (CrtZ) and β,β-carotenoid 4,4′-ketolase (4,4′-oxygenase; CrtW) from a marine bacterium Brevundimonas sp. strain SD212, and isopentenyl diphosphate isomerase from a marine bacterium Paracoccus sp. strain N81106. Constructed transplastomic lettuce plants were able to grow on soil at a growth rate similar to that of non-transformed lettuce cv. Berkeley and generate flowers and seeds. The germination ratio of the lettuce transformants (T₀) (98.8 %) was higher than that of non-transformed lettuce (93.1 %). The transplastomic lettuce (T₁) leaves produced the astaxanthin fatty acid (myristate or palmitate) diester (49.2 % of total carotenoids), astaxanthin monoester (18.2 %), and the free forms of astaxanthin (10.0 %) and the other ketocarotenoids (17.5 %), which indicated that artificial ketocarotenoids corresponded to 94.9 % of total carotenoids (230 μg/g fresh weight). Native carotenoids were there lactucaxanthin (3.8 %) and lutein (1.3 %) only. This is the first report to structurally identify the astaxanthin esters biosynthesized in transgenic or transplastomic plants producing astaxanthin. The singlet oxygen-quenching activity of the total carotenoids extracted from the transplastomic leaves was similar to that of astaxanthin (mostly esterified) from the green algae Haematococcus pluvialis.     

Assessing contamination of microalgal astaxanthin producer <Emphasis Type="Italic">Haematococcus</Emphasis> cultures with high-resolution melting curve analysis

Due to its superior antioxidant capabilities and higher activity than other carotenoids, astaxanthin is used widely in the nutraceutical and medicine industries. The most prolific natural producer of astaxanthin is the unicellular green microalga Haematococcus pluvialis. The correct identification of any contaminants in H. pluvialis cultures is both essential and nontrivial for several reasons. Firstly, while it is possible to distinguish the main microalgal contaminant Coelastrella sp. (in H. pluvialis cultures), in practice, it is frequently a daunting and error-prone task for personnel without extensive experience in the microscopic identification of algal species. Secondly, the undetected contaminants may decrease or stop production of astaxanthin. Lastly, the presence of other contaminants such as fungi can eventually infect and destroy the whole algae collection. In this study, high-resolution melting (HRM) analysis was developed to detect microalgal and fungal contamination. The developed diagnostic procedure allowed to distinguish pure H. pluvialis samples from cultures contaminated with low amounts (1.25 ng/ml) of microalgal DNA and fungal DNA (2.5 ng/ml). Such discrimination is not possible with the use of microscopy observations and allows fast and efficient collection testing.     

Preparation of astaxanthin by lipase-catalyzed hydrolysis from its esters in a slug-flow microchannel reactor

Natural astaxanthin is widely used as a food and cosmetics additive because of its multiple biological activities. However, astaxanthin produced by Haematococcus pluvialis is generally esterified, and its activity is far less than that of free astaxanthin. Hydrolysis of astaxanthin esters to free astaxanthin by enzymes can overcome the drawbacks of chemical saponification methods. In this paper, a slug-flow microchannel reactor was constructed and tested in enzymatic hydrolysis of astaxanthin esters. The reactor consists of a “T” slug-flow generator, a stainless-steel microchannel, two constant-flow pumps, and a temperature controller. The reactor has the advantages of simple configuration and easy scale-up, and is suitable for two-phase biochemical reactions. Using the microchannel reactor, astaxanthin esters in H. pluvialis oil were efficiently hydrolyzed to free astaxanthin by lipase from Aspergillus niger. After hydrolysis, the content of free astaxanthin in H. pluvialis oil was 18.8 mg/L, 7.83-times higher than that before hydrolysis (2.13 mg/L). The hydrolysis rate reached 75.4 %. These results indicate that the microchannel reactor can be useful for the production of free astaxanthin from its esters.     

Concentration and distribution characteristics of airborne fungi in indoor and outdoor air of Tehran subway stations

The concentration and distribution characteristics of airborne fungi were investigated in indoor and outdoor air of two metro stations (Imam Khomeini and Sadeghiyeh stations) in Tehran subway. Samples were taken from indoor air at each station from platform and ticket office area also from adjacent outdoor air of each station. Indoor sampling was conducted for two types of trains, old and new. The concentration of airborne fungi ranged from 21 CFU/m³ at the outdoor air of Imam Khomeini station to 1,402 CFU/m³ in the air samples collected from the platform of this station. Results showed that airborne fungi concentrations at indoor air were higher than the outdoor air (p < 0.05), and fungal levels significantly correlated with the number of passengers (p < 0.05; r = 0.68) and RH % (p < 0.05; r = 0.43). Sixteen genera of fungi were isolated in all sampled environments. The predominant genera identified in indoor and outdoor air were Penicillium spp. (34.88 % of total airborne fungi) and Alternaria spp. (29.33 % of total airborne fungi), respectively. The results of this study showed that the indoor air quality in subway is worse than the outdoor air.     

A novel low-cost thin-film flat plate photobioreactor for microalgae cultivation

In this study, a novel thin-film flat plate photobioreactor (FPPBR) mounted with baffles and a 61.2 m² (2,000 L) photobioreactor system based on the FPPBR were developed. The flow of the fluid in the thinfilm photobioreactor was investigated by means of computational fluid dynamics (CFD). The cultivation of Chlorella sp. and Scenedesmus dimorphus in the thin-film FPPBR was carried out outdoors. The results showed that the flow of culture medium in different channels was uniform. In outdoor cultivation, the biomass productivity in the FPPBR with baffles was 25.2% higher than that in the FPPBR without baffles. In the pilot-scale FPPBR system, the maximum area productivity of Scenedesmus dimorphus reached 21.9 g/m²/day. When the service time of the photobioreactor was 1 and 3 years, the capital cost of the photobioreactor was 4.72 and 2.45 $ kg_algae, respectively. The results demonstrated that the thin-film FPPBR was cost effective, and it has the potential to be used for mass cultivation of microalgae.     

Astaxanthin production from the green alga Haematococcus pluvialis with different stress conditions

Summary Haematococcus pluvialis was induced to produce the astaxanthin pigment. A factorial design was carried out with three sodium acetate concentrations, 0.025, 0.05, 0.1 (g/l), and three NaCl concentrations (0.1, 0.2 and 0.4 %).The best conditions in algal culture for astaxanthin production were 0.2 % NaCl, 0.025 g/l sodium acetate and 0.05 g/l sodium acetate, each a 3.0, 1.83 and 1.78 % of astaxanthin, production in base to total dry weight, respectively. The higher astaxanthin production by bioreactor was 18.6 mg/l in the condition with 0.2 % NaCl.     

Utilization of gaseous emissions from a methanol plant for cultivation of <Emphasis Type="Italic">Acutodesmus obliquus</Emphasis>

This study aimed to culture the green alga Acutodesmus obliquus utilizing the gaseous emissions containing a high concentration of CO₂ (99.13 %) from a methanol plant and study the tolerance of microalgae. The effect of CO₂ concentration, aeration rate, inoculum concentration, intermittent sparging, and nitrogen sources on the growth of A. obliquus was examined. Acutodesmus obliquus also was cultivated in a 500-L pilot outdoor tubular photobioreactor (OTP) to advance the laboratory scale system to outdoor scale-up applications. The results showed that A. obliquus could tolerate high CO₂ concentrations of 50 %, and a maximum biomass of 0.935 g L^?1 (dry weight) was achieved at 20 % CO₂. An aeration rate of 500 mL min^?1, inoculum concentration (optical density at 680 nm [OD₆₈₀]?=?0.3), and intermittent sparging of 10 min per 2 h enhanced growth to the optimum and influenced culture pH and photosynthesis. Urea as a nitrogen source was shown to be more beneficial to cell growth. A urea concentration of 0.3 g L^?1 and an N/P ratio of 15 led to maximum biomass accumulation thus enhancing the gaseous emission utilization efficiency. In conclusion, this work demonstrated that gaseous emissions containing high concentration of CO₂ from a methanol plant could be directly introduced into A. obliquus cultures and that A. obliquus was suitable well for large-scale outdoor cultivation in a tubular photobiorecator.     

Screening for a low-cost Haematococcus pluvialis medium reveals an unexpected impact of a low N/P ratio on vegetative growth

The green alga Haematococcus pluvialis is the current best source of natural astaxanthin, a high-value carotenoid. Traditionally, the production process of astaxanthin by this algae is achieved by a two-stage system: during the first stage, vegetative “green” cells are produced and then converted, in the second stage, into cysts that accumulate astaxanthin. In this work, a medium screening strategy based on the mixing of a three-component hydroponic fertilizer was applied to identify a new formulation optimized for the vegetative stage. A maximal and high cell density of 2?×?10⁶ cells mL^?1 was obtained in a medium containing a high level of phosphate relative to nitrate, resulting in a N/P ratio much lower than commonly used media for H. pluvialis. In this medium, cells remained at the vegetative and motile stage during a prolonged period of time. Both high cell density culture and motile stage persistence was proved to be related to the N/P feature of this medium. We conclude that the macrozoid stage of H. pluvialis is favored under high-P and low-N supply and that low-cost hydroponic fertilizers can be successfully used for achieving high density cultures of vegetative cells of H. pluvialis.     

Influence of microalgae cell wall characteristics on protein extractability and determination of nitrogen-to-protein conversion factors

Additional evidence about the influence of the cell wall physical and chemical characteristics on protein extractability was determined by calculating the conversion factors of five different microalgae known to have different cell wall composition, and their protein extracts. The conversion factors obtained for crude rigid cell walled Chlorella vulgaris, Nannochloropsis oculata and Haematococcus pluvialis were 6.35, 6.28 and 6.25, respectively, but for their protein extracts the values were lower with 5.96, 5.86 and 5.63. On the other hand, conversion factor obtained for fragile cell walled microalgae Porphyridium cruentum and Athrospira platensis was 6.35 for the former and 6.27 for the latter, with no significant difference for their protein extract with 6.34 for the former and 6.21 for the latter. In addition, the highest hydro-soluble protein percentage recovered from total protein was for P. cruentum 80.3 % and A. platensis 69.5 % but lower for C. vulgaris with 43.3 %, N. oculata with 33.3 % and H. pluvialis with 27.5 %. The study spotted the light on the influence of the cell wall on evaluating the conversion factor and protein extractability. In addition, it showed the necessity of finding the conversion factor every time accurate protein quantification is required, and proved that there is not a universal conversion factor that can be recommended.