Multistage operation of airlift photobioreactor for increased production of astaxanthin from Haematococcus pluvialis

An internally radiating photobioreactor was applied for the production of astaxanthin using the unicellular green alga Haematococcus pluvialis. The cellular morphology of H. pluvialis was significantly affected by the intensity of irradiance of the photobioreactor. Small green cells were widespread under lower light intensity, whereas big reddish cells were predominant under high light intensity. For these reasons, growth reflected by cell number or dry weight varied markedly with light conditions. Even under internal illumination of the photobioreactor, light penetration was significantly decreased as algal cells grew. Therefore, we employed a multistage process by gradually increasing the internal illuminations for astaxanthin production. Our results revealed that a multistage process might be essential to the successful operation of a photobioreactor for astaxnthin production using H. pluvialis.     

Photoautotrophic high-density cultivation of vegetative cells of Haematococcus pluvialis in airlift bioreactor

This work aimed to investigate the effects of the bioreactor configurations and their design variables on the cultivation of vegetative cells Haematococcus pluvialis to achieve sustainable high cell density. The addition of vitamin B to F1 growth medium could appreciably enhance the final cell density. Employing this medium, the cultivation in the airlift bioreactor was demonstrated to outperform the bubble column at the same operating conditions. Aeration was crucial for a proper growth of the alga in the airlift bioreactor, but it must be maintained at low level to minimize shear stress. The most appropriate aeration velocity (superficial velocity) was at the lower limit of the pump, i.e. 0.4 cm s(-1) and a smaller riser was shown to have positive influence on the cell growth. A 1% CO(2) supplement to the air supply considerably enhanced the growth rate of H. pluvialis and the most suitable light intensity for the growth was at 20 micromol photon m(-2) s(-1). The semi-continuous culture was successfully implemented with the optimal airlift bioreactor design and under optimal conditions the harvest could be performed every four days with the specific growth rate of 0.31 d(-1).     

High efficiency production of astaxanthin by autotrophic cultivation of Haematococcus pluvialis in a bubble column photobioreactor

Haematococcus pluvialis was cultivated under photoautotrophic conditions in a bubble column with fed-batch addition of nutrients, especially nitrate, and a cell number above 5 × 10⁶ cells mL⁻¹ was attained after 300 h.The reduction of nutrient concentrations accompanied by dilution of the fermentation broth and an increase in the light intensity enhanced accumulation of astaxanthin. The final astaxanthin concentration of 390 mg L⁻¹ was several times higher than ever reported. This combination of fed-batch addition of nutrients and dilution of broth for nutrient deficiency is a promising method for attainment of high cell and astaxanthin concentrations in a bubble column photobioreactor.     

A novel double-layered photobioreactor for simultaneous Haematococcus pluvialis cell growth and astaxanthin accumulation

This study proposes a novel double-region photobioreactor to simplify the commercial two-stage process of astaxanthin production by the cultivation of Haematococcus pluvialis. The feasibility of the double-region photobioreactor has been investigated and found to achieve high biomass yield in the inner core region and simultaneous astaxanthin accumulation in the outer jacket region. Among many environmental factors, light condition and nitrate level were manipulated for selective cell growth and astaxanthin production. In the outer jacket region, efficient astaxanthin production was accomplished by excessive irradiation (770+/-20 microE m(-2)s(-1)) and nitrate starvation, resulting in a dramatic increase of astaxanthin productivity (357 mg l(-1)). Meanwhile, attenuated light energy (40+/-3 microE m(-2)s(-1)) and sufficient nitrates were supplied to the vegetative cells in the inner core region, which continued to grow to a high cell concentration of 4.0 x 10(5) cells ml(-1). The sequential batch run was performed by utilizing the high-density vegetative cells as inoculum for the next batch run. The cultivation results exhibited similar trends as the previous run, reaching high cell density (4.3 x 10(5) cells ml(-1)) in the inner core region and high astaxanthin content (5.79% on a dry weight basis) in the outer jacket region. The present study indicates that the double-region photobioreactor and its method of operation possess a good potential for commercial production of astaxanthin by H. pluvialis.     

Hydrodynamic behaviour of animal cell microcarrier suspensions in split-cylinder airlift bioreactors

Hydrodynamic characteristics of suspensions of microcarriers used for culturing anchorage dependent animal cells are reported in split-cylinder internal-loop airlift bioreactors. Cell culture media are simulated using salt solutions that duplicate the ionic strengths of typical media. Effects of solids loading (0–30 kg·m^–3), microcarrier particle size (150–300×10^–6 m diameter) and density (1030–1050 kg·m^–3) on gas induced circulation of the slurry, mixing time, gas holdup and gas velocity requirements to attain complete suspension of solids are discussed for two reactors with aspect ratios of 7.6 and 14.5, but equal riser-to-downcomer cross-sectional area ratios of 1.0, aerated at low air flow rates (0–8×10^–6 m³·s^–1) through a sintered glass sparger with 110×10^–6 m diameter pores. The study covers the ranges of solids concentrations, types, densities, particle sizes and aeration rates that are of relevance in animal cell culture applications.Airlift bioreactors displayed suitable hydrodynamic characteristics for potentially supporting anchorage dependent cell cultures on microcarriers at carrier loadings similar to those that are currently used in stirred tank bioreactors. The minimum gas flow rates and the induced liquid circulation rates necessary to achieve and maintain suspension of the heaviest and the largest microcarriers were well within practicable limits, limits which have been shown to be withstood by animal cells in non-anchorage dependent suspension culture in airlift bioreactors. No floatation problems were encountered with the carriers, nor was sedimentation a problem so long as the identified minimum suspension criteria were met. Chisti's liquid circulation equation, originally intended for two-phase flow, applied to the three-phase gas-liquid-microcarrier systems.     

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.

     

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.     

Enhancement of astaxanthin production from Haematococcus pluvialis under autotrophic growth conditions by a sequential stress strategy

Abstract

The study of microalgal culture has been growing in recent decades, because the cellular structure of microalgae has diverse highly valuable metabolites that have attract attention of numerous companies and research groups. The pigment astaxanthin is considered one of the most powerful antioxidants in nature. The microalga Haematococcus pluvialis was proposed as one of the best natural astaxanthin sources, because it can accumulate high amount of the pigment. In this work, we studied different stress treatments on H. pluvialis growth cultures as well as astaxanthin production under autotrophic growth conditions. The results showed that extending nitrogen starvation before increasing radiation intensity up to 110?μmol photons m^?2 s^?1 during late the palmella cell phase incremented the astaxanthin concentration up to 2.7% of dry biomass with an efficient light energy utilization during the stress stage.     

TheCaesalpinia peltophoroides cell cycle under different aeration conditions

Summary Cell cycle parameters were studied inCaesalpinia peltophoroides meristems proliferating under different oxygen tensions. This species has been selected for mixed planting in degraded areas in Brazil, some of which are occasionally flooded. As the species’ adaptation to oxygen deprivation during flooding is not fully understood, the objective of this study was to characterize the meristematic activity of root cells under various oxygen regimes. Synchronous binucleate cells, induced by a pulse of caffeine, showed a cell-cycle time constant under both control (5.6 mg of O₂ per l) and oxygenated conditions (7.9 and 3.2 mg of O₂ per l). The whole cell cycle lasted 10 h, although the relative duration of metaphase and anaphase/early telophase increased in more hypoxic conditions. The species appeared to utilise oxygen diffusing from the shoot to the root system to maintain cell division and root growth.     

Effect of cultivation parameters on growth and pigment biosynthesis in flagellated cells of Haematococcus pluvialis

The volvocalean microalga Haematococcus pluvialis is used as a sourceof the ketocarotenoid astaxanthin for applications in aquaculture and thepharmaceutical and cosmetic industries. This green alga accumulatesastaxanthin, mostly esterified, canthaxanthin and echinenone in lipid vesiclesoutside the chloroplast. This accumulation process normally but notexclusively accompanies formation of the resting state in the developmentalcycle. With regard to increased bioavailability of the accumulated secondarycarotenoids, the fragility of the extracellular matrix makes the flagellatedstate of H. pluvialis an interesting alternative to the thick-walledaplanospore state. A two-step batch cultivation scheme was developed thatleads to accumulation of secondary carotenoids in flagellated cells of H. pluvialis (strain 192.80, Göttingen, Germany). Germination ofgreen aplanospores during the first step of cultivation proceeded optimallyunder 30 mol photon m^-2 s^-1 of whitefluorescent light at 20 °C. For optimal induction and enhancementof carotenoid biosynthesis, the flagellated cells formed were then exposedto a decreased level of nitrate (0.4 mM KNO₃) and to enhancedirradiance (150 mol photon m^-2 s^-1). Under theseconditions, which still permitted cell division and chlorophyll synthesisduring the first two days of exposure, carotenoid accumulation in theflagellated cells reached 2° of dry mass at the fourth day of exposure. Asa mixotrophic carbon source, addition of acetate at a concentration nothigher than 10 mM increased carotenoid synthesis only slightly whereaspartial or complete phosphate deficiency or salt stress (40 mM NaCl) didnot.     

Improving bioreactor cultivation conditions for sensitive cell lines by dynamic membrane aeration

Although the importance of animal cell culture for the industrial (large scale) production of pharmaceutical products is continuously increasing, the sensibility of the cells towards their cultivation environment is still a challenging issue. In comparison to microbial cultures, cell cultures which are not protected by a cell wall are much more sensitive to shear stress and foam formation. Reactor design as well as the selection of ‘robust’ cell lines is particularly important for these circumstances. Nevertheless, even ‘sensitive’ cell lines are selected for certain pharmaceutical processes due to various reasons. These sensitive cell lines have even higher requirements regarding their cultivation environment. Important characteristics for the corresponding reactor design are a high (volumetric) gas mass transfer coefficient, low volumetric power input, low shear stress, low susceptibility to bio-fouling, the ability to cultivate sticky cells and sufficient mixing properties. Membrane aeration has been a long-known possibility to meet some of these requirements, but has not often been applied in recent years. The reasons lie mainly in low gas mass transfer rates, a limited installable volume-specific membrane surface area, restrictions in scalability and problems with membrane fouling. The dynamic membrane aeration bioreactor aeration is a simple concept for bubble-free oxygen supply of such sensitive cultures. It overcomes limitations and draw-backs of previous systems. Consisting of an oscillating, centrally arranged rotor (stirrer) that is wrapped with silicone membrane tubing, it enables doubling the gas mass transfer at the same shear stress in the investigated cultivation scales of 12, 20, 100, and 200 L. Continuous cultivation at these scales allows the same product output as fed-batch cultivation does at tremendously larger reactor volumes. Apart from introducing this novel technology, the presentation comprises selected cultivation results obtained for blood coagulation factor VIII in continuous mode and a therapeutic monoclonal antibody in fed-batch mode in comparison to reference trials.     

Large-scale cultivation of Spirulina in a floating horizontal photobioreactor without aeration or an agitation device

A low-cost floating photobioreactor (PBR) without the use of aeration and/or an agitation device, in which carbon was supplied in the form of bicarbonate and only wave energy was utilized for mixing, was developed in our previous study. Scaling up is a common challenge in the practical application of PBRs and has not yet been demonstrated for this new design. To fill this gap, cultivation of Spirulina platensis was conducted in this study. The results demonstrated that S. platensis had the highest productivity at 0.3 mol L⁻¹ sodium bicarbonate, but the highest carbon utilization (104 ± 2.6%) was obtained at 0.1 mol L⁻¹. Culture of Spirulina aerated with pure oxygen resulted in only minor inhibition of growth, indicating that its productivity will not be significantly reduced even if dissolved oxygen is accumulated to a high level due to intermittent mixing resulting from the use of wave energy. In cultivation using a floating horizontal photobioreactor at the 1.0 m² scale, the highest biomass concentration of 2.24 ± 0.05 g L⁻¹ was obtained with a culture depth of 5.0 cm and the highest biomass productivity of 18.9 g m⁻² day⁻¹ was obtained with a depth of 10.0 cm. This PBR was scaled up to 10 m² (1000 L) with few challenges; biomass concentration and productivity during ocean testing were little different than those at the 1.0 m² (100 L) scale. However, the larger PBR had an apparent carbon utilization efficiency of 45.0 ± 2.8%, significantly higher than the 39.4 ± 0.9% obtained at the 1 m² scale. These results verified the ease of scaling up floating horizontal photobioreactors and showed their great potential in commercial applications.

     

Comparison of the accumulation of astaxanthin in Haematococcus pluvialis and other green microalgae under N-starvation and high light conditions

Haematococcus pluvialis gave the highest astaxanthin accumulation rate (2.7 mg l^–1 day^–1) and total astaxanthin content ( 22.7 mg g^–1 biomass). Astaxanthin accumulation in Neochloris wimmeri, Protosiphon botryoides, Scotiellopsis oocystiformis, Chorella zofingiensis and Scenedesmus vacuolatus was, respectively, 19.2, 14.3, 10.9, 6.8 and 2.7 mg astaxanthin g^–1 biomass, respectively.     

外源2,6-二叔丁基对甲酚对非生物胁迫条件下雨生红球藻虾青素积累的影响

【背景】雨生红球藻是天然虾青素的最佳来源,广泛应用于虾青素的工业化生产。【目的】探究外源添加不同浓度的2,6-二叔丁基对甲酚(Butylated hydroxytoluene,BHT)对雨生红球藻虾青素积累的影响,以期建立BHT提高雨生红球藻虾青素产量的技术体系。【方法】选用不含硝态氮的BBM培养基,辅以强光照,培养雨生红球藻(Haematococcus pluvialis)LUGU,测试不同浓度BHT对雨生红球藻生物量、虾青素含量、活性氧、抗氧化系统和虾青素合成相关酶基因的影响。【结果】在0-3 mg/L BHT范围内,2 mg/L BHT对雨生红球藻虾青素积累的促进效果最佳,达到31.66 mg/g。2 mg/L BHT有效降低了雨生红球藻内的活性氧水平,增加了细胞内NO水平,提高了藻细胞内过氧化氢酶(Catalase,CAT)、过氧化物酶(Peroxidase,POD)和超氧化物歧化酶(Superoxidedismutase,SOD)活性以及谷胱甘肽(Glutathione,GSH)的含量,诱导了虾青素合成关键酶基因chy和lcy的高效表达。【结论】非生物胁迫条件下,外源添加适量的BHT能促进雨生红球藻中虾青素的积累,且与藻细胞内的信号分子活性氧(Reactive oxygen species,ROS)、NO水平及虾青素合成相关基因的表达调控相关。     

Outdoor microalgae cultivation in airlift photobioreactor at high irradiance and temperature conditions: effect of batch and fed-batch strategies,photoinhibition, and temperature stress

The microalgae Scenedesmus abundans cultivated in five identical airlift photobioreactors (PBRs) in batch and fed-batch modes at the outdoor tropical condition. The microalgae strain S. abundans was found to tolerate high temperature (35–45 °C) and high light intensity (770–1690 µmol m^− 2 s^− 1). The highest biomass productivities were 152.5–162.5 mg L^− 1 day^− 1 for fed-batch strategy. The biomass productivity was drastically reduced due to photoinhibition effect at a culture temperature of > 45 °C. The lipid compositions showed fatty acids mainly in the form of saturated and monounsaturated fatty acids (> 80%) in all PBRs with Cetane number more than 51. The fed-batch strategies efficiently produced higher biomass and lipid productivities at harsh outdoor conditions. Furthermore, the microalgae also accumulated omega-3 fatty acid (C18:3) up to 14% (w/w) of total fatty acid at given outdoor condition.

     

Bacterial cellulose production under oxygen-enriched air at different fructose concentrations in a 50-liter, internal-loop airlift reactor

Bacterial cellulose (BC) production by Acetobacter xylinum subsp. sucrofermentans BPR2001 was carried out in a 50-1 internal-loop airlift reactor in air at an initial fructose concentration of 40 g/l. The BC production rate was 0.059 g/l per h. When oxygen-enriched air was supplied instead of air, the BC production rate increased to 0.093 g/l per h, and the BC yield was enhanced from 11% in air to 18%. When the initial fructose concentrations were varied from 30 to 70 g/l, the highest BC yield (35%) the highest production rate (0.22 g/l x per h), and the highest concentration of BC produced (10.4 g/l) were observed at 60-70 g/l fructose. From the carbon mass balance calculated at the final stage of cultivation, it was observed that enhanced BC production was reflected as a decrease in both CO2 evolution and the concentration of other unknown substances, suggesting the efficient utilization of energy for BC synthesis despite O2 limitation.     

Dynamics of luminescence characteristics of Haematococcus lacustris cultures in different cultivation conditions

Luminescence of microalgae cultures is a valuable property for the fast diagnostics of their physiological state; however, it has been rarely used in algaculture practice. In this work, luminescence spectrum characteristics of two-stage batch cultures of the green carotenogenic microalga Haematococcus lacustris (Girod-Chantrans) Rostafinski 1875 (Chlorophyceae, Chlamydomonadales) under conditions of autotrophic and mixotrophic growth were investigated. The dynamics of the heterotrophy indices in cultures at different stages of their development in different growth media was determined. The transition of H. lacustris cultures from the initially autotrophic to mixotrophic growth regime was registered during the induction of the astaxanthin biosynthesis by complex physicochemical stressing, including nutritional deficiencies, exposure to high concentrations of sodium acetate and chloride and increased illuminance and temperature. The applicability of luminescence spectrometry in vivo for a rapid assessment of the state of H. lacustris cultures in various growth media and with different methods of secondary carotenogenesis induction was shown. The results obtained can be used in experimental studies on optimizing cultivation methods for this species, as well as for the express control of the physiological state of its industrial cultures.