Two-stage Cultivation Strategy Research Articles

Role of cultivation parameters in carbohydrate accretion for production of bioethanol and C-phycocyanin from a marine cyanobacterium Leptolyngbya valderiana BDU 41001: A sustainable approach

The investigation aimed to augment carbohydrate accumulation in the marine cyanobacterium Leptolyngbya valderiana BDU 41001 to facilitate bioethanol production. Under the standardised physiochemical condition (SPC), i.e. 90 µmol photon m−2 s−1 light intensity, initial culture pH 8.5, 35 °C temperature and mixing at 150 rpm increased the carbohydrate productivity ∼70 % than the control, while a 47 % rise in content was obtained under the nitrate (N)-starved condition. Therefore, a two-stage cultivation strategy was implemented, combining SPC at the 1st stage and N starvation at the 2nd stage, resulting in 80 % augmentation of carbohydrate yield, which enhanced the bioethanol yield by ∼86 % as compared to the control employing immobilised yeast fermentation. Moreover, biomass utilisation was maximised by extracting C-phycocyanin, where a ∼77 % rise in productivity was recorded under the SPC. This study highlights the potential of L. valderiana for pilot-scale biorefinery applications, advancing the understanding of sustainable biofuel production.

Optimization of continuous astaxanthin production by Haematococcus pluvialis in nitrogen-limited photobioreactor

The industrial production of astaxanthin from Haematococcus pluvialis is mainly operated following a two-stage cultivation strategy in photobioreactors (PBRs) operating in batch mode. This study provides a strategy for optimizing continuous astaxanthin production in a one-stage approach, by optimizing both astaxanthin accumulation and biomass productivity of H. pluvialis under nitrogen-limited condition. To achieve the good balance in culture conditions to maintain growth under nitrogen-limited conditions, while triggering significant astaxanthin accumulation in reddish vegetative cells, the role of light absorption, as represented by the mean rate of photon absorption (MRPA), and nitrate concentration was especially investigated. For that purpose, H. pluvialis cultures were grown in a flat-panel PBR with constant dilution rate D of 0.015 h−1, different photon flux densities PFDs ranging from 75 to 750 μmolhν·m−2·s−1 and different nitrogen concentration levels in the feeding medium [NO3−] of 1, 3 and 8.8 mM. A parabolic relationship between MRPA and astaxanthin production rate was obtained. This indicated that astaxanthin synthesis was limited by the MRPA, opening further optimization by adjusting incident light intensity, nitrogen concentration in the feeding medium or culture dilution rate. After optimization of operating conditions, we demonstrated that a large quantity of astaxanthin can be produced in continuous mode, following then a one-stage strategy which may be advantageous for industrial use. A maximum astaxanthin productivity of 1.27 ± 0.03 g·m−2·d−1 with an astaxanthin accumulation of 3.9 ± 0.2 % DW was reached for the culture featuring MRPA of 9000 μmolhν·kgx−1·s−1. The process was also proved reversible, allowing to tailor the biomass composition and physiological state (from green to red cells enriched in astaxanthin) by adjusting operating conditions, opening perspectives from an optimized coupling with downstream processing steps.

Enhanced biomass and lipid production from olive processing wastewater using Scenedesmus obliquus in a two-stage cultivation strategy under salt stress

The study enhanced Scenedesmus obliquus cultivation via optimization of C/N and C/P ratios in synthetic media and evaluation of a two-stage cultivation strategy through application of salinity stress in olive processing wastewater-based feedstocks. S. obliquus under 40/1 C/N ratio performed biomass and lipid productivity that reached 0.15 and 34.1 mg L−1 d−1 respectively, demonstrating high glucose removal efficiency. Application of a two-stage cultivation strategy employing 0, 10, 20 and 30 g L−1 NaCl during the second bioprocess segment resulted in elevated biomass productivity (0.14–0.19 g L−1 d−1), which was reduced under batch mode yielding 0.11 g L−1 d−1. Two-stage cultivation triggered higher lipid productivity (4.2%-156.9%) following 3 d and 6 d upon the onset of the second stage (42.7–55.2 mg L−1 d−1) as compared to batch conditions, while the utilization of reducing sugars in exhausted olive pomace hydrolysates and table olive processing wastewater reached 53.6%-61.2%. Proline, glycerol and reactive oxygen species accumulated at considerable levels employing 20 and 30 g L−1 NaCl, indicating that S. obliquus expressed cellular stress under elevated salinity content, which was not notably stimulated using 10 g L−1 NaCl. The use of olive processing wastewater in a two-stage cultivation strategy under stress conditions enables future development of algal biorefinery systems for production of S. obliquus biomass and polyunsaturated fatty acids as high added-value products from biowaste.

A new isolate cold-adapted Ankistrodesmus sp. OR119838: influence of light, temperature, and nitrogen concentration on growth characteristics and biochemical composition using the two-stage cultivation strategy.

Natural-based chemicals from microalgae such as lipids and pigments are the interests in industries and the bioeconomy. Cold-adapted Ankistrodesmus sp. OR119838, an isolated strain from Cheshmeh-Sabz Lake in northeastern Iran, was cultivated using a two-stage culture strategy under different environmental conditions. With doubling the nitrate concentration at the vegetative stage (170mg/L) and increasing the light intensity (180µmol photons/m2/s) the highest specific growth rate (0.61 ± 0.02 per day) and biomass productivity (121.1 ± 7.2 mg/L/day) were observed at 25°C. In the optimal growth condition Chl a and Chl b contents of Ankistrodesmus sp. OR119838 reached the highest amount (11.07 ± 0.14 and 11.23 ± 0.29 µg/mL, respectively) at 25°C. While carotenoid content correlated negatively with optimum biomass productivity (-0.708) and had the best value (12.23 ± 0.29µg/mL) in nitrogen deficiency (42 mg/L) and intense light conditions (180µmol photons/m2/s) at 15°C. Lipid content was increased with declined nitrate concentration (42mg/L), high light intensity, and 180µmol photons/m2/s at 25°C. The highest percentage of polyunsaturated fatty acids (71.94%) and α-linolenic acid (57.73 ± 6.63%) was observed in conditions with 170mg/L nitrate concentration and low light intensity (40µmol photons/m2/ s) at the low temperature (15°C). While saturated fatty acids content (43.27%) and palmitic acid reached the highest amount under 40µmol photons/m2/s, 42mg/L nitrate at 25°C (35.02 ± 5.33%). Biomass productivity of Ankistrodesmus sp. OR119838, as a cold-adapted strain, decreased by only 8.2% with a 10-degree decline in temperature. Therefore, this strain has good potential to grow in open ponds by tolerating the daily temperature fluctuations.

Sustainable Lutein Production from Chlorella sorokiniana NIES-2168 by Using Aquaculture Wastewater with Two-Stage Cultivation Strategies

Unnecessary discharge of nutrient-rich aquaculture wastewater is a significant threat to the environment, exacerbating massive resource wasting worldwide. Microalgae-based circular economy strategies utilize atmospheric CO2 and aquaculture wastewater nutrients and convert them into valuable compounds. Lutein, a natural pigment, is a nutritional supplement for eye protection, anti-cancer and anti-inflammatory effects, and other health benefits. It is widely utilized in the food and pharmaceutical industries. The primary purpose of this study is to reuse aquaculture wastewater to grow microalgae and optimize conditions to achieve a high yield of lutein as well as the removal of nutrients from wastewater. When cultured in 1.0× BG11 nutrient-added aquaculture wastewater and aerated using 2% CO2, the biomass concentration and lutein content of Chlorella sorokiniana NIES-2168 increased to 1.78 g L−1 and 7.43 mg g−1, respectively. A two-stage culture strategy further increased the lutein content and yield of microalgae. The highest lutein content of 13.95 mg g−1 and lutein productivity of 3.63 mg L−1 d−1 in the second stage aligned with other phototrophic microalgae currently used for lutein production. C. sorokiniana NIES-2168 also showed exceptional nitrogen and phosphorus removal efficiency, with nitrate and phosphate removal rates reaching 96.07% and 96.75% during the two-stage culture process.

A novel two-stage culture strategy to enhance the C-phycocyanin productivity and purity of Arthrospira platensis

The inconsistent light intensity requirements for C-phycocyanin accumulation and algal growth have greatly hampered the productivity and quality of this high-value pigment protein. This study aimed to improve both the production efficiency and extract quality of C-phycocyanin in the cyanobacterium Arthrospira platensis HN5, by adjusting light intensity and nitrogen supply at different cultivation stages. We established an optimized two-stage culture strategy consisting of a 4-d phase for algal growth under 320 μmol m−2 s−1 and a subsequent 2-d period using 160 μmol m−2 s−1 illumination and NaNO3 supplement to promote C-phycocyanin accumulation. By adopting this strategy, up to 18.86% content and 59.72 mg L−1 d−1 productivity of C-phycocyanin were obtained. Our results indicate that the increased C-phycocyanin content improved the quality of C-phycocyanin extract, and reactive grade C-phycocyanin (A620/A280 = 3.16) was recovered through a one-step ammonium sulfate precipitation. The established two-stage culture strategy, as illustrated herein, could be a highly promising strategy for improving the content and productivity of C-phycocyanin and effectively enhancing the quality of C-phycocyanin products, which could be particularly conducive to the large-scale development of this algal pigment in the food industry sector.

Enhanced phycocyanin production from Spirulina subsalsa via freshwater and marine cultivation with optimized light source and temperature

To find out optimum and cost-efficient strategy for phycocyanin production, the effect of light source and temperature on Spirulina subsalsa growth were studied in chemically defined freshwater medium and seawater supplied with wastewater from glutamic acid fermentation tank. Maximum growth rate and the highest phycocyanin content were obtained by 35 °C and green light, respectively. A two-stage cultivation strategy was proposed and applied, which combines biomass accumulation at 35 °C and phycocyanin synthesis simulated under green light. As a result, phycocyanin production reached 70 mg/L/d and 11 mg/L/d from freshwater and seawater medium, respectively. With all tested conditions, a strong correlation between biomass and phycocyanin/chlorophyll ratio, rather than phycocyanin, revealed the dependence of Spirulina subsalsa growth on coordinating regulation of photosynthetic pigments. The relationship between growth and phycocyanin production under various light and temperature can be a good basis for improving phycocyanin production from Spirulina subsalsa with or without freshwater consumption.

Two-stage lipid induction in the microalga Tetraselmis striata CTP4 upon exposure to different abiotic stresses

Tetraselmis striata CTP4 is a euryhaline, robust, fast-growing microalga suitable for wastewater treatment and industrial production. Lipid production was induced through a two-stage cultivation strategy: a 1st stage under standard growth-promoting conditions (100 μmol photons m−2 s−1, salinity 36 ppt and 20 °C) to achieve high biomass concentration and a 2nd stage of 6 days for lipid induction by the application of abiotic stresses such as nutrient depletion, high light intensity (200 and 400 μmol photons m−2 s−1), high salinity (75 and 100 ppt), and extreme temperatures (5 and 35 °C). Although nutrient depletion always resulted in a decrease in biomass productivity, it had also the highest impact on lipid induction. The highest lipid content (43.2%) and lipid productivity (29.2 mg L−1 d−1) were obtained using a combination of nutrient depletion and high light intensity (400 μmol m−2 s−1). The fatty acid profile was mainly composed of C16:0 (palmitic), C18:1 (oleic) and C18:2 (linoleic) acids. The low content of unsaturated fatty acids and absence of C18:3 (linolenic) acid render the oil of this microalga suitable for biodiesel production, a renewable source of energy.

Enhanced production of bioactive compounds from marine microalgae Tetraselmis tetrathele under salinity and light stresses: A two-stage cultivation strategy

This study leveraged the salinity and light intensity stresses during the stationary phase for enhancing the pigment contents and antioxidant capacity of Tetraselmis tetrathele. The highest pigments content was obtained in cultures under salinity stress (40 g L−1) illuminated using fluorescent light. Furthermore, the best inhibitory concentration (IC50) for scavenging the 2, 2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radicals was found as 79.53 µg mL−1 in ethanol extract and cultures under red LED light stress (300 µmol m−2 s−1). The highest antioxidant capacity in a ferric-reducing antioxidant power (FRAP) assay (1,778.6 µM Fe+2) was found in ethanol extract and cultures under salinity stress illuminated using fluorescent light. Maximum scavenging of the 2.2-diphenyl-1-picrylhydrazyl (DPPH) radical was found in ethyl acetate extracts under light and salinity stresses. These results indicated that abiotic stresses could enhance the pigment and antioxidant components of T. tetrathele, which are value-added compounds in the pharmaceutical, cosmetic, and food industries.

Exopolysaccharide production by Anabaena sp. PCC 7120: physicochemical parameter optimization and two-stage cultivation strategy to maximize the product yield

Exopolysaccharide production by Anabaena sp. PCC 7120: physicochemical parameter optimization and two-stage cultivation strategy to maximize the product yield

Exploration of two-stage cultivation strategy using nitrogen limited and phosphorus sufficient to simultaneously improve the biomass and lipid productivity in Desmodesmus intermedius Z8

In this work, the effects of nine different nitrogen and phosphorus conditions on the growth and lipid production of Desmodesmus intermedius Z8 were investigated. The highest lipid concentration of 0.89 g·L−1 was obtained at 6 days under the Nlim&P (nitrogen limited and phosphorus sufficient) condition. To further enhance the total lipid production, the two-stage cultivation strategy was performed. The results show that the addition of 750.0 mg·L−1 of nitrogen and 40.00 mg·L−1 of phosphorus at the end of the second day of incubation could effectively improve the lipid productivity. Under this condition, they could reach 244.69 mg·(L·d)−1, which was 1.66-folds higher than that of the control group. Furthermore, the fatty acid composition analysis indicates that the lipid could meet the standards of biodiesel. Therefore, the two-stage regulation strategy, according to the results in this study, is an effective and simple method to realize high lipid production and quality, and then showing its potential for biodiesel production.

A Two-Stage Culture Strategy for Scenedesmus sp. FSP3 for CO2 Fixation and the Simultaneous Production of Lutein under Light and Salt Stress.

In this study, Scenedesmus sp. FSP3 was cultured using a two-stage culture strategy for CO2 fixation and lutein production. During the first stage, propylene carbonate was added to the medium, with 5% CO2 introduced to promote the rapid growth and CO2 fixation of the microalgae. During the second stage of cultivation, a NaCl concentration of 156 mmol L-1 and a light intensity of 160 μmol m-2 s-1 were used to stimulate the accumulation of lutein in the microalgal cells. By using this culture method, high lutein production and CO2 fixation were simultaneously achieved. The biomass productivity and carbon fixation rate of Scenedesmus sp. FSP3 reached 0.58 g L-1 d-1 and 1.09 g L-1 d-1, with a lutein content and yield as high as 6.45 mg g-1 and 2.30 mg L-1 d-1, respectively. The results reveal a commercially feasible way to integrate microalgal lutein production with CO2 fixation processes.

A novel and effective two-stage cultivation strategy for enhanced lutein production with Chlorella sorokiniana

Microalgae are currently being recognized as a sustainable resource for valuable products such as pigments for their superior antioxidant activities. Chlorella sorokiniana MB-1-M12 is a lutein-rich mutant capable of growth and lutein production in photoautotrophic, mixotrophic, heterotrophic, and photoheterotrophic metabolism. Despite being capable of heterotrophic growth, light and CO2 were found to be critical for lutein accumulation in MB-1- M12. Thus, an innovative two-stage heterotrophic-mixotrophic process (TSHM) was developed and when performed under optimal conditions, about 10 g/L biomass and 7.42 mg/g lutein were obtained. The integration of fed-batch and semi-batch operational strategies and scale-up in a 5 L bioreactor increased biomass production, lutein content, and lutein production to 20 g/L, 8.71 mg/g, and 181.11 mg/L, respectively. This work demonstrated that the integration of the TSHM process with appropriate operational strategies (the integrated fed-batch and semi-batch operation) enhanced lutein accumulation in MB-1-M12, making it apt for commercial lutein production.

Cold stress combined with salt or abscisic acid supplementation enhances lipogenesis and carotenogenesis in Phaeodactylum tricornutum (Bacillariophyceae).

Compounds from microalgae such as ω3-fatty acids or carotenoid are commercially exploited within the pharmacology, nutraceutical, or cosmetic sectors. The co-stimulation of several compounds of interest may improve the cost-effectiveness of microalgal biorefinery pipelines. This study focussed on Phaeodactylum tricornutum to investigate the effects on lipogenesis and carotenogenesis of combined stressors, here cold temperature and addition of NaCl salt or the phytohormone abscisic acid, using a two-stage cultivation strategy. Cold stress with NaCl or phytohormone addition increased the neutral lipid content of the biomass (20 to 35%). These treatments also enhanced the proportions of EPA (22% greater than control) in the fatty acid profile. Also, these treatments had a stimulatory effect on carotenogenesis, especially the combination of cold stress with NaCl addition, which returned the highest production of fucoxanthin (33% increase). The gene expression of diacylglycerol acyltransferase (DGAT) and the ω-3 desaturase precursor (PTD15) were enhanced 4- and 16-fold relative to the control, respectively. In addition, zeaxanthin epoxidase 3 (ZEP3), was downregulated at low temperature when combined with abscisic acid. These results highlight the benefits of applying a combination of low temperature and salinity stress, to simultaneously enhance the yields of the valuable metabolites EPA and fucoxanthin in Phaeodactylum tricornutum.

Docosahexaenoic acid production of the marine microalga Isochrysis galbana cultivated on renewable substrates from food processing waste under CO2 enrichment

Leftover dough is a starch-rich food processing waste of Chinese steamed bread. Leftover dough hydrolysates enriched with glucose and amino acids were used to cultivate the marine microalga Isochrysis galbana to produce docosahexaenoic acid (DHA) under CO2 enrichment. Isochrysis galbana could use mixed carbon sources (CO2, glucose, and amino acids) synchronously to grow and accumulate DHA. Cell growth, the uptake of glucose and amino acids, and DHA production were significantly affected by CO2 enrichment. The maximum biomass concentration of 3.85 g L−1 was achieved with 3 % CO2. And the maximum DHA yield was 65.5 mg L−1 d−1. To enhance DHA production, a two-stage cultivation strategy was successfully developed by this work. The maximum DHA yield of the two-stage culture was elevated by 2.3-fold. It is feasible to produce DHA by Isochrysis galbana using leftover dough under CO2 enrichment.

Low-frequency ultrasound and nitrogen limitation induced enhancement in biomass production and lipid accumulation of Tetradesmus obliquus FACHB-12

The two-stage cultivation strategy was optimized in this study to simultaneously promote the growth and lipid accumulation of Tetradesmus obliquus. Results showed that the optimal dual-stress conditions were nitrogen concentration at 25 mg N·L−1 and low-frequency ultrasound at 200 Watt, 1 min, and 8 h interval. The biomass and lipid content of Tetradesmus obliquus were increased by 32.1% and 44.5%, respectively, comparing to the control, and the lipid productivity reached 86.97 mg−1·L−1·d−1 at the end of the cultivation period. The protein and photosynthetic pigment contents of microalgae decreased by 22.4% and 14.0% under dual stress comparing to the control environment. In addition, dual stress cultivation of microalgae presented higher level of antioxidant capacity to balance to oxidation level in microalgal cells. This study provides a new insight for microalgae growth and lipid accumulation with dual stress stimulation.

Effective Two-Stage Heterotrophic Cultivation of the Unicellular Green Microalga Chromochloris zofingiensis Enabled Ultrahigh Biomass and Astaxanthin Production.

Chromochloris zofingiensis has obtained particular interest as a promising candidate for natural astaxanthin production. In this study, we established a two-stage heterotrophic cultivation process, by using which both the growth of C. zofingiensis and astaxanthin accumulation are substantially enhanced. Specifically, the ultrahigh biomass concentration of 221.3 g L−1 was achieved under the optimum culture conditions in 7.5 L fermenter during 12 days. When scaled-up in the 500 L fermentor, the biomass yield reached 182.3 g L−1 in 9 days, while the astaxanthin content was 0.068% of DW. To further promote astaxanthin accumulation, gibberellic Acid-3 (GA3) was screened from a variety of phytohormones and was combined with increased C/N ratio and NaCl concentration for induction. When C. zofingiensis was grown with the two-stage cultivation strategy, the astaxanthin yield reached 0.318 g L−1, of which the biomass yield was 235.4 g L−1 and astaxanthin content was 0.144% of DW. The content of the total fatty acids increased from 23 to 42% of DW simultaneously. Such an astaxanthin yield was 5.4-fold higher than the reported highest record and surpassed the level of Haematococcus pluvialis. This study demonstrated that heterotrophic cultivation of C. zofingiensis is competitive for industrial astaxanthin production.

Newly isolated native microalgal strains producing polyhydroxybutyrate and energy storage precursors simultaneously: Targeting microalgal biorefinery

With escalating urbanization and ever-increasing population, plastic pollution has become the problem of hour. Owing to its deep roots in our daily life, it is imperative to find an alternative. Sustainable production of poly hydroxy butyrate (PHB) can be our way out from this soup of plastic pollution. Microalgae, being photoautotrophs, helps in bioremediation and can metabolize the waste products into biodiesel, bioethanol, protein, and also bioplastics in the form of PHB. The present study reports the accumulation of PHB in Coelastrella sp., Ettlia texensis and Pecatinodesmus sp. isolated from nearby regions of Indore under nutrient stress and mixotrophic conditions. The individual effect of nutrients was studied, and concluded that under mixotrophic cultivation, Coelastrella sp. can accumulate up to 151.8 ± 12.1 μg/mg dry cell weight (DCW) of PHB under N1.5P0.04Ga10 supplementation in 72 h of cultivation. A two-stage cultivation strategy enabled the accumulation of high PHB content in microalgae without compromising the microalgal growth, which was noted in terms of chlorophyll and biomass accumulation. Principle component analysis (PCA) revealed close relation between biomass and poly hydroxybutyrate (PHB) accumulation with organic carbon presence having a great impact. These results pave the way for sustainable PHA/PHB production using three microalgal species.

Improving carbohydrate accumulation in Chlamydomonas debaryana induced by sulfur starvation using response surface methodology.

Most methods that promote carbohydrate production negatively affect cell growth and microalgal biomass production. This study explores, in a two-stage cultivation strategy, in Chlamydomonas debaryana the optimization of certain culture conditions for high carbohydrate production without loss of biomass. In the first stage, the interaction between sodium bicarbonate supplementation, aeration, and different growth periods was optimized using the response surface methodology (RMS). The 3-factor Box-Behnken design (BBD) was applied, and a second-order polynomial regression analysis was used to analyze the experimental data. The results showed that 0.45g L-1 of sodium bicarbonate combined with a good aerated agitation (0.6 L min-1) and a cultivation period of 18days are optimal to produce 5.02g L-1 of biomass containing 43% of carbohydrates.Under these optimized growth conditions, accumulation of carbohydrates was studied using different modes of nutritional stress. The results indicated that carbohydrate content was improved and the maximum accumulation (about 60% of the dry weight) was recorded under sulfur starvation with only a 14% reduction in biomass as compared to control. This study showed promising results as to biomass production and carbohydrate yield by the microalgae C. debaryana in view of production of third-generation biofuels.

Sodium chloride stimulates the biomass and astaxanthin production by Haematococcus pluvialis via a two-stage cultivation strategy

A major challenge facing by astaxanthin industrialization is the low productivity and high production costs. This study established a two-stage cultivation strategy based on the application of NaCl to improve the production of biomass and astaxanthin by Haematococcus pluvialis. During the first growth stage, 12.5 mg L-1 NaCl led to a remarkable enhancement in biomass, which was 1.28 times compared with the control. Moreover, 2 g L-1 NaCl stimulated the astaxanthin content from 12.18 mg g−1 to 25.92 mg g−1 during the second induction stage. Simultaneously, salinity stress application increased the lipids and GABA contents, as well as the levels of Ca2+ and carotenogenic genes’ expression, but suppressed the contents of carbohydrate and protein and high-light induced-ROS. This study proposed a simple and convenient strategy for efficient coproduction of biomass and astaxanthin and provides insights into the underlying mechanism of astaxanthin biosynthesis in H. pluvialis induced by salinity stress.