Two-stage Culture Research Articles

Phytohormone augmented biomass and lipid production in Dunaliella salina under two-stage cultivation strategy: A comprehensive characterization of biomass and lipid using DLS, FTIR, CHNS and NMR for bioenergy prospects

Increasing population, climatic shifts, and decreasing fossil-fuels worldwide led scientists to explore microalgae as a renewable energy resource. The present study unravels the impact of gibberellic acid (GA3) on biomass production, enzymatic activities, macromolecular synthesis, and metabolome analysis of Dunaliella salina using two-stage cultivation strategy. The results indicated a marked increase in biomass (1.44-fold), photosynthetic yield (Fv/Fm 0.68), along with RuBisCO activity (1.66-fold), with an increased in absolute value of zeta potential(ζ) in cultures treated with 10 µM GA3 compared to controls (Stage I). Interestingly, 15 µM GA3-treated cells significantly enhanced lipid content (42.36 %), and carbohydrates (16.56 %) with elevated antioxidative enzymes (SOD, CAT, and APX) activities (Stage II). Enhanced lipid content was further confirmed through FTIR spectra (2928 cm-1, and 1740 cm-1) and 1H NMR signals (1.0– 2, 3.5–4.5 ppm), indicating an increase in both saturated (SFA) and polyunsaturated fatty acids (PUFAs). Elemental analysis demonstrated higher carbon and hydrogen percentages, reflected in higher heating value (HHV) and H/C ratio at 15 µM GA3. The increased lipid content was found to be significantly correlated with ACCase and GPAT activities, suggesting the allocation of carbon flux toward lipid biosynthesis pathways. To substantiate the carbon flux, HR-MS-based metabolomic analysis was performed, indicating a reduction in TCA and Calvin cycle intermediates with a significant rise in carotenoid biosynthesis, as well as metabolic shift towards fatty acid biosynthesis reflecting in the enhanced SFA and PUFAs. Thus, these findings highlighted GA3 significant role in the carbon flux allocation towards the neutral lipid biosynthesis for bioenergy prospects.

Efficient Solar-Powered Bioremediation of Hexavalent Chromium in Contaminated Waters by Chlorella sp. MQ-1

Microalgae are known for their efficient removal of hexavalent chromium (Cr(VI)) through biosorption and bioaccumulation, yet the subsequent release of Cr(VI) upon cell death remains a challenge. The reduction of Cr(VI) to the less toxic trivalent chromium [Cr(III)] is another critical remediation strategy that mitigates the risk of Cr(VI) re-release, but research on microalgal reduction of Cr(VI) is scarce. In this study, a microalgal strain designated as MQ-1 was isolated from chromium-contaminated mine effluent, demonstrating the capability to tolerate and remove Cr(VI). Phylogenetic analysis revealed that MQ-1 is closely related to the genus Chlorella; hence, it is classified as Chlorella sp. MQ-1. This strain exhibited robust growth at Cr(VI) concentrations below 2 mg/L, achieving a removal rate higher than 82% for initial Cr(VI) concentrations between 0.5 and 1 mg/L after a 5-day incubation period. Mechanistic studies revealed that MQ-1 promoted the removal of Cr(VI) mainly through intracellular bioreduction and bioaccumulation processes, in which more than 60% of Cr(VI) was reduced to the less toxic Cr(III) and stocked in the cells. A two-stage cultivation strategy, involving initial biomass accumulation followed by Cr(VI) treatment, significantly enhanced the removal efficiency, which was further accelerated under illuminated conditions. Notably, MQ-1 cultures with initial OD680 values of 4 and 6 accomplished 84.28% and 91.31% Cr(VI) removal from 2 mg/L solutions, respectively, within 30 hours under light exposure. These findings highlight the potential of MQ-1 to utilize renewable solar energy to reduce Cr(VI) and to mitigate the risk of its re-release into the environment. This characteristic positions MQ-1 as a potentially sustainable and cost-effective solution for Cr(VI) remediation and suggests its significant potential for large-scale implementation in bioremediation strategies aimed at Cr(VI)-contaminated waters.

Synergistic microalgal co-cultivation for treatment of municipal wastewater using a two-stage cultivation system and biomass valorization

The present study aimed to develop different microalgal co-cultivation Tetraselmis indica BDUG001 (TS) and Picochlorum sp. (PC) for the treatment and recycling of raw municipal wastewater (MWW). The nutrient removal and biomolecules production, 75 % raw MWW + 25 % ASN-III, 50 % secondary treated municipal wastewater (STMWW) + 50 % ASN-III, and 50 % TS-treated MWW (TSTMWW) + 50 % ASN-III was used. Among them, co-cultivation 2TS:1PC and 2PC:1TS exhibited maximum nutrient removal efficiency (NRE) and biomass and biomolecules production in 75 % raw MWW + 25 % ASN-III, and 50 % TSTMWW + 50 % ASN-III, respectively. Based on the above outcomes, two-stage cultivation systems (TSCSs) were developed to maximize the NRE and biomolecules production using the micro vertical glass photobioreactor (MVG-PBR). 2TS:1PC and 2PC:1TS co-cultivation ratio was used for first and second stage of cultivation and noted as 2TS:1PC (S1) and 2PC:TS (S2), respectively. The co-cultivation ratios were compared with monoculture TS (S1) and PC (S2), and the cost analysis was performed to assess the viability of the process. The obtained results showed that during the first stage of TSCSs, co-cultivation 2TS:1PC (S1) and TS (S1) exhibited NRE of COD (95 and 91.42 %), TN (93.06 and 89.54 %) and TP (100 and 93.87 %), and generated dry biomass (3.11 and 2.75 g/L), lipid content (46 and 42.17 %) having FAME content (82.25 and 32.01 %), astaxanthin (1.12 and 0.93 mg/g DCW) and β-carotene (7.01 and 3.30 mg/g DCW), respectively. During the second stage of TSCSs, co-cultivation 2PC:1TS (S2) and PC (S2) exhibited NRE of COD (95.50 and 93.28 %), TN (98.97 and 94.38 %) and TP (100 and 94.50 %), and generated biomass (3.24 and 2.78 g/L), lipid content (48.50 and 40.70 %) having FAME content (86.78 and 44.20 %), astaxanthin (1.15 and 0.71 mg/g DCW) and β-carotene (5.14 and 5.33 mg/g DCW), respectively. The cost analysis suggested that co-cultivation-assisted TSSCSs generated more revenue compared to monoculture-based TSCSs.

Enhancing carotenoid accumulation in Dunaliella bardawil by combined treatments with fulvic acid and optimized culture conditions

Natural carotenoids from microalgae have received more attention as an alternative source. In this study, fulvic acid (FA), a plant growth regulator, was used to enhance carotenoid accumulation in microalgae Dunaliella bardawil rich in lutein. However, the addition of FA promoted pigment synthesis but also exhibited an inhibitory effect on biomass. Therefore, the optimization of culture conditions was performed to further enhance carotenoid accumulation, including high light stress (10,000 lx) and the two-stage cultivation comprising 1-aminocyclopropane-1-carboxylic acid (ACC) and FA. Under both culture conditions, the growth inhibition caused by FA was alleviated, leading to a further increase in the contents of chlorophylls and carotenoids. HPLC analysis revealed that the production of lutein, α-carotene and β-carotene increased by 0.44-, 0.37- and 0.54-fold under the treatment of 400 mg/L FA with high light intensity and 0.91-, 1.15–0.29-fold under the two-stage cultivation comprising 11 mM ACC and 500 mg/L FA. Furthermore, algal cells under FA treatment and the two-stage cultivation stained with Bodipy505/515 emitted stronger fluorescence under a laser confocal microscope, suggesting that lipid accumulation was increased. Additionally, the transcription levels of carotenogenic genes were also found to be up-regulated by qRT-PCR. These results indicated an enhancement in both the storage capacity and synthesis of carotenoids in D. bardawil. This study revealed the potential application of plant growth regulators in promoting carotenoid accumulation in D. bardawil which could be further improved by optimizing the culture conditions, providing a reference for efficient carotenoid production in microalgae.

Salinity as an Abiotic Stressor for Eliciting Bioactive Compounds in Marine Microalgae.

This study investigated the impact of culture medium salinity (5-50 PSU) on the growth and maximum photochemical yield of photosystem II (Fv/Fm) and the composition of carotenoids, fatty acids, and bioactive substances in three marine microalgae (Chrysochromulina rotalis, Amphidinium carterae, and Heterosigma akashiwo). The microalgae were photoautotrophically cultured in discontinuous mode in a single stage (S1) and a two-stage culture with salt shock (S2). A growth model was developed to link biomass productivity with salinity for each species. C. rotalis achieved a maximum biomass productivity (Pmax) of 15.85 ± 0.32 mg·L-1·day-1 in S1 and 16.12 ± 0.13 mg·L-1·day-1 in S2. The salt shock in S2 notably enhanced carotenoid production, particularly in C. rotalis and H. akashiwo, where fucoxanthin was the main carotenoid, while peridinin dominated in A. carterae. H. akashiwo also exhibited increased fatty acid productivity in S2. Salinity changes affected the proportions of saturated, monounsaturated, and polyunsaturated fatty acids in all three species. Additionally, hyposaline conditions boosted the production of haemolytic substances in A. carterae and C. rotalis.

Oscillation-static cycle cultivation enhances the synthesis of triterpenes and mycelium activity in Ganoderma lucidum

Ganoderma lucidum is a precious fungus with both edible and medicinal values and has a long history of medical use. Triterpenes as the main active components endow G. lucidum with anti-tumor, antioxidant, and other pharmacological activities. The present study endeavors to establish a proficient liquid-state fermentation technology for the enhanced production of triterpenes. In view of the limitations inherent in conventional submerged fermentation and oscillation-static two-stage cultivation, this study established an oscillation-static cycle cultivation process and optimized the cultivation conditions by building an artificial neural network model based on genetic algorithms. The cultivation conditions for the high-yield production of triterpenes were optimized as follows: 2.8 days of oscillation, 7.3 days of static cultivation, 0.2 day of oscillation, and 0.3 day of static cultivation. Under these conditions, the content of triterpenes reached 20.82 mg/g. The yield of triterpenes reached 129.09 mg/L, showing a remarkable increase of 324.78% compared with that of the Z10J0 method. Moreover, the established method shortened the cultivation cycle by 10.6 days. The mycelia cultivated under this regimen exhibited commendable anti-tumor and antioxidant activities. This study not only presents an economical liquid-state fermentation approach but also streamlines the fermentation flow, reduces fermentation duration, and effectively ameliorates drawbacks associated with conventional cultivation methods. In addition, this study gives valuable insights into the scaled application of liquid-state fermentation in the high-yield production of triterpenes, which showcases broad prospects.

Bioactivity of Amphidinol-Containing Extracts of Amphidinium carterae Grown Under Varying Cultivation Conditions.

Microalgae are of great interest due to their ability to produce valuable compounds, such as pigments, omega-3 fatty acids, antioxidants, and antimicrobials. The dinoflagellate genus Amphidinium is particularly notable for its amphidinol-like compounds, which exhibit antibacterial and antifungal properties. This study utilized a two-stage cultivation method to grow Amphidinium carterae CCAP 1102/8 under varying conditions, such as blue LED light, increased salinity, and the addition of sodium carbonate or hydrogen peroxide. After cultivation, the biomass was extracted and fractionated using solid-phase extraction, yielding six fractions per treatment. These fractions were analyzed using Liquid Chromatography-High-Resolution Mass Spectrometry (LC-HRMS/MS) to identify their chemical components. Key amphidinol compounds (AM-B, AM-C, AM-22, and AM-A) were identified, with AM-B being the most abundant in Fraction 4, followed by AM-C. Fraction 5 also contained a significant amount of AM-C along with an unknown compound. Fraction 4 returned the highest antimicrobial activity against the pathogens Staphylococcus aureus, Enterococcus faecalis, and Candida albicans, with Minimal Biocidal Concentrations (MBCs) ranging from 1 to 512µg/mL. Results indicate that the modulation of both amphidinol profile and fraction bioactivity can be induced by adjusting the cultivation parameters used to grow two-stage batch cultures of A. carterae.

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.

Effects of Temperature and Nitrogen Limitation on Growth and Lipid Production of Oleaginous Microalgae from Hot Springs

Oleaginous microalgae have gained increasing attention as an alternative feedstock for biodiesel production due to the increasing demand of fuel, climate change, and global warming. This study aimed to isolate and screen robust microalgal strains from hot springs for cultivation in subtropical and tropical areas. The newly isolated oleaginous microalgae were cultivated at 30, 35 and 40 °C. Among mesophilic and thermophilic strains tested, Chlamydomonas sp. HP59 is considered the most robust strain as it showed high cell growth in a broad range of temperatures (30 - 40 °C), with the maximum dried cell weight at 40 °C. Un-optimal temperatures for cell growth did improve lipid content by 2 - 4 folds. To increase lipid production, the 2-stage cultivation, in which nitrogen-rich was applied to promote cell growth in the 1st stage and nitrogen-limitation was applied to stimulate lipid accumulation in the 2nd stage, was performed. The high temperature combined with nitrogen-limitation did improve lipid production by all microalgae. With this strategy, Chlamydomonas sp. HP59 showed the highest dried cell weight of 4.33 g/L and lipid production of 1.72 g/L. This study has shown the potential use of the newly isolated oleaginous microalgae from hot springs to be cultivated at high temperatures and under nitrogen-limited conditions for the production of biodiesel feedstocks. HIGHLIGHTS Mesophilic and thermophilic oleaginous microalgae were isolated from hot springs. Un-optimal temperatures for cell growth did improve lipid content by 2 - 4 folds. Combined effect of high temperature and nitrogen limitation effectively increased lipid content. Two-stage cultivation gave high dried cell weight and hence overall high lipid production. GRAPHICAL ABSTRACT

Sequential two-stage cultivation system using novel microalga consortia for treatment of municipal wastewater and simultaneous biomass production: Sustainable environmental management

Monoculture-based microalgae cultivation systems to treat wastewater are well-reported. Despite that, this method has some limitations in terms of nutrient removal potential, environment adaptation, and low biomass productivity. Conversely, microalgae co-cultivation and a two-stage sequential cultivation system (TSSCS) recently emerged as a promising approach to improve the treatment process and biomass productivity through better adaptation to the environment. However, no outdoor large-scale experiments were reported using this approach which hinders the viability of the process. Thus, in the present study, a sequential two-stage large-scale outdoor novel microalgae consortia experiment was developed. In first stage consortia-assisted sequential cultivation, two ratios of Tetraselmis indica (TS) and one ratio of Picochlorum sp. (PC) (2 TS:1 PC) were cultivated in a 1000-L pond containing 75%-municipal wastewater (MWW) + 25%-ASN-III, while in the second stage, 2 PC:1 TS was cultivated in two different ponds, and each containing 375-L 2 TS:1 PC-treated water + 375-L ASN-III. Outdoor parameters and nutrient removal efficiency (NRE), biomass, and biomolecule productivity such as lipid, photosynthetic pigments, astaxanthin, and β-carotene were quantified, and cost analysis was performed. At the end of the first and second stages, 2 TS:1 PC and 2 PC:1 TS showed maximum NRE of COD (68.71 and 86.40%), TN (66.98 and 94.73%), and TP (82.70 and 94.36%), respectively. Moreover, 2 TS:1 PC and 2 PC:1 TS Pond 1 and 2 produced maximum dry biomass production; 2.41 and ∼2.54 g/L contained lipid content; 36.89 and 34.90% that have 86.50 and 55.79% FAME content respectively. Similarly, 2 TS:1 PC and 2 PC:1 TS biomass exhibited valuable pigments production of astaxanthin i.e., 0.56 and 0.35 mg/g, and β-carotene; 4.65 and 2.82 mg/g, respectively. The cost analysis suggested that only microalgal-based MWW treatment was unfeasible, while valorization of produced biomass into co-products could offset the operation costs and could allow the option for the microalgal-based sustainable approach for the treatment of MWW and recovery of valuable resources.

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.

Enhancing the biosynthesis of polyunsaturated fatty acids by Rhodotorula mucilaginosa and Lodderomyces elongisporus

Single-cell oils (SCO) produced by oleaginous yeast hold promise as a sustainable alternative for producing nutritionally and pharmaceutically valuable lipids. However, the accumulation of oils varies substantially between yeast spp. Consequently, identifying well-suited producers with a high innate capacity for lipids biosynthesis is paramount. Equally important is optimizing culturing and processing conditions to realize the total lipids production potential of selected strains. The marine Rhodotorula mucilaginosa and Lodderomyces elongisporus yeast were investigated to explore their potential for polyunsaturated fatty acids (PUFAs) production on high glucose media (HGM) using two-stage culture mode. Both strains accumulated > 20% (w/w) of their dry cell weight as lipids when grown on HGM using a two-stage culture system. Both yeast isolates exhibited a maximal lipid/biomass coefficient (YL/X) of 0.58–0.66 mg/mg at 7 °C and 0.49–0.53 mg/mg at 26 °C when grown on 8% glucose and produced monounsaturated and PUFAs similar to that of Menhaden and Salmon marine oils. For the first time, significant amounts of Eicosapentaenoic acid (19%) and Eicosadienoic acid (19.6%) were produced by L. elongisporus and R. mucilaginosa, respectively. Thus, the SCO derived from these wild strains possesses significant potential as a substitute source for the industrial-scale production of long-chain PUFAs, making them a promising contender in the market.Graphical

Effect of adaptive laboratory evolution of engineered Escherichia coli in acetate on the biosynthesis of succinic acid from glucose in two-stage cultivation

Escherichia coli MLB (MG1655 ΔpflB ΔldhA), which can hardly grow on glucose with little succinate accumulation under anaerobic conditions. Two-stage fermentation is a fermentation in which the first stage is used for cell growth and the second stage is used for product production. The ability of glucose consumption and succinate production of MLB under anaerobic conditions can be improved significantly by using acetate as the solo carbon source under aerobic condition during the two-stage fermentation. Then, the adaptive laboratory evolution (ALE) of growing on acetate was applied here. We assumed that the activities of succinate production related enzymes might be further improved in this study. E. coli MLB46-05 evolved from MLB and it had an improved growth phenotype on acetate. Interestingly, in MLB46-05, the yield and tolerance of succinic acid in the anaerobic condition of two-stage fermentation were improved significantly. According to transcriptome analysis, upregulation of the glyoxylate cycle and the activity of stress regulatory factors are the possible reasons for the elevated yield. And the increased tolerance to acetate made it more tolerant to high concentrations of glucose and succinate. Finally, strain MLB46-05 produced 111 g/L of succinic acid with a product yield of 0.74 g/g glucose.Synopsis

Development of robust in vitro propagation protocol and cyto-genetic fidelity assessment of Siraitia grosvenorii (monk fruit)

Siraitia grosvenorii (Swingle) C. Jeffrey of the family Cucurbitaceae is a unique and precious plant of medicinal and economic importance in the international market. Due to the huge market potential and to narrow down the gap between demand and supply, an efficient in vitro propagation system using nodal explants was established. Shoot nodal segments were cultured in medium supplemented with different concentrations of 6-benzylaminopurine (BAP) and kinetin (Kn 1.0–7.5 µM). A two-stage culture is recommended for micropropagation of S. grosvenorii. The highest shoot multiplication was observed in BAP-supplemented medium (5.0 µM), whereas rooting was observed in half-strength MS medium supplemented with 8.75 µM of indole-3-butyric acid (IBA). Fully developed plantlets were acclimatized with 100 % survival in sterilized sand. These hardened 15-day-old plants were successfully established under shade net conditions in fields. In vitro raised plants were found genetically stable using molecular markers, including RAPD and ISSR. The 2C nuclear DNA content using flow cytometry was observed to be 0.73–0.76 pg. Hence, the protocol developed for in vitro propagation of S. grosvenorii is cost-effective and can be efficiently used for the industrial production of this natural sweetener and pharmaceutically important plants.

Enhancing lipid production in Chlorella under successive stresses of periodic micro-current and salinity: Performance and contribution

Two-stage cultivation of Chlorella with periodic micro-current coupled with NaCl stress was investigated to analyze the performance and contribution of increased lipid production. In the growth stage, periodic micro-current of 3 mA resulted in an escalation of 290.06 mg/L of Chlorella’s biomass by 61.25 % in comparison to the control. During the induction stage, under a NaCl concentration of 8 g/L, the highest lipid content and yield attained were 42.11 % and 199.32 mg/L, revealing an increment of 53.13 % and 39.11 % respectively when compared to the control. Additionally, the polyunsaturated fatty acids content (PUFA) decreased from 30.11 % to 23.09 %, while the saturated fatty acids content (SFA) increased from 31.86 % to 42.39 %. Chlorella demonstrated adaptability to the alterations in the cultivation environment by an initial upsurge and later a reduction in SOD and CAT enzyme activities, and concurrently facilitated lipid synthesis by similar changes in the activities of ACCase and DGAT. The above cultivation enabled a synergistic effect of swift growth and efficient lipid accumulation in Chlorella, providing groundwork for large-scale biofuel production.

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.

Optimization of stage conversion time and modification of cell metabolism to enhance lipid production of Auxenochlorella pyrenoidosa in two-stage cultivation

Traditionally, the time of maximum biomass concentration in stage I is the widely adopted stage conversion time in two-stage microalgae culture. This study challenges this conventional approach, demonstrating that the optimal stage conversion time in stage I is 72 h rather than 120 h for achieving maximum biomass concentration. A comparison of cell characteristics revealed that algal cells at 72 h exhibited better growth potential, leading to a higher biomass concentration after transfer to stage II and, consequently, increased lipid productivity. Moreover, the use of phosphorus repletion (5-fold) in stage II directed carbon flux toward biomass growth and lipid accumulation, thereby enhancing lipid productivity. By optimizing the stage conversion time and implementing phosphorus repletion, the mean lipid productivity of Auxenochlorella pyrenoidosa cultured under autotrophy-nitrogen starvation and autotrophy-high light conditions increased by 31 % and 60 %, respectively. This study underscores the importance of reevaluating the currently widely used stage conversion time.

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.

Carotenoids from wastewater-grown microalgae biomass: Life cycle assessment and techno-economical analysis

Microalgae stand out as a valuable source of carotenoids, with potential applications in various industrial sectors. Cultivating microalgae in wastewater emerges as an alternative to mitigate the high costs associated with the synthetic production of these compounds while contributing to sanitation resource recovery. Previous studies demonstrated the potential for obtaining lutein and betacarotene from biomass cultivated in domestic sewage and agro-industrial effluent. This study conducted comprehensive economic and life cycle analyses to evaluate cultivation reactors (high-rate pond and hybrid system) and extraction methods (ethyl acetate solvent and supercritical fluid) to acquire 1 kg of carotenoids efficiently. For all evaluated scenarios, higher environmental impacts were observed in the Human carcinogenic toxicity category and human health environmental damage associated with ethyl acetate and electricity consumption. Ethyl acetate also stood out with higher costs. The high-rate pond and hybrid system showed similar environmental impacts in scenarios with ethyl acetate extraction; however, when supercritical fluid was used, cultivation in the first reactor resulted in 2.2–6.2 times lower environmental impacts in Midpoint categories. Regarding the extraction method, the use of supercritical fluid demonstrated superior economic and environmental performance despite registering a 53% higher energy consumption than solvent extraction. Thus, for the viability of obtaining carotenoids from microalgae biomass, we recommend that two-stage cultivation approaches, incorporating solvent recovery steps and biomass valorization in a cascade manner within a biorefinery context, be considered in future studies.

Влияние температурных режимов процесса культивирования на синтез экзополисахаридов консорциумом пробиотических микроорганизмов

In the development of food technologies, an urgent direction is the expansion of the possibility of using natural polysaccharides, which are vital products of probiotic microorganisms formed in the process of biomass synthesis, which increase the adhesion activity of lacto- and bifidobacteria on mucous surfaces of the gastrointestinal tract and contribute to the formation of anticancerogenic, antiviral and immunomodulatory properties of probiotic products. It is known that the synthesis of water-binding metabolites by probiotic microorganisms is activated under adverse conditions for biomass growth. Experimental studies of the activity of synthesis of exopolysaccharides by a consortium of probiotic microorganisms based on Str. thermophiles, B. bifidum, B. longum, B. adolescentis, B. breve, L. acidophilus, L. plantarum, L. fermentum. The temperature modes of cultivation have been studied on the basis of combining the steps of optimal and suboptimal conditions for the development of microorganisms corresponding to temperature variation in the range of higher (47 ± 2) °C or lower (32 ± 2) °C in order to intensify the synthesis of water-binding metabolites of polysaccharide nature. It is possible to achieve lactic acid concentration sufficient for acid coagulation of the cultured mixture proteins, probiotic microorganism concentration at the level of not less than 108 CFU/g. The results of optical density studies in the range 632–643 A and exopolysaccharide concentrations for biomass samples of the consortium of lacto- and bifidobacteria within the range and 87,5–89,5 mg/mL (273–277 A and 37,5–38,5 mg/mL in the control sample, respectively) are presented. The maximum activity of synthesis of moisture-binding metabolites was found to correspond to a two-stage cultivation process at temperature (32 ± 2) °C (4–5 hours), (40 ± 2) °C (4–5 hours), followed by cooling to (4 ± 2) °C.