Discovery of two novel bioactive algicidal substances from Brevibacillus sp. via metabolomics profiling and back-validation.

Identifying potent bacterial algicidal agents is essential for the development of effective, safe, and economically viable algaecides. Challenges in isolating and purifying these substances from complex secretions have impeded progress in this field. Metabolomics profiling, an efficient strategy for identifying metabolites, was pioneered in identifying bacterial algicidal substances in this study. Extracellular secretions from different generations of the algicidal bacterium Brevibacillus sp. were isolated for comprehensive analysis. Specifically, a higher algicidal efficacy was observed in the secretion from Generation 3 (G3) of Brevibacillus sp. compared to Generation 1 (G1). Subsequent metabolomics profiling comparing G3 and 1 revealed 83 significantly up-regulated metabolites, of which 9 were identified as potential algicidal candidates. Back-validation highlighted the potency of 4-acetamidobutanoic acid (4-ABC) and 8-hydroxyquinoline (8-HQL), which exhibited robust algicidal activity with 3d-EC50 values of 6.40 mg/L and 92.90 µg/L, respectively. These substances disrupted photosynthetic activity in M. aeruginosa by ceasing electron transfer in PSⅡ, like the impact exerted by Brevibacillus sp. secretion. These findings confirmed that 4-ABC and 8-HQL were the main algicidal components derived from Brevibacillus sp.. Thus, this study presents a streamlined strategy for identifying bacterial algicidal substances and unveils two novel and highly active algicidal substances. ENVIRONMENTAL IMPLICATION: Harmful cyanobacterial blooms (HCBs) pose significant environmental problems and health effects to humans and other organisms. The increasing frequency of HCBs has emerged as a pressing global concern. Bacterial-derived algicidal substances are expected to serve as effective, safe, and economically viable algaecides against HCBs. This study presents a streamlined strategy for identifying bacterial algicidal substances and unveils two novel substances (4-ABC and 8-HQL). These two substances demonstrate remarkable algicidal activity and disrupt the photosynthetic system in M. aeruginosa. They hold potential as prospective algaecides for addressing HCBs.

Harmony in detoxification: Microalgae unleashing the potential of lignocellulosic pretreatment wastewater for resource utilization.

Lignocellulosic biomass is a pivotal renewable resource in biorefinery process, requiring pretreatment, primarily chemical pretreatment, for effective depolymerization and subsequent transformation. This process yields solid residue for saccharification and lignocellulosic pretreatment wastewater (LPW), which comprises sugars and inhibitors such as phenols and furans. This study explored the microalgal capacity to treat LPW, focusing on two key hydrolysate inhibitors: furfural and vanillin, which impact the growth of six green microalgae. Chlorella sorokiniana exhibited higher tolerance to furfural and vanillin. However, both inhibitors hindered the growth of C. sorokiniana and disrupted algal photosynthetic system, with vanillin displaying superior inhibition. A synergistic inhibitory effect (Q < 0.85) was observed with furfural and vanillin on algal growth. Furfural transformation to low-toxic furfuryl alcohol was rapid, yet the addition of vanillin hindered this process. Vanillin stimulated carbohydrate accumulation, with 50.48 % observed in the 0.1 g/L furfural + 0.1 g/L vanillin group. Additionally, vanillin enhanced the accumulation of C16: 0 and C18: 2, reaching 21.71 % and 40.36 %, respectively, with 0.1 g/L vanillin. This study proposed a microalgae-based detoxification and resource utilization approach for LPW, enhancing the comprehensive utilization of lignocellulosic components. The observed biomass modifications also suggested potential applications for biofuel production, contributing to the evolving landscape of sustainable biorefinery processes.

Treatment of mixed wastewater by vertical rotating microalgae-bacteria symbiotic biofilm reactor.

A novel vertical rotating microalgae-bacteria symbiotic biofilm reactor was built to treat the mixed wastewater containing municipal and soybean soaking wastewater. The reactor was operated in both sequential batch and semi-continuous modes. Under the sequential batch operation mode, the maximum removal rates for Chemical Oxygen Demand (COD), Total Nitrogen (TN), Total Phosphorus (TP), and Ammonia Nitrogen (NH4+-N) of the mixed wastewater were 95.6 %, 96.1 %, 97.6 %, and 100 %, respectively. During the semi-continuous operation, the water discharge indices decreased gradually and eventually stabilized. At stabilization, the removal rates of COD, TN, and NH4+-N achieved 98 %, 95 %, and 99.9 %, respectively. The maximum biomass productivity of the biofilm was 2.69 g·m-2·d-1. Additionally, the carbohydrate, protein and lipid comprised approximately 22 %, 51 % and 10 % of the dry weight of Chlorella. This study demonstrates the great potential of the microalgae-bacteria symbiotic biofilm system to treat food and domestic wastewater while harvesting microalgal biomass.

Applications-oriented algicidal efficacy research and in-depth mechanism of a novel strain Brevibacillus sp. on Microcystis aeruginosa.

The utilization of algicidal bacteria for the control of harmful algal blooms (HABs) is a promising technology for ecological remediation. In our most recent publication, a novel strain of Brevibacillus sp. was isolated and proved to have significant algicidal activity and stability against Microcystis aeruginosa. In order to verify the algicidal effect of the strain in the practical application scenario, the algicidal efficacy of Brevibacillus sp. under conditions close to water in the environment was investigated. Results indicated that the algicidal threshold of Brevibacillus sp. culture was 3‰ inoculation concentration, and the removal rate of M. aeruginosa reached 100%. The process of Chl-a degradation followed a first-order kinetic model, which could be used to predict the degradation effect of M. aeruginosa in practical applications. Additionally, the inoculation of Brevibacillus sp. culture introduced additional nutrients, some of which remained in the water. Furthermore, the algicidal substances demonstrated good sustainability, with a removal rate of up to 78.53% at 144 h after three repeated uses. At 12 h, the algicidal substances caused a 78.65% increase in malondialdehyde (MDA) content in M. aeruginosa compared to the control group, thereby triggering the antioxidant system of M. aeruginosa. Moreover, algal cell fragments were observed to aggregate. This study provides a promising direction for treating cyanobacterial blooms using algicidal bacteria in practical applications.

Single and combined toxicity assessment of primary or UV-aged microplastics and adsorbed organic pollutants on microalga Chlorella pyrenoidosa.

Microplastics (MPs), an emerging pollutant, have been increasingly raising concern due to the potential impacts on aquatic organisms. Moreover, the environmental aged MPs always exhibit different environmental behavior and interaction effect with organic pollutants from virgin MPs. In this work, the single and combined toxicity impact on Chlorella pyrenoidosa, a symbiont representative, has been investigated between MPs (e.g., polyamide microplastic (PA6), 75 µm) and organic pollutants (e.g., sulfamethoxazole (SMX) and dicamba (DCB)). Growth inhibition, chlorophyll accumulation, superoxide dismutase (SOD), malondialdehyde (MDA), and catalase (CAT) were investigated with the primary or UV-aged PA6. Above 0.5 g/L PA6 (primary or UV-aged) inhibited cell growth and chlorophyll accumulation after 96 h cultivation as compared with the control. Besides, the inhibition impacts have enhanced as the UV-aging time extending in the single PA6 systems. The algae growth inhibition rate after 96 h cultivation in both the system i.e., single (PA6: 6.9%) and combined (PA6-SMX: 14.2%, PA6-DCB: 14.9%) was slightly lower than that of exposing in organic pollutants alone (SMX: 23.9%, DCB: 25.0%), while the chl. b concentration in 60 days UV-aged PA6 combined with SMX (1.19 mg/L) or DCB (1.40 mg/L) systems were higher than in single SMX (1.04 mg/L) or DCB (1.33 mg/L) system. In addition, there were several differences of the cellular oxidative stress in the combined system of SMX and DCB. Specially, it was not noticeable of three enzymatic activities for SMX exposing in the presence of primary or UV-aged PA6. While SOD, CAT, and MDA activities was obviously increasing after exposing in PA6 and DCB combined system, indicating the significant synergistic effect on algae cells damage. This research verified the remarkable combined toxicity between UV-aged MPs and organic pollutants on microalgae.

New insights into the degradation and detoxification of methylene blue using heterogeneous-Fenton catalyzed by sustainable siderite.

The huge application of synthetic dyes caused a severe impact in the environment. In the present study, a physico-chemical strategy of heterogeneous-Fenton catalyzed by the natural ferrous ore has been established for toxic chemical degradation, of which the complex and high-expense repetitive pH adjustment procedures were escaping. And this natural heterogeneous catalyst also could be recycled and sustainable for toxic substances treatment involved in synergetic adsorption and oxidation. The siderite, served as an adsorbent and catalyst for the degradation of methylene blue (MB). Siderite exhibited a better adsorption capacity with a saturated adsorption capacity of ∼11.08 mg/g. Batch adsorption experiments have verified that adsorption rate and adsorption equilibrium followed pseudo-second-order rate model and Langmuir isotherm equation, respectively. The combination with H2O2, showed significant enhancement of MB degradation without any pH adjustment. The effect of siderite dosage, H2O2 dosage, MB concentration, initial pH, and reaction temperature on MB degradation was investigated, which also has indicated the excellent catalytic performance of siderite. About 99.71% of MB was degraded in 480 min with initial pH of 7.0, reaction temperature of 25 °C, siderite, and H2O2 dosage of 2.5 g/L and 122.38 mM, respectively. It was found that siderite could be reused and remained high degradation efficiency on MB after 5 times reutilization, which also could demonstrate the sustainable and effective process to degrade organic pollution. The generation of reactive species including ·OH and O2·- have been confirmed based on scavenger test and electron spin resonance (ESR) analysis, which was dominated by heterogeneous reaction. The possible degradation mechanisms of MB have been predicted based on spectrum scanning and GC-MS analysis. Moreover, acute toxicity assessment with marine photobacterium Vibrio fisheri was conducted to investigate the toxicity change in the adsorption/oxidation coupled process. This sustainable heterogeneous-Fenton technology has been verified as a promising and applicable process for toxic organic chemicals removal due to effective mineralization and detoxification assisted with the natural ore mineral through the simple operation and mild condtions.

Integrated strategies for robust growth of Chlorella vulgaris on undiluted dairy farm liquid digestate and pollutant removal.

Undiluted dairy farm liquid digestate contains high levels of organic matters, chromaticity and total ammonia nitrogen (TAN), resulting in inhibition to microalgal growth. In this study, a novel cascade pretreatment with ozonation and ammonia stripping (O + S) was employed to remove these inhibitors, and was compared with single pretreatment approach. The optimum parameters for ozonation and ammonia stripping were obtained and the mechanisms of inhibition elimination were investigated. The results show that ozonation contributed to the degradation of non-fluorescent chromophoric organics through the direct molecular ozone attack, which mitigated the inhibition of chromaticity to microalgae, while ammonia stripping relieved the inhibition of high TAN to microalgae. After cascade pretreatment, TAN, total nitrogen (TN), COD and chromaticity were reduced by 80.2 %, 75.4 %, 20.6 % and 75.8 % respectively. When C. vulgaris was cultured on different pretreated digestate, it was found that cascade pretreatment was beneficial for retaining high PSII activity and synergistically improved microalgal growth. The highest biomass increment and productivity achieved 5.40 g L-1 and 900 mg L-1 d-1 respectively in the integration system of cascade pretreatment with microalgae cultivation (O + S + M). After O + S + M treatment, the removal efficiencies of TAN, TN, COD and total phosphorus (TP) were 100 %, 92.8 %, 46.7 % and 99.6 %, respectively. This work provided a promising strategy (O + S + M) for sustainable liquid digestate treatment, along with nutrient recovery and value-added biomass production.

Treatment and resource utilization of dairy liquid digestate by nitrification of biological aerated filter coupled with assimilation of Chlorella pyrenoidosa.

Ammonia inhibition is considered a key issue when using liquid digestate for microalgae cultivation. To study the effect of pretreatment with a biological aerated filter (BAF) on microalgae culture with dairy liquid digestate, nitrification characteristics of BAFs under different hydraulic retention time (HRT) and the growth characteristics of Chlorella pyrenoidosa in effluents of BAFs were investigated. Results showed that the BAFs can rapidly nitrify ammonia nitrogen and significantly improve the light transmittance of liquid digestate (the maximum promotion rate was ~260%), and the effect improved as the HRT increased. Pretreatment of liquid digestate with BAFs can eliminate ammonia inhibition for C. pyrenoidosa. Furthermore, lipid, crude protein, and higher heating value (HHV) output were also not affected by HRT. The similar removal of nitrate nitrogen in microalgae culture systems using effluents with 6-h and 12-h HRT (21.59% and 21.07%, respectively) were recorded. The results suggested that BAF coupled with microalgae culture is a novel option on the resource utilization of dairy liquid digestate.

Treatment of Synthetic Ammonium Sulfate Wastewater by Mixed Culture of Chlorella pyrenoidosa and Enriched Nitrobacteria.

Ammonium sulfate wastewater can cause eutrophication and black odor of water body. Although ammonia nitrogen can be used as nutrient of microalgae, high ammonia nitrogen levels could inhibit the growth of microalgae. Nitrobacteria can transform ammonia nitrogen into nitrate nitrogen. In this study, mono Chlorella pyrenoidosa culture (mono-C.py), synchronous mixed culture (mixed-a), and asynchronous mixed culture (mixed-b) systems were examined for their ability to treat ammonium sulfate wastewater. Nitrogen removal rate of mixed-b at the end of culture (52.96%) was higher than that of the mono-C.py (46.37%) and the mixed-a (39.11%). Higher total suspended solid concentration (2.40 g/L), crude protein yield (0.76 g/L), and heating value yield (35.73 kJ/L) were obtained in mixed-b, meanwhile with excellent settlement performance (91.43 ± 0.51%). Mechanism analysis of settlement showed that the relative abundance of floc-forming-related bacteria Sphingopyxis and Acidovorax were increased generally, while nitrification/denitrifying members were decreased in mixed-b along with the culture proceeding.

Facile synthesis of lignin-based epoxy resins with excellent thermal-mechanical performance.

Up to now, various approaches have been used to fabricate lignin-based epoxy thermosets by utilizing lignin or lignin-derivatives, but there is still lack of a simple, effective and environmental-friendly pathway for producing lignin-based epoxy resins from industrial lignin. In this work, a novel strategy - one-pot to synthesize phenolated lignin incorporated novolac epoxy networks (PLIENs) was proposed. As expected, PLIENs obtained from the novel route exhibited preferable mechanical and thermal properties compared with the epoxy resins which obtained from common route. Moreover, increasing the loading of lignin did not significantly deteriorate the thermal-mechanical performance of cured epoxy resins. However, the Tg of PLIENs was slightly lowered compared with conventional petroleum-based epoxy resins (DGEBA). Nonetheless, the flexural strength and storage modulus of PLIENs were higher than that of DGEBA. Especially, the char yield of PLIENs at 800 °C was up to 28.9%, much higher than that of DGEBA (only 6.9%), which indicated that lignin has a certain promoting effect on the flame retardancy of epoxy resins. This research provides a new insight for producing commercially viable lignin-based epoxy thermosets.

The joint effect of ammonium and pH on the growth of Chlorella vulgaris and ammonium removal in artificial liquid digestate.

Although ammonium containing digestate is an ideal alternative medium for microalgae cultivation, high ammonium or unfavorable pH may inhibit microalgal growth. In this study, the joint effect of ammonium and pH on the growth of C. vulgaris and nutrient removal in artificial digestate was investigated. Our results show that ammonium and pH both affected algal growth, but free ammonia (FA) was the main actual inhibitory factor. Algal specific growth rate presented a negative correlation with FA and their relationship was well fitted by a linear regression model. Microalgal growth was little affected below 36.8 mg L-1 FA, while the obvious inhibition occurred at 184 mg L-1 FA (EC50), indicating a high tolerance to FA. Ammonium removal was well described by a first-order kinetics model. FA stress stimulated the production of extracellular organic matters (EOMs), which was good for microalgae adaptation but adverse to pollutant removal.

Biological nitrogen and phosphorus removal by a phosphorus-accumulating bacteria Acinetobacter sp. strain C-13 with the ability of heterotrophic nitrification-aerobic denitrification.

Strain C-13, identified as an Acinetobacter sp. by homology searches, exhibited efficient simultaneous heterotrophic nitrification-aerobic denitrification phosphorus removal (SNDPR) abilities by nitrogen balance analysis and further confirmation of successful amplification of functional genes ppk, napA, and nirS. In addition, strain C-13 could utilize NH4+-N, NO3--N, and NO2--N as nitrogen sources, among which NH4+-N was indicated to be an excellent nitrogen source for assimilation and heterotrophic nitrification. Besides, the optimum conditions for nutrient removal were determined as follows: sodium acetate as the sole carbon source, C/N/P ratio of 100/10/2, pH = 7.5, and temperature of 30 °C. Meanwhile, the strain also showed the traditional features, such as release and the excess uptake of phosphate under anaerobic/aerobic conditions, with the highest phosphorus content of 5.01% after cultivation. Strain C-13 presents promising prospects for application in biologicalnutrient removal in wastewater treatment.

Performance of a microalgal-bacterial consortium system for the treatment of dairy-derived liquid digestate and biomass production.

To enhance the treatment performance of dairy-derived liquid digestate (DLD) using microalgal-bacterial consortium system composed of Chlorella vulgaris and indigenous bacteria (CV), activated sludge was introduced to form a new microalgal-bacterial consortium system (Co-culture). The activated sludge shortened the lag phase and increased the specific growth rate of C. vulgaris (0.56 d-1). The biomass yield in the Co-culture was 2.72 g L-1, which was lower than that in the CV (3.24 g L-1), but the Co-culture had an improved COD (chemical oxygen demand) removal (25.26%) compared to the CV (13.59%). Quantitative PCR and metagenomic analyses demonstrated that microalgae also promoted bacterial growth, but influenced differently on the bacterial communities of indigenous bacteria and activated sludge. Compared with indigenous bacteria, activated sludge was more prone to forming a favorable symbiosis with C. vulgaris. These findings contribute to the construction of efficient microalgal-bacterial consortium system in wastewater treatment.

Effects of different nitrogen sources and light paths of flat plate photobioreactors on the growth and lipid accumulation of Chlorella sp. GN1 outdoors.

To assess the potential of Chlorella sp. GN1 for producing biodiesel raw materials in flat plate photobioreactors (FPPs) outdoors, we optimized the nitrogen sources and concentrations for the growth of the algae. The effects of different light paths of FPPs on the growth, lipid accumulation, and fatty acids of Chlorella sp. GN1 were also studied. As the light path of the FPPs was reduced, the alga could accumulate lipids rapidly, achieving high lipid content and lipid productivity outdoors. The highest lipid content obtained was 53.5%, when the light path was 5 cm. In addition, the lipid productivity was 66.7 mg L-1 day-1. The main fatty acids were C16/C18, accounting more than 90% of the total fatty acids. Results showed that Chlorella sp. GN1 had the ability to accumulate large quantities of lipids in FPPs outdoors and was a promising microalgal species for biofuel production.

Cultivation of Chlorella vulgaris on unsterilized dairy-derived liquid digestate for simultaneous biofuels feedstock production and pollutant removal.

In order to assess viability of microalgae cultivation using unsterilized dairy-derived liquid digestate (DLD) for simultaneous biofuels feedstock production and contaminant removal, four DLD concentrations (25%, 50%, 75% and 100%) were used to grow Chlorella vulgaris in batch photobioreactors (PBRs). The 25% DLD was an ideal alternative medium in that high growth rate (0.69 d-1), high lipid productivity (112.9 mg L-1 d-1) as well as high nutrient removal were attained. The high DLD concentration caused inhibition of microalgal growth, where COD was more inhibitive than ammonium. The presence of bacteria did not influence microalgae production because of limited growth. Microalgal growth reduced the richness and diversity of bacterial community. Furthermore, the species of Bacteroidetes, Candidatus Saccharibacteria, and Chlamydiae rather than Proteobacteria benefited microalgal-bacterial symbiosis. These findings contribute to better application of microalgal-bacterial system for large-scale microalgae cultivation as well as environmental sustainability.

Treatment of low C/N ratio wastewater and biomass production using co-culture of Chlorella vulgaris and activated sludge in a batch photobioreactor.

The aim of this work was to study the performance of pollutants removal and biomass production by co-culture of Chlorella vulgaris and activated sludge in a batch photobioreactor (PBR), compared with their single system to treat a low C/N ratio (COD/N = 4.3) wastewater. The co-culture system surpassed activated sludge system in terms of nutrients removal and outperformed microalgae alone system in regard to COD removal. Biomass productivity of the co-culture system was 343.3 mg L-1 d-1, and the harvested biomass could be developed as biofuels, animal feeds or soil conditioners due to the improved calorific value and cellular composition compared with activated sludge. The low C/N ratio wastewater enabled bacteria to maintain a relatively low level, hence in favor of microalgae enrichment and nutrient recovery.

The roles of starch and lipid in Chlorella sp. during cell recovery from nitrogen starvation.

The influence of N-replenishment on cell growth, chlorophyll content, nitrogen uptake, and accumulation of starch and lipid was studied in Chlorella sp. N-starved algal cells were fully recovered within 2days after N-replenishment. Stored starch in N-deficient culture was degraded immediately during recovery process. However, lipid response had a period of delay when suffered from long starvation. During the recovery process, neutral lipid was reduced accompanied by an increase in membrane lipid. It was speculated that starch served as a source of carbon and energy catabolized to support quick recovery of the culture, while lipid preferred to play a structural role that specific fatty acid species were released from storage lipid and as building blocks for quick synthesis of membrane lipid. In light of rapid growth recovery and no net degradation in total fatty acids, a semi-continuous cultivation process might be a potential way to enhance lipid productivity.

Water-saving analysis on an effective water reuse system in biodiesel feedstock production based on Chlorella zofingiensis fed-batch cultivation.

The micralgae-based biofuel obtained from dairy wastewater (DWW) is considered a promising source of energy. However, this process consumes water due to the concentration of wastewater being normally too high for some micoralgae cultivation, and dilution is always needed. In this work, the cultivation of microalgae has been examined in non-recirculated water (NR) and recirculated water systems (R). The growth of Chlorella zofingiensis and the nutrient removal of DWW have been recorded. The comparison indicates the R had a little more advantage in biomass and lipid output (1.55, 0.22 g, respectively) than the NR (1.51, 0.20 g, respectively). However, the total chemical oxygen demand (COD), total Kjeldahl nitrogen (TKN), and total phosphorus (TP) removals of the R were lower than those of the NR system during the culture. The highest removal of total COD, TKN, and TP were 85.05%, 93.64%, and 98.45%, respectively. Furthermore, no significant difference has been observed in the higher heating value and lipid content of the biomass of the R and NR. The results show the R can save 30% of the total water input during the culture. All above results indicate the R system has great potential in industry.

Luxury uptake of phosphorus changes the accumulation of starch and lipid in Chlorella sp. under nitrogen depletion.

The aim of this research was to study the effect of phosphorus supply on starch and lipid production under nitrogen starvation using Chlorella sp. as a model. High phosphate level had marginal effect on cell density but increased biomass growth. Massive phosphorus was assimilated quickly and mainly stored in the form of polyphosphate. The algal cells ceased phosphorus uptake when intracellular phosphorus reached a certain level. 5mM phosphate in the culture rendered a 16.7% decrease of starch synthesis and a 22.4% increase of lipid synthesis relative to low phosphate (0.17 mM). It is plausible that phosphate can regulate carbon partitioning between starch and lipid synthesis pathway by influencing ADP-glucose pyrophosphorylase activity. Moreover, high phosphate concentration enhanced the abundance of oleic acid, improving oil quality for biodiesel production. It is a promising cultivation strategy by integration of phosphorus removal from wastewater with biodiesel production for this alga.

Characterization of lipid and fatty acids composition of Chlorella zofingiensis in response to nitrogen starvation.

Cellular biochemical composition of the microalga Chlorella zofingiensis was studied under favorable and nitrogen starvation conditions, with special emphasis on lipid classes and fatty acids distribution. When algal cells were grown in nitrogen-free medium (N stress), the increase in the contents of lipid and carbohydrate while a decrease in protein content was detected. Glycolipids were the major lipid fraction (50.7% of total lipids) under control condition, while neutral lipids increased to be predominant (86.7% of total lipids) under N stress condition. Triacylglycerol (TAG) content in N stressed cells was 27.3% dw, which was over three times higher than that obtained under control condition. Within neutral lipids fraction, monounsaturated fatty acids (MUFA) were the main group (40.6%) upon N stress, in which oleic acid was the most representative fatty acids (34.5%). Contrarily, glycolipids and phospholipids showed a higher percentage of polyunsaturated fatty acids (PUFA). Lipid quality assessment indicated the potential of this alga as a biodiesel feedstock when its neutral lipids were a principal lipid fraction. The results demonstrate that the neutral lipids content is key to determine the suitability of the microalga for biodiesel, and the stress cultivation is essential for lipid quality.