Lutein Production Research Articles

Enhancing production of lutein by a mixotrophic cultivation system using microalga Scenedesmus obliquus CWL-1

This work studies a series of strategies in the production of lutein by Scenedesmus obliquus CWL-1 under mixotrophic cultivation. Our experimental results revealed that the optimal conditions associated with light-related strategies were 12 h light period followed by a 12 h dark period and blue to red light under mixotrophic cultivation. Under such conditions, the biomass, lutein content and lutein productivity were maximized to 9.88 (g/L), 1.78 (mg/g) and 1.43 (mg/L/day), respectively. Moreover, the assimilation of 4.5 g/L of calcium nitrate into S. obliquus CWL-1 increased the maximal biomass (12.73 g/L) and the highest maximal lutein productivity (3.06 mg/L/day), while the assimilation of 1.5 g/L of calcium nitrate yielded the highest maximal lutein content of 2.45 mg/g. The highest maximal lutein productivity of 4.96 (mg/L/day) was obtained when fed-batch fermentation was conducted, and this value was approximately 11-folds that obtained using the batch system.

Hybrid physics‐based and data‐driven modeling for bioprocess online simulation and optimization

Model-based online optimization has not been widely applied to bioprocesses due to the challenges of modeling complex biological behaviors, low-quality industrial measurements, and lack of visualization techniques for ongoing processes. This study proposes an innovative hybrid modeling framework which takes advantages of both physics-based and data-driven modeling for bioprocess online monitoring, prediction, and optimization. The framework initially generates high-quality data by correcting raw process measurements via a physics-based noise filter (a generally available simple kinetic model with high fitting but low predictive performance); then constructs a predictive data-driven model to identify optimal control actions and predict discrete future bioprocess behaviors. Continuous future process trajectories are subsequently visualized by re-fitting the simple kinetic model (soft sensor) using the data-driven model predicted discrete future data points, enabling the accurate monitoring of ongoing processes at any operating time. This framework was tested to maximize fed-batch microalgal lutein production by combining with different online optimization schemes and compared against the conventional open-loop optimization technique. The optimal results using the proposed framework were found to be comparable to the theoretically best production, demonstrating its high predictive and flexible capabilities as well as its potential for industrial application.

Exploring biostimulation of plant hormones and nitrate supplement to effectively enhance biomass growth and lutein production with thermo-tolerant Desmodesmus sp. F51

In this study, the interactive effect of plant hormone-salicylic acid and succinic acid on biomass growth, lutein content, and productivity of Desmodesmus sp. F51 were investigated. The results demonstrated that the synergistic action of salicylic acid and succinic acid could effectively enhance the assimilation of nitrate and significantly improve lutein production. The maximal lutein content 7.01 mg/g and productivity 5.11 mg/L/d could be obtained with a supplement of 100 µM salicylic acid and 2.5 mM succinic acid in batch culture. Furthermore, operation strategy of nitrate fed-batch coupled with supplementation for succinic acid and salicylic acid resulted in further enhancement of lutein content and productivity by 7.50 mg/g and 5.78 mg/L/d, respectively. The performance is better than most of the previously reported values.

Bench-Scale Cultivation of Microalgae Scenedesmus almeriensis for CO2 Capture and Lutein Production

In this study, Scenedesmus almeriensis as green microalga was cultivated on bench-scale for carbon dioxide (CO2) capture and lutein production. The autotrophic cultivation of S. almeriensis was carried out by using a vertical bubble column photo-bioreactor (VBC-PBR) with a continuous flow of a gaseous mixture of oxygen (O2), nitrogen (N2), and CO2, the latter in content of 0.0–3.0 %v/v. The liquid phase was batch. S. almeriensis growth was optimized. In addition, lutein extraction was carried out by using accelerated solvent extraction with ethanol as Generally Recognized as Safe (GRAS) solvent at 67 °C and 10 MPa. Upon optimization of CO2 concentration, the maximum biomass productivity, equal to 129.24 mg·L−1·d−1, was achieved during the cultivation by using a content of CO2 equal to 3.0 %v/v and it allowed to obtain a lutein content of 8.54 mg·g−1, which was 5.6-fold higher in comparison to the analogous process carried out without CO2 addition. The ion chemical analysis in the growth medium showed that by gradually increasing CO2 content, the nutrient consumption during the growth phase also increased. This study may be of potential interest for lutein extraction at industrial scale, since it is focused on pigment production from a natural source with a concomitantly CO2 capture.

Bioprocess operation strategies with mixotrophy/photoinduction to enhance lutein production of microalga Chlorella sorokiniana FZU60

This study isolated and identified the lutein-enriching microalga Chlorella sorokiniana FZU60. Different types of media and concentrations of sodium acetate and nitrate were evaluated to improve mixotrophic growth and lutein production. Highest lutein content, production, and productivity were obtained in BG11 medium with 1 g/L acetate and 0.75 g/L nitrate. Additionally, pulse feeding with 1 g/L acetate every 48 h led to the alternation between mixotrophy and photoinduction, resulting in a lutein production of 33.6 mg/L. Most notably, excellent lutein content (9.57 mg/g) and productivity (11.57 mg/L/d) were obtained using a new multi-operation integrated strategy, and the achieved levels exceed those reported in most related studies. This work demonstrates the synergistic integration of simple and effective strategies for the enhancement of lutein production in the indigenous microalga C. sorokiniana FZU60 and provides new insight into the highly efficient microalgae-based lutein production.

A novel process for the mixotrophic production of lutein with Chlorella sorokiniana MB-1-M12 using aquaculture wastewater

In this study, microalgal cultivation was applied as a feasible strategy for treating shrimp culture wastewater (SCW) from a shrimp farm in southern Tainan. Chlorella sorokiniana MB-1-M12 was first grown on BG-11 medium with 0.5% salinity, obtaining a biomass concentration and productivity of 4.35 g/L and 1.56 g/L/d, respectively. When 80% of BG-11 nutrients were added to 75% strength SCW, lutein content and productivity increased to 5.19 mg/g and 5.55 mg/L/d, respectively. A novel operation strategy involving periodic exchange of freshwater and SCW was designed for semi-continuous cultivation of MB-1-M12 strain for optimal biomass and lutein production. The average biomass concentration, productivity, lutein content, and productivity were 3.5 g/L, 1.3 g/L/d, 3.89 mg/g and 5.0 mg/L/d, respectively. Although microalgae have been considered as an alternative natural source of lutein, this work is among the earliest reports describing lutein production from microalgae cultivated with wastewater via a circular economy concept.

Purification of lutein from the green microalgae Chlorella vulgaris by integrated use of a new extraction protocol and a multi-injection high performance counter-current chromatography (HPCCC)

Lutein is a natural carotenoid with health promoting properties whose commercial source is the yellow Marigold flower, but its production is limited by climate, planting area and high labor costs. The microalga Chlorella vulgaris is a promising lutein source; however, no commercial lutein production uses microalgae. In the present study, the lower phase (LP4) of the biphasic solvent system composed of n-heptane–ethanol–water (5:4:1.5, v/v/v) was used both as a solvent for microalgae biomass extraction and as a mobile phase for lutein isolation using high performance counter-current chromatography (HPCCC). The ultrasound-assisted extraction of biomass with LP4 for 30 min led to an extract with a lutein content (3.20 mg/g dried biomass) that was higher than those obtained by the reference solvents tetrahydrofuran and dichloromethane. A multi-injection HPCCC method was developed for lutein isolation, where LP4 was pumped as the mobile phase at a flow rate of 8 mL/min, whereas its upper phase was the stationary phase. In total, 2 g of C. vulgaris extract were processed affording 60 mg of lutein (92% purity), which was further cleaned up by gel permeation chromatography yielding 50 mg of lutein (97% purity). The chemical identity of the purified compound was confirmed by mass spectrometry and absorption spectroscopy in comparison with an authentic standard. The developed HPCCC method may serve as a reference for promoting the development of a new production model of microalgae-based lutein.

Growth medium and nitrogen stress sparked biochemical and carotenogenic alterations in Scenedesmus sp. CCNM 1028

Microalgae are a spring of valuable compounds finding its wide applicability as cosmo-nutraceuticals, a renewable substitute for bioenergy and bioremediation. The present study utilizes Scenedesmus sp. CCNM 1028, freshwater microalgae, to understand the influence of various microalgal growth media, like BG-11, Chu's 10, Bold's basal medium (BBM) and Zarrouk's media (ZM), on the growth and lutein production. Furthermore, two-stage nitrogen starvation studies were carried out using the selected BG-11 media and its effect on the lutein and biochemical parameters of Scenedesmus sp. CCNM 1028 after 0, 6, 9, 15 and 21 days (d) of nitrogen starvation were recorded. Lutein was accumulated till 6d of nitrogen starvation. Additionally, ~3-fold increase of carbohydrate content (%w/w) was observed after 21d of nitrogen starvation with respect to the control. Thus, this study proposes two-stage nitrogen starvation as a capable strategy to cultivate Scenedesmus sp. CCNM 1028 for lutein (6d) and carbohydrate production (21d).

Performance evaluation of an outdoor algal biorefinery for sustainable production of biomass, lipid and lutein valorizing flue-gas carbon dioxide and wastewater cocktail

We evaluated wastewater remediation and CO2 utilization potential of Chlorella minutissima from flue gas in a raceway pond, while synthesizing lutein and lipid for potential healthcare and biofuel application. A mix of 20% kitchen waste, 10 g L−1 of poultry litter waste and 5% flue gas was maintained while cultivating the microalga. Complete removal of nitrate, nitrite and ammonium, 85% carbon and 91% phosphorus was observed. An average areal biomass productivity of 4.06 ± 0.12 g m−2 day−1 with a specific growth rate of 0.34 ± 0.03 day−1 was observed within 9 days. Biomass productivity of 6.21 ± 0.16 g m−2 day−1 with a specific growth rate of 0.34 ± 0.03 day−1 was achieved during winter. Furthermore, lipid content with appropriate fatty acid composition 1.04:1 (saturation:unsaturation) increased from 25% to 58%. Additionally, lutein productivity of 1.2 ± 0.08 mgL−1 day−1, while utilizing 80.74 ± 0.07 mg L−1 day−1 of CO2 from 5% flue gas was obtained.

Manipulating Nutritional Conditions and Salinity-Gradient Stress for Enhanced Lutein Production in Marine Microalga Chlamydomonas sp.

Marine microalgae has great potential for lutein production with the advantage of saving fresh water resource. Thus, marine microalga Chlamydomonas sp. JSC4 is investigated as a potential lutein producer in this study. The medium types, nitrate-N and sea salt concentration are individually investigated to promote the cell growth rate and lutein production of JSC4. In Modified Bold Basal 3N medium, cell growth and lutein content are optimal at the nitrate-N concentration of 1000 mg L-1 and sea salt concentration of 2%. In addition, an innovative salinity-gradient strategy is operated to dramatically enhance biomass productivity (560 mg/L/d) and lutein content (3.42 mg g-1 ), resulting in the optimal lutein productivity (1.92 mg/L/d). Overall, this study clearly demonstrates that salinity is a significant inducer of lutein accumulation by strain JSC4 and that lutein production can be successfully optimized using the salinity-gradient strategy, which is beneficial for the outdoor large-scale lutein production in the future.

Enhancing lutein production with mixotrophic cultivation of Chlorella sorokiniana MB-1-M12 using different bioprocess operation strategies

A lutein-enriched mutant, Chlorella sorokiniana MB-1-M12 was grown mixotrophically for lutein production. The lutein production efficiency of the strain was enhanced via optimizing the operating strategies. The results show that using semi-continuous cultivation with a medium replacement ratio of 75% resulted in a higher lutein productivity and lutein concentration of 6.24 mg/L/d and 50.6 mg/L, respectively, which were markedly higher than those obtained from batch and fed-batch cultivation. Cultivation under simulated outdoor cultivation conditions (i.e., temperature of 35 °C/25 °C for a 12 h/12 h light/dark cycle) could achieve the highest lutein productivity and lutein concentration of 3.34 mg/L/d and 30.8 mg/L, respectively. Lutein production via outdoor cultivation of MB-1-M12 strain with a 60-L tubular photobioreactor was performed using semi-continuous operation. With a medium replacement ratio of 75%, a good lutein productivity (4.46 mg/L/d) and concentration (27.4 mg/L) was obtained, indicating the feasibility of producing lutein under outdoor cultivation of the microalgal strain.

Manipulation of Environmental Stress Towards Lutein Production in Chlorella fusca Cell Culture

All carotenoids originate from a single, common precursor, phytoene. The colour of carotenoids is determinedby desaturation, isomerization, cyclization, hydroxylation and epoxidation of the 40-carbon phytoene. The conjugated double-bond structure and nature of end ring groups confer on the carotenoids properties such as colour and antioxidant activity. Algae may become major sources of carotenoids but the extent of environmental stress and genetic influences on algae carotenoid biosynthesis are poorly understood. Carotenoid biosynthesis can be influenced by many aspects and is liable to geometric isomerization with the existence of oxygen, light and heat which affect the colour degradation and oxidation. Therefore, in this study carotenoid biogenesis is investigated in cell culture of Chlorella fusca as a potential model system for rapid initiation, and extraction of carotenoids by providing stringent control of genetic, developmental and environmental factors. The value of this experimental system for investigating key factors controlling the carotenoid accumulation is then tested by assessing the effects of environmental variables, such as drought stress, light intensity, nutrient strength and media formulation on carotenoid accumulation. Our findings revealed that the conversion of violaxanthin to lutein is due to irradiance stress condition, nutrient strength as well as drought stress. As a result, manipulation of environmental variables will up-regulate lutein concentration. This reaction will restrict the supply of precursors for ABA biosynthesis and the algae cell culture responds by increasing carotenogenic metabolic flux to compensate for this restriction. In conclusion, selecting the appropriate algae species for the appropriate environmental conditions is not only important for yield production, but also for nutritional value quality of carotenoid.

Enhancing lutein productivity of Chlamydomonas sp. via high-intensity light exposure with corresponding carotenogenic genes expression profiles

The marine microalga Chlamydomonas sp. JSC4 is a potential lutein source with high light tolerance. In this study, light intensity was manipulated to enhance cell growth and lutein production of this microalga. High lutein productivity (5.08 mg/L/d) was achieved under high light irradiation of 625 μmol/m2/s. Further increase in light intensity to 750 μmol/m2/s enhanced the biomass productivity to 1821.5 mg/L/d, but led to a decrease in lutein content. Under high light conditions, most carotenoids and chlorophyll contents decreased, while zeaxanthin and antheraxanthin contents increased. Inspection of gene expression profile shows that the lut1 and zep genes, responsible for lutein synthesis and flow of zeaxanthin into violaxanthin, respectively, were downregulated, while zeaxanthin biosynthesis gene crtZ was upregulated when the microalga was exposed to a high light intensity. This is consistent with the decrease in lutein content and increase in zeaxanthin content under high light exposure.

Strategies related to light quality and temperature to improve lutein production of marine microalga Chlamydomonas sp.

The marine microalga Chlamydomonas sp. JSC4 was examined for its potential as a lutein producer. Environmental conditions, including light quality, temperature and light wavelength mixing ratio, were individually altered to enhance the cell growth rate and lutein production in strain JSC4. Results showed that optimal cell growth was obtained under white light and a temperature of 35°C, while the optimal lutein content was obtained under blue light and a lower temperature of 20-25°C. The best lutein production occurred when using a mixing ratio of 3:1 (white light: blue light). Strategies related to light quality and temperature (namely, temperature-gradient and two-stage strategies) were then used to further improve lutein production. Among them, the two-stage strategy proved to be effective markedly improving lutein content from 2.52 to 4.24mg/g and resulting in the highest lutein productivity of 3.25mg/L/day.

Chromate Resistance Mechanisms in Leucobacter chromiiresistens.

Chromate is one of the major anthropogenic contaminants on Earth. Leucobacter chromiiresistens is a highly chromate-resistant strain, tolerating chromate concentrations in LB medium of up to 400 mM. In response to chromate stress, L. chromiiresistens forms biofilms, which are held together via extracellular DNA. Inhibition of biofilm formation leads to drastically decreased chromate tolerance. Moreover, chromate is reduced intracellularly to the less-toxic Cr(III). The oxidation status and localization of chromium in cell aggregates were analyzed by energy-dispersive X-ray spectroscopy coupled to scanning transmission electron microscopy and X-ray absorption spectroscopy measurements. Most of the heavy metal is localized as Cr(III) at the cytoplasmic membrane. As a new cellular response to chromate stress, we observed an increased production of the carotenoid lutein. Carotenoid production could increase membrane stability and reduce the concentration of reactive oxygen species. Bioinformatic analysis of the L. chromiiresistens genome revealed several gene clusters that could enable heavy-metal resistance. The extreme chromate tolerance and the unique set of resistance factors suggest the use of L. chromiiresistens as a new model organism to study microbial chromate resistance.IMPORTANCE Chromate is a highly toxic oxyanion. Extensive industrial use and inadequate waste management has caused the toxic pollution of several field sites. Understanding the chromate resistance mechanisms that enable organisms to thrive under these conditions is fundamental to develop (micro)biological strategies and applications aiming at bioremediation of contaminated soils or waters. Potential detoxifying microorganisms are often not sufficient in their resistance characteristics to effectively perform, e.g., chromate reduction or biosorption. In this study, we describe the manifold strategies of L. chromiiresistens to establish an extremely high level of chromate resistance. The multitude of mechanisms conferring it make this organism suitable for consideration as a new model organism to study chromate resistance.

Recombinant Synechococcus elongatus PCC 7942 for improved zeaxanthin production under natural light conditions

Cyanobacteria are globally recognized as potential photosynthetic platform cell factories for production of value-added chemicals. Carotenoids are industrially important fine chemicals used in food, pharmaceutical and health-care products. Zeaxanthin has emerged as a high value xanthophyll carotenoid that exhibits superior antioxidant properties over many other carotenoids. Traditionally sourced from plants like marigold flowers wherein, zeaxanthin co-occurs with lutein. Therefore, there is a need for isolated production system for zeaxanthin on account of difficulties in separation of zeaxanthin from lutein due to their very similar molecular structures. Synechococcus elongatus PCC 7942 (PCC 7942) is a cyanobacterium that is known to synthesize zeaxanthin as one of the predominant cellular carotenoids and does not possess pathway genes for lutein production. In order to construct a system capable of industrial production of zeaxanthin, we genetically modified PCC 7942 (Synechococcus 79R48) to improve β‑carotene flux towards zeaxanthin synthesis by cloning CrtR (β‑carotene oxygenase) gene from Synechococcus elongatus PCC 7002 through homologous recombination. This strategy effectively enhanced the yield (mg/g DCW) of zeaxanthin in the transformants. Moreover, to increase zeaxanthin titer (mg/L), an operon construct was synthesized where a heterologous gene GalP (Hexose-H+ symporter) from E. coli MG1655 was cloned downstream of CrtR (Synechococcus 79RG48) which facilitated inherently obligate photoautotrophic WT PCC 7942 cells to uptake extracellular glucose in transformants, thereby increasing biomass productivity. Autotrophically grown cells yielded 9.02 ± 1.10 mg/g DCW of zeaxanthin while under mixotrophy the yield was 8.09 ± 0.19 mg/g DCW, which was 2-fold improvement over the wild type. The volumetric productivities of the transformants were 1.18 ± 0.17 mg/L·d and 1.8 ± 0.06 mg/L·d under autotrophic and mixotrophic conditions, respectively while WT produced 0.58 ± 0.02 mg/L·d. The work was successfully able to demonstrate that the modifications resulted in, enhanced production of lutein-free zeaxanthin in the cyanobacterial system.

Photosynthetic Accumulation of Lutein in Auxenochlorella protothecoides after Heterotrophic Growth.

In order to enhance lutein accumulation and to explain the reasons for the difference in lutein accumulation under photoautotrophic and heterotrophic conditions, different culture modes and the associated transcriptome profiles were investigated in Auxenochlorella protothecoides. The heterotrophic-photoautotrophic transition culture mode was investigated for lutein accumulation, changing from organic carbon to increase biomass in dark fermentation to irradiation under nitrogen rich conditions. This strategy increased the lutein content 10 times along with chloroplast regeneration and little biomass loss in 48 h. The highest lutein productivity and production in the heterotrophic-photoautotrophic transition culture reached 12.36 mg/L/day and 34.13 mg/L respectively within seven days. Furthermore, compared to the photoautotrophic conditions, most genes involved in lutein biosynthesis and photosystem generation were down-regulated during heterotrophic growth. By contrast, two β-ring hydroxylases were transiently upregulated, while violaxanthin de-epoxidase and zeaxanthin epoxidase were mostly downregulated, which explained the extremely low lutein content of heterotrophic cells. Nevertheless, the lutein proportion in total carotenoids reached nearly 100%. This study is the first to our knowledge to report on a comparative transcriptome analysis of lutein biosynthesis, and it provides a promising strategy to boost lutein production in A. protothecoides.

Dynamic modeling and optimization of sustainable algal production with uncertainty using multivariate Gaussian processes

Dynamic modeling is an important tool to gain better understanding of complex bioprocesses and to determine optimal operating conditions for process control. Currently, two modeling methodologies have been applied to biosystems: kinetic modeling, which necessitates deep mechanistic knowledge, and artificial neural networks (ANN), which in most cases cannot incorporate process uncertainty. The goal of this study is to introduce an alternative modeling strategy, namely Gaussian processes (GP), which incorporates uncertainty but does not require complicated kinetic information. To test the performance of this strategy, GPs were applied to model microalgae growth and lutein production based on existing experimental datasets and compared against the results of previous ANNs. Furthermore, a dynamic optimization under uncertainty is performed, avoiding over-optimistic optimization outside of the model’s validity. The results show that GPs possess comparable prediction capabilities to ANNs for long-term dynamic bioprocess modeling, while accounting for model uncertainty. This strongly suggests their potential applications in bioprocess systems engineering.

Indigenous microalga Parachlorella sp. JD-076 as a potential source for lutein production: Optimization of lutein productivity via regulation of light intensity and carbon source

Lutein has attracted attention because of its beneficial roles, including alleviating cardiovascular diseases. We compared lutein production of diverse indigenous algal candidates, and selected Parachlorella sp. JD-076 as a lutein producer. The strain was cultivated under different types of photo-bioreactors (PBRs). Maximal growth was obtained from the tubular type with 2.7-fold higher dry cell weight (DCW) than in the cylindrical type. To optimize lutein production, Parachlorella sp. was exposed to various levels of illumination (100–1000 μmol m−2 s−1) and combinations of glucose and CO2 in the tubular-type PBR. High-light intensity efficiently increased DCW and lutein productivity in an intensity-dependent manner, and 5% CO2 resulted in the maximum biomass and lutein productivity compared to that of other conditions, with 2.1 g L−1 day−1 and 25 mg L−1 day−1, respectively. We speculated that the strain Parachlorella sp. JD-076 will provide a cost-effective lutein bioprocess via regulation of illumination and CO2 supply.

High lutein production by a halo-tolerant strain of Dunaliella sp. (Chlorophyceae) isolated from solar salterns in Central Italy

High lutein production by a halo-tolerant strain of Dunaliella sp. (Chlorophyceae) isolated from solar salterns in Central Italy